Latest Trends in Bioinoculant Production Technology

Biofertilizers improve soil texture and yield of plants.
They do not allow pathogens to flourish.
They are eco-friendly and cost-effective.
Biofertilizers protect the environment from pollutants since they are natural fertilizers.
They destroy many harmful substances present in the soil
that can cause plant diseases.
Biofertilizers are proved to be effective even under semi- arid conditions.

1. COLLEGE OF A GRIC U L TUR A L SCIENCES Latest Trends in Bioinoculant Production Technology M. MELVIN JOE DEPT OF AGRICULTURAL MICROBIOLOGY

2. Biofertilizers improve soil texture and yield of plants. They do not allow pathogens to flourish. They are eco-friendly and cost-effective. Biofertilizers protect the environment from pollutants since they are natural fertilizers. They destroy many harmful substances present in the soil that can cause plant diseases. Biofertilizers are proved to be effective even under semi- arid conditions. “Biofertilizers are substances that contain microorganisms, which when added to the soil increase its fertility and promotes plant growth.” Biofertilizers are important for the following reasons:

3. Agricultural M i cro b io l ogy Associative Symbiotic Nitrogen Fixation- Azospirillum sp. Symbiotic Nitrogen Fixation- Rhizobium , Frankia sp. Symbiotic Nitrogen Fixation- Azotobacter sp. Phosph ate Solubilizing Microbes- Bacillus sp., Pseudomonas sp. Phosphate Mobilizing Microbes -Mycorrhizae Potash mobilizing bacteria - Frateuria aurentia Silicate solubilizing bacteria - Bacillus sp. Zinc Solubilizing Bacteria- Pseudomonas fragi

4. Non-symbiotic nitrogen fixing forms such as cyanobacteria Anabaena, Nostoc , Azospirillum, and Azotobacter, etc. 180 × 10 6 metric tons per year globally 78% N 2 unavailable to plants Symbiotic N 2 -fixing bacteria -symbiosis with legumes- Rhizobium , Sinorhizobium , plants and trees – Frankia sp. Nitrogen fixing Bacteria

5. Convert organic and inorganic insoluble to soluble P Bacteria- Bacillus megaterium, Pseudomonas striata; Fungi- Aspergillus niger, Penicillium sp. 30 million tons of P fertilizers- actual uptake efficiency only 20% by plant system. Mycorrhizae absorb phosphorus, mobilizing it from wide soil surfaces into the plant Phosphate Solubilizing Microorganisms P Solubilization as shown by clearance Microbe mediated P Solubilization

6. K, Zn and Si s o lubiliz a tion Potash mobilizing bacteria Frateuria aurentia – mobilizing bacteria - increase the availability of more potash in usable form to the plants. Silicate solubilizing bacteria Bacillus sp. converts insoluble inorganic (Ca, Al, K and Mg) silicates and biogenic materials to available Si Zinc Solubilizing Bacteria Zinc solubilizing bacteria Pseudomonas fragi and Pantoea dispersa convert inorganic zinc to available forms.

7. Pink Pigmented Facultative Methylotrophs Methylobacterium spp. dominate the phyllosphere bacterial population Endophytic and epiphytic association with the plants Produce exopolysaccharide (EPS), Indole acetic acid (IAA) and aminocyclopropane-1-carboxylic acid (ACC) deaminase protect plants against flooding, drought, salt, flower wilting, metals, organic contaminants, and pathogens IAA signaling molecule and promotes plant growth EPS maintain moisture content and growth of plants under severe drought ACC deaminase lower plant stress ethylene

8. Cyanobacteria and Azolla Cyanobacteria are oxygenic photosynthetic capable of fixing atmospheric nitrogen. Cyanobacteria such as Nostoc, and Anabaena differ in many respects from the legume nodules fix N through heterocyst Azolla; pteridophyte that lives in symbiosis with cyanobacterium Rice with Azolla “blooms” fix up to 600 Kg N ha -1 yr -1 during the growing season Azolla cultivation

9. The bacterial symbionts are Gram negative, non-spore forming, aerobic rods and the bacterial species are grouped into the genera Rhizobium, Bradyrhizobium, Mesorhizobium, Photorhizobium, Sinorhizobium and Azorhizobium . Isolation of R h izobi u m F r om Ro o t Nod u les

10. Uproot the plant and wash roots thoroughly. Collect nodules from plant without damaging it. Select pink and healthy nodules. Surface sterilize the nodules by immersing in 70% ethanol for 1-3 min Rinse the nodules in sterile water. Surface sterilize the nodules again with 0.1% (W/V) mercuric chloride for 2-3 min Rinse the nodules 3 times in sterile water. Prepare nodule suspension by crushing the nodule in a small quantity of sterile water using a glass rod. Transfer 1 ml nodule suspension on to a sterile Petri plate and add 15-20 ml of Congo Red Yeast Extract Mannitol Agar Medium and incubate at room temperature for 3-5 days. Appearance of white translucent colonies indicates Rhizobium. Red colored, small colonies are Agrobacterium .

