nodulation capability, this is well known that this nitrogen-fixing bacteria can not fix nitrogen unless it is symbiotically harboured in the nodule of leguminous plant.

## Application of Microbial Biofertilizers

Application of the microbial biofertilizer is an important step in the Biofertilizer Technology. If the microbial inoculant is not applied properly, the benefits from the biofertilizer may not be obtained. During application one should always remember that the most of the microbial biofertilizers are heterotropic, i.e. they can not prepare their own food and depend upon the organic carbon of soil for their energy requirement and growth. So, they either colonise in rhizosphere zone or live symbiotically within the root of higher plants. The bacteria which are colonised in the rhizosphere zone obtain their organic carbon compounds from the root exudes of the higher plants. The symbiotic ones obtain organic carbon directly from the root. So, microbial inoculants must be applied in such a way that the bacteria will be adhered with the root surface. So, in case of transplanting crops, the inoculant are applied through roots, and in case of the crops in which seeds are sown directly in the field, the inoculants are applied through the seeds so that they can colonize in the rhizosphere region when the young roots are emerged after germination of seed. On the basis of the above principal, the following inoculation methods have been developed:

1. Inoculation of the seeds by slurry inoculating technique

2. Inoculation of seeds by seed pelleting technique

3. Inoculation of the seedlings

4. Inoculation of the soil by solid inoculation technique

5. Inoculation of soil by liquid inoculation technique
# Mass Production of Carrier Based and Liquid Based Biofertilizers

Dr. AJEET KUMAR<fnref n="19" />

## Media Preparation and Starter Culture

Bacteria require different nutrients for their growth. These include: a) organic carbons source, b) nitrogen source and c) a variety of other elements dissolved in water. Blue Green Algae (BGA) that can fix atmospheric carbon dioxide, does not require any carbon source and the nitrogen-fixing bacteria, which can fix atmospheric nitrogen, do not require any nitrogen source. A medium is an aquatic solution of a variety of organic and inorganic compounds that can supplement the above requirements for the growth of different microorganisms. Generally, media are of two types:

a) General media and

b) Specific media.

General media is constituted for the growth of most of the microorganisms. Such a medium contains all the ingredients required for the growth of any microorganism. A specific medium is constituted for the growth of specific or specific group of microorganisms. During constitution of such medium, one or few additional components are added and /or one or few components are deleted from the general media depending upon the requirement of the specific microorganism. Again, according to the physical appearance, media are of two types:

a) Liquid media and

b) Solid media.

The liquid medium is solidified by the addition of solidifying agent-agar-agar. Liquid medium can harbour bacterial growth suspended in the media, whereas solid medium harbours microbial growth on the surface. Solid media may be prepared as slant or plate.

## Sterilization of medium in autoclave:

The media is then autoclaved at 121°C temperature and 15lb pressure. The process is as follows

* ✓ Check the water level of the autoclave to note whether the heating coil is completely immersed in water, add water, if necessary.

* ✓ Put the conical flasks, petridishes and the beaker with test tubes inside the basket of the autoclave.

* ✓ Set the lid, tie the screws of the autoclave and switch on the power supply.

* ✓ Keep the outlet of the steam open and wait until the inside air is completely released from the autoclave and only steam is coming out.

<footnote n="19">¹⁹ Assistant Professor-cum-Scientist (Soil Science-Soil Microbiology, Soil fertility, Soil Chemistry), Department of Microbiology, College of Basic Sciences & Humanities (CBS&H), RPCAU, Pusa-848125, Bihar. E-mail: ajeet.sri@rpcau.ac.in</footnote>
✓ When the inside air is completely released close the outlet and observe the meter indicating the inside pressure.

✓ When the pressure rises to 15 lbs, adjust the regulating valve to keep the pressure constant.

✓ Keep this pressure for 30 min and then close the regulating valve and switch off the power supply

✓ When the pressure comes down to zero, open the regulatory valve to release the steam and then open the lid and take out the contents.

## Prepare slants and plates:

The slants are necessary for culture storage. The slant preparation process is as follows:

<figure><img src="image_122.png" /></figure>

Put the culture tubes containing medium in slanting position on the table with the help of a wooden blade and allow cooling down. The medium will be solid after cooling down and thus slants are prepared.

<figure><img src="image_123.png" /></figure>

Take the conical flask containing molten media with agar-agar and the petridishes in the laminar flow cabinet and allow the medium to cool down to 50°C.

<figure><img src="image_124.png" /></figure>

Open each petridish by slightly lifting the upper lid, pour 15-20 ml of medium and close the lid.

<figure><img src="image_125.png" /></figure>

Keep to cool down and solidify and thus plates are prepared.

## Preparation of Starter Culture:

❖ The starter culture is a little amount of bacterial suspension, which is added to the medium to start the growth of that bacterium. Twin flask is a pair of flasks of identical size joined together by a latex tube. For the preparation of starter culture, this type of flask is used. Each flask contains a side arm below the neck position. The latex tube joining the two flasks is held together by this glass tube. The benefit of the use of the twin flask is that contamination can be avoided.

## Fermentation:

<figure><img src="image_126.png" /></figure>

A fermenter is a device in which the optimum conditions for the microbial growth and activity is established artificially. This device may be used for the production of microbial metabolites such as antibiotics or enzymes; it may also be used for the growth of microorganisms i.e. production of microorganism itself.

