Biofertilizer Production

Sustainable agriculture via Biofertilizer application

Biofertilizer Production

Lesson: Siderophore Producing Microorganisms

  1. What are siderophores?

Siderophores are low molecular weight (MW ranging from 400 – 1500 Da) iron binding proteins (ferric ion specific chelating agents) secreted by microorganisms in response to iron starvation. They enable the organism to procure iron from insoluble forms by mineralization and sequestration. Siderophores scavenge iron from the environment and make the almost always essential mineral, available to the microbial cell.

Four types of siderophores have been reported: hydroxamate, catecholate, salicylate and carboxylate.

  1. What is the importance of siderophore producing microorganisms ?

Siderophores produced by rhizosphere inhabitants have been reported to facilitate absorption of iron by the plant and play a role in antagonism against phytopathogens. If such organisms are included as components of biofertilizers, application of such biofertilizers to seeds or agricultural soil can enhance the growth of the plants/ crops. Siderophores have applications in clinical, agricultural and environmental fields and they

have been related to virulence mechanisms in microorganisms pathogenic to both animals and plants.

 

  1. What are the examples of siderophore producing microorganisms?

 

Siderophores are synthesized by bacteria, actinomycetes, fungi and certain algae growing under iron starvation conditions.

Some important siderophore producing bacteria are Pseudomonas species, Bacillus sp., Escherichia coli, Salmonella, Klebsiella pneumoniae, Vibrio cholerae, Vibrio anguillarum, Aeromonas, Aerobacter aerogens, Enterobacter, Yersinia and Mycobacterium species.

Some important siderophore producing fungi include Aspergillus nidulans, A. versicolor, Penicillium chrysogenum, P. citrinum, Mucor, Rhizopus, Trametes versicolor, Ustilago sphaerogina, Saccharomyces cerivisiae, Rhodotorula minuta and Debaromyces species. Actinomycetes are aerobic gram positive filamentous bacteria with high guanine + cytosine (G+C) content and form asexual spores. Actinomycetes are usually saprophytic and prefer complex substrate for their growth. They are able to tolerate certain metals at high concentrations. Examples of siderophore producing actinomycetes are Actinomadura madurae, Streptomyces griseus and Nocardia asteroids. Actinomycetes produce both hydroxymate and salicylate types of siderophores.

Algae that produce siderophores: Schizokinen, a dihydroxamate type of siderophore, produced by Anabaena sp reported to facilitate iron uptake. Anabaena flosaquae and Anabaena cylindrica produce copper accumulating siderophores.

  1. How would you detect siderophore production?

All glassware used for detection of siderophore production are to be treated with/ immersed in 6N HCl overnight to remove residual iron and washed with double distilled water. All media and solutions are to be prepared in double distilled water. Chrome Azurol S solution [60.5 mg Chrome Azurol S (sulphonate) in 50 mL distilled water (D.W.)] and Hexadecyltrimethyl ammonium bromide solution (HDTMA 72.9 mg in 40 mL D.W.) are mixed and then the mixture is added to 10 mL of 1mM FeCl3.6H2O solution prepared in 10 mM HCl. The solution thus obtained is autoclaved, cooled and then added to 900 mL of molten sterile Nutrient Agar (for some organisms such as Pseudomonas sp. King’s B medium is used) base. This medium is then aseptically poured into sterile petri plates. A loopful of the culture inoculum is spot inoculated on sterile Chrome azurol S (CAS) agar plates containing nutrient agar as basal medium and incubated at 30 oC for 4 days. Uninoculated sterile medium will serve as the control. Development of orange halo around the growing bacterial colony indicatessiderophore production.

 

  1. What is the composition of Nutrient agar medium ?

 

  • Composition of Nutrient agar medium:

Ingredients Gms / Litre: Peptone 10.0, Beef extract 10.0, Sodium chloride 5.0. The pH of the medium is adjusted to 7.0-7.2. Agar-agar 25 g/ L is added to the medium before autoclaving it.

 

  1. How do you sterilize these media?

Autoclave the medium at 15 PSI (ie lbs/ in2), at 121.6°C for 15-20 min. Cool the medium 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.

 

  1. How would determine the type and quantity of the siderophores produced?

Hydroxamate, catecholate and carboxylate nature of siderophore is determined by examining absorption maxima (max) in a uv-vis spectrophotometer.