11. Agrobacterium Rhizobium Differentiation between Agrobacterium and Rhizobium

12. Isolation of Azospirillum microaerophilic, Gram negative, associative nitrogen fixing bacterium. Brazilian microbiologist, Johanna Dobereiner, first isolated the organism. There are 12 species including A. brasilense, A. lipoferum, A. halopra eferense, A. amazonense, A. irakense,A.oryzae, A.serapedicae, A. dobereinerae, A. form osense, A. rugosum, A. picis and A. zeae . N-Free Semisolid Malic Acid Medium Malic acid Potassium hydroxide : 5.0g : 4.0g Dipotassium hydrogen orthophosphate : . 5 g Magnesium sulphate Sodium chloride Calcium chloride : 0.2g : 0.1g : 0.2g Fe-EDTA (1.64% w/v aqueous) : 4.0 ml Trace element solution : 2.0 ml BTB (0.5% alcoholic solution) : 2.0 ml Agar Distilled water pH : 1.75 g : 1000 ml : 6.8

13. Procedure Prepare semisolid malic acid medium in test tubes in 5 ml quantity and sterilize @ 121 o C (15 psi) for 15 min. Collect fresh root samples from any graminaceous plant Wash the roots in tap water to remove the adhering soil particles. Using sterilized knife/ blade, cut the roots into small bits of 1-2 cm size. Surface sterilize the root bits by immersing them in either 80% ethanol or 0.1% mercuric chloride for 1 min. Wash the root bits with sterile distilled water 3-4 times to remove the excess ethanol or mercuric chloride. Using sterile forceps transfer aseptically 2-3 root bits to the test tubes containing N-free semisolid malic acid medium. Incubate the tubes under room temperature 28  2 o C for 2-3 days Maintain one tube as control without root bits

14. Pellicle formation in Azospirillum brasilense TEM image of Azospirillum brasilense

15. Isolation of Phosphobacteria They include different groups of microorganisms such as bacteria and fungi, which convert insoluble inorganic phosphate compounds into soluble form, e.g., Pseudomonas striata , Bacillus megaterium var.phosphaticum, Bacillus subtilis , Penicillium digitatum and Aspergillus awamori . Pikovskaya’s Broth : 10.0 g 5.0 g 0.5 g 0.2 g : 0.1 g : T race T race Glucose Ca 3 (PO 4 ) 2 : (NH 4 ) 2 SO 4 : KCl : MgSO 4 . 7H 2 O MnSO 4 FeSO 4 : Yeast Extract D i stil l ed W at e r : : 0.5 g 1000 ml Sperber’sHydroxy Apatite Medium Soil Extract : 100 ml Gluc o se Agar : 1.0 g : 2.0 g

16. Fig. Clear zone in soil extract calcium phosphate medium supplemented with: (A) Congo red; (B) Bromocresol green; (C) Bromothymol blue with the plates incubated at 28 ± 2 °C, for an incubation period of 7 days.

17. The benefits are as follows: ability to fix atmospheric nitrogen; secrete plant growth hormones (viz, IAA, GA) and growth factors (viz., thiamine, riboflavin etc.) and produces some antifungal antibiotics. A. chroococcum - Produces black pigments (melanin) A. vinelandii - Produces yellow pigments A. beijerinckii - Produces green yellow fluorescent pigments Isolation of Azotobacter W a k sm a n No : 77 M edium Mannitol CaCO 3 K 2 HPO 4 MgSO 4 .7H 2 O : NaCl : F e r ric c hlorid e : MnSO 4 .4H 2 O : : 10.0 g : 5.0 g : 0.5 g 0.2 g 0.2 g T race T r ace N - f r ee wash e d Aga r : 15.0 g pH : 7.0 D is t ill e d W ater : 1000 m l

18. Weigh one g of sample and put in the 100 ml water blank and mix thoroughly Shake for 15 min for complete dispersion ( This gives 10 -2 dilution) Transfer one ml of the suspension to 9 ml water blank (This gives 10 -3 dilution) Transfer 1 ml of appropriate dilutions (10 -3 ) to Petri dishes Maintain 2 or 3 replications for each dilution Pour melted and cooled media (just before solidification) of about 15 ml and mix well by shaking clock wise and anti clock wise for 3 or 4 times and allow it for complete solidification Incubate the plates in inverted position at room temperature fo 3-4 days for appearance of Azotobacter colonies.

19. Isolation of Plant Growth Promoting Rhizobacteria(PGPR) ( Pseudomonas sp.) Rhizosphere bacteria that favor plant growth are termed as Plant Growth Promoting Rhizobacteria (PGPR). They improve plant growth directly by producing plant growth regulators such as auxins, gibbrellins and cytokinins; by eliciting root metabolic activities and / or by supplying biologically fixed nitrogen.

20. Add 1g sample to 100ml sterile water blank. This will give a dilution of 1:100 (10 -2 ) Transfer 1 ml of the supernatant from 10 -2 dilution to 9 ml blank with a sterile 1.0 ml pipette which will give a dilution of 10 -3 3. Repeat the above process with the second 9.0 ml blank, so that the second tube have a dilution of 10 -4 – 4 4. Transfer 1.0 ml aliquot from 10 -3 and 10 to sterile Petri- d i sh e s. D i l ut i o n s sh o u ld b e p l at e d i n trip l ic a te for greater accuracy. Add approximately 15 to 20 ml molten and cooled medium (45 C) to each Petri dish and mix the inoculum by gently rotating the Petri dish. Incubate the plates at room temperature for 3-5 days. King’s B Medium Glycerol : 10.0 ml Protease peptone :20.0 g K 2 HPO4 : 1.5 g MgSO4. 7H2O :1.5 g Distilled Water :1000 ml pH : 7.2 Agar : 20.0 g

21. Ma ss p r oduction o f B io f e r ti lizer

22. Sterilization of growth medium in the Fermentor Close the air outlet valve and allow the pressure to build up inside the vessel Maintain a pressure of 15 1b / in2 at 121°C for 20 minutes Switch off the fermentor and cool the medium by circulating cool water. Mass culturing in Fermentor Spray the inoculation port with alcohol and flame thoroughly Allow the port to cool, inoculate the media in the fermentor vessel with the log phase culture grown in 5 litre flask. Usually 1 -2 % inoculum is sufficient, however inoculation is done up to 5% depending on the growth of the culture in the larger flasks. Turn on the air pump, open the air outlet valve- regulate the air flow to 3-10 lit of air per hour per lit. of the medium. harvest the broth with the population load of 10 9 cells ml -1 after incubation period through the sampling port. There should not be any fungal or any other bacterial contamination at 10 -6 dilution level

23. Carrier-based biofertilizers A Good carrier should have the following qualities: Highly absorptive (water-holding capacity) and easy to process; Non-toxic to microorganisms; Easy to sterilize effectively; Available in adequate amounts and low-cost; Provide good adhesion to seeds; Has good buffering capacity; High organic matter content and water-holding capacity of more than 50%.

24. Natural and synthetic carriers

25. Quality Control in Biofertilizer production

26. Name of the Product Name and address of the Manufacturer Carrier used Batch number Date of manufacture Date of expiry Net Mass Crop for which Intended Storage Instructions Application Instructions (viii) Marking on the Pocket: The Inoculant Packet shall have the following information:

27. Application-Method of applying biofertilizers Seedling root dip: This method is usually applicable for rice crops. The seedlings are sowed in the bed of water and kept for 8-10 hours. Seed Treatment: The seeds are soaked in the mixture of nitrogen and phosphorus fertilizers. These seeds are then left to dry and are sown as soon as possible. Soil Treatment: The biofertilizers along with the compost fertilizers are blended and kept for one night. This mixture is then scattered on the soil where the seeds have to be sown.

28. Curing unit Packing unit W ashing Room Chemi cal and glass war e sto ra ge R o om Storage unit Refrigerator Hot air o v en 50 f t 30 f t Layout for the proposed biofertilizer production unit 100 s q. f t 80 s q. f t F ermenter I ncubato r La m ina r air f l o w W orking A r ea Autoc l a v e Microwave oven, colony counter, microscope, w eighing balanc e , sti r r e r , pH m eter D is t illat i on unit

29. Non-recuring Cost in INR Laminar air f l o w cha m ber wit h gas attach m ent 1 1,50,000.00 Hot air o v en 1 50,000.00 Incubator 1 60,000.00 F ermenter 50 L 1 10,00,000.00 Refrigerator 1 20,000.00 R o tato r y sha k er 1 40,000.00 Microscope 2 1,00,000.0 Weighing balance, packing machine, water bath, stirrer, pH mete r , D is t illat i on unit 2 70,000.00 Chemical Racks 2 1,00,000.0 Glassware, plasticware and other miscellaneous As per r equi r ement 1,50,000.0 Recurring Manpower T echn i cia n - 1 @ 25 k pm Labour -1@15 K pm 48,000.00 Chemicals and m o ther cultu r e As per r equi r ement 1,00,000.00 T otal cost 18,88,000.00 Budget estimate for the proposed biofertilizer production unit

30. Economics for the biofertilizer production unit Total production capacity per year Total Units to be produced Expected income per year Azospirillum -Carrier 600 kg @ 200 per K g 1,20,000.00 Azospirillum-Liquid 100 L i t @ 300 per L i t 30,000.00 Rhizobium-Carrier 600 kg @ 250 per K g 1,50,000.00 Rh i z o bium L iquid 100 L i t @ 300 per L i t 30,000.00 Phosphobacteria 600 kg @ 200 per K g 1,20,000.00 Azotobacter 300 Kg @ 200 per Kg 60,000.00 Total 5,10,000.00

31. The key constraints that such a framework will combat include: Inadequate or incomplete policies and guidelines for regulation of biofertilizers and biopesticides; Multiple and often overlapping regulatory mandates by responsible authorities; Limited capacity, including staff, skills and laboratory for product monitoring; Inadequate enforcement of quality control for biofertilizers and biopesticides; Lack of biofertilizer- and biopesticide-specific regulations, standards and guidelines; Weak institutional arrangements with limited collaboration between relevant authorities .

32. Thank You