Upon addition of 1 mL of 2% aqueous FeCl3 to 1 mL of cell-free culture filtrate:

  1. a peak between 420-450 nm indicates the presence of ferric hydroxamates;
  2. a peak at 495 nm indicates the presence of ferric catecholate.
  3. A peak at 320, 250 and 210 nm indicates the presence of catecholate siderophore.
  4. A peak at 190-280 nm indicates the presence of copper-carboxylates.
  5. Chemical nature of siderophore produced can be confirmed by:
    1. Tetrazolium salt test (Snow, 1954) for hydroxamate nature,
    2. Arnow (1937) test for catecholate nature and
  • Vogel (1992) test for carboxylate nature can be performed.
  1. For quantification, 1 mL of culture filtrate is mixed with 1 mL of CAS assay solution and the A630 is read after 1 h of incubation at room temperature (Fekete et al., 1989). Simultaneously, 1mL of CAS assay solution is also added to compounds:
    1. deferrioxamine mesylate (standard for hydroxamate siderophores),
    2. 2, 3 dihydroxybenzoic acid (standard for catecholates) and
  • rhizoferrin (standard for carboxylates).

The A630 is read after 15 min of incubation at room temperature. The reference solution contains 1 mL of uninoculated medium and 1 mL of CAS solution. The amount of siderophore produced is extrapolated from the standard curve and denoted as μg/mL.

References

  1. http://www.jocpr.com/articles/studies-on-siderophore-production-by-microbial-isolates-obtained-from-rhizosphere-soil-and-its-antibacterial-activity.pdf
  2. Yeole, R.D., B.P. Dave and H.C. Dube, 2001. Siderophore production by fluorescent pseudomonads colonizing roots of certain crop plants. Indian J. Exp. Biol., 39: 464-464.
    PubMed|  http://nopr.niscair.res.in/bitstream/123456789/23753/1/IJEB%2039(5)%20464-468.pdf
  3. Schwyn, B. and J.B. Neilands, 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem., 160: 47-56.
  4. Neilands, J.D., 1995. Siderophores: Structure and function of microbial iron transport compounds. J. Biol. Chem., 270: 26723-26726.
    CrossRef|  Direct Link  |
  5. Arnow, L.E., 1937. Colorimetric determination of the components of 3,4-dihydroxyphenylalaninetyrosine mixtures. J. Biol. Chem., 118: 531-537.
    Direct Link|
  6. 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.
  7. Snow, G.A., 1954. A growth factor for Mycobacterium johneiII-degradation and identification of fragments. J. Chem. Soc., 49: 2588-2596.
  8. Vogel, A.L., 1992. Class Reactions (Reactions for Functional Groups). CBS Publishers, New Delhi, India, pp: 190.
  9. PGPR in biocontrol:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571425/pdf/gmb-35-1044.pdf

 

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12 thoughts on “Biofertilizer Production”

  1. Sir, I want to join this. I am assistant professor in microbiology. Whats the process to join

    1. Dear Sir,
      The course is being finalized and soon to be launched. I shall inform you once it is launched so that you and your colleagues and students can join.
      Thanks and regards,
      Mrs. Sneha Ogale

  2. I am working as Assistant professor microbiology (chb).. Interested in this activity..

    1. Dear Madam,
      The course is being finalized and soon to be launched. I shall inform you once it is launched so that you, your colleagues and students can join.
      Thanks and regards,
      Mrs. Sneha Ogale

  3. First of all congratulations Ma’am.
    It’s a very good initiative and need of society.
    Ma’am Please let me know once you launch this course.
    Will share the link among my friends and those who are interested.

    1. Hello Shahen,
      Thank you so much! The course is being finalized and soon to be launched. I shall inform you once it is launched so that you can share the link.
      Thanks and regards,
      Mrs. Sneha Ogale

  4. Congratulations Madam for taking this initiative.
    Let me know that how will I know whether I m registered or not?

    1. Dear Sir,
      Thank you for your interest. Welcome aboard. I am adding you to the whatsapp group of registered participants in order to coordinate your participation.
      Thanks and regards,
      Sneha Ogale

  5. Congratulations mam and thank you for lunching such an open resource.
    It will be helpful to farmer’s & I am one of them, eager to join.
    Please let me know when & how to join.

    1. Dear Milind,
      Welcome aboard. I am adding you to the whatsapp group of registered participants in order to coordinate your participation.
      Thanks and regards,
      Sneha Ogale

  6. Dear Sneha Ogale,

    My name is Chau – greenhouse grower in Vietnam.
    I’d like to take your Biofertilizer production course online. I’ve tried to sign up at Edmodo website using the group code written in this page. Howerver, it wasn’t a correct code.
    Please advise how to register for this course.

    Thank you very much for your sharing and helping.

    Regards,

    Chau

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