<figure><img src="image_127.png" /></figure>

A low-cost production unit has been developed for the production of microbial inoculants to be used as biofertilizers such as Rhizobium, Azotobacter, and Phosphate Solubilising Bacteria.

<figure><img src="image_128.png" /></figure>

This device can be prepared by investing small amount of money as compared to the scientific fermenter used in laboratories for research purposes

## Sterilization of the fermenter:
Fermenter is a metallic vessel for moist sterilization of any article. The principle of moist sterilization lies in the fact that when water is boiled in a closed system, the water vapour produced due to boiling accumulates within the vessel and increases the inside pressure. Thus, the boiling point of water increases beyond 100°C, which is the boiling point of water in normal atmospheric pressure. In this condition, the steam, released from the boiling water is of higher temperature. If any article placed in this vessel in such condition, the high temperature destroys the microorganisms present in or on the article.

## Inoculation, Growth, Quality Testing and Termination of growth:

Inoculation means addition of starter culture to the medium in the fermenter. For the production of microbial biofertilizers a small amount of suspension of the desired bacterium in pure form is inoculated to the medium. Care should be taken to maintain the quality of starter culture, as extent of purity (no contaminants should be allowed), size of the starter culture (in terms of culture volume and density of cell) and stage of growth.

Greater the size of starter culture, lesser the chance of contamination. If the starter culture is inoculated in its log phase, rapid initial growth will occur. Maintenance of proper physical and chemical environment inside the fermenter is essential for proper growth of microorganism.

Quality testing, in this case, is enumeration of cell density and its purity in the broth. When the cell density reaches the desired level, growth is terminated and the culture is ready for mixing with carrier. The time period required for optimum cell density is thus standardized.

## Carrier Preparation

Carrier is a medium, which can carry the microorganisms in sufficient quantities and keep them viable under specified conditions and easy to supply to the farmers. 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 amount and low cost.

* Provide good adhesion to seed.

* Has good buffering capacity.

Different carriers are available in the market like, Charcoal, Peat, Lignite, Rice husk etc.

* But considering all the above qualities Azolla powder is the most suitable carrier in this region. This is due to

* It has high water holding capacity (360%).

* It has good pH buffering capacity.

* It contains nutrient so bacteria can remain viable for a long period.

* It is easily available in this region.
# Biofertilizers-Storage, Shelf Life and Marketing

Dr. AJEET KUMAR<fnref n="20" />

## Biofertilizers: Storage, Shelf Life, and Marketing

Biofertilizers are microbial inoculants containing living microorganisms that enhance soil fertility and plant growth by fixing atmospheric nitrogen, solubilizing phosphorus, or producing growth-promoting substances. Proper **storage, shelf-life management, and effective marketing** are crucial for maintaining their efficacy and commercial success.

### 1. Storage of Biofertilizers

Proper storage ensures microbial viability and product effectiveness. Key factors include:

#### A. Temperature Control

* ✓ **Optimal Storage Temp:** 4°C to 10°C (refrigeration preferred).

* ✓ **Avoid High Temperatures:** >30°C kills microbes.

* ✓ **Avoid Freezing:** Can rupture microbial cells.

#### B. Moisture & Humidity

* ✓ **Dry Conditions:** Moisture encourages contaminant growth.

* ✓ **Airtight Packaging:** Prevents moisture absorption.

#### C. Light Exposure

* ✓ **UV Light Harmful:** Store in **dark or opaque containers.**

#### D. Packaging Material

* ✓ **Polyethylene bags** (for carrier-based biofertilizers).

* ✓ **Glass/Plastic Bottles** (for liquid formulations).

<footnote n="20">²⁰ Assistant Professor-cum-Scientist (Soil Science-Soil Microbiology, Soil fertility, Soil Chemistry), Department of Microbiology, College of Basic Sciences & Humanities (CBS&H), RPCAU, Pusa-848125, Bihar. E-mail: ajeet.sri@rpcau.ac.in</footnote>
## 2. Shelf Life of Biofertilizers

The shelf life depends on the microbial strain, carrier material, and storage conditions.

<table><thead><tr><td>Biofertilizer Type</td><td>Shelf Life (Months)</td><td>Storage Condition</td></tr></thead><tbody><tr><td>Rhizobium</td><td>6-12</td><td>4-10°C (Refrigerated)</td></tr><tr><td>Azotobacter / Azospirillum</td><td>6-9</td><td>Room Temp (≤30°C)</td></tr><tr><td>Phosphate Solubilizers (PSB)</td><td>6-12</td><td>Cool & Dry Place</td></tr><tr><td>Liquid Biofertilizers</td><td>12-24</td><td>Room Temp (Avoid Sun)</td></tr></tbody></table>

* ✓ **Liquid formulations** generally last longer than carrier-based ones.

* ✓ **Expiry Date:** Should be clearly mentioned on the package.

## 3. Marketing of Biofertilizers

* ✓ Effective marketing strategies are essential for farmer adoption and commercial success.

### A. Target Audience

* ✓ **Farmers** (organic & conventional).

* ✓ **Horticulturists & Floriculturists.**

* ✓ **Government Agricultural Schemes.**

### B. Marketing Strategies

#### 1. Awareness Campaigns

* o Farmer training programs, workshops, and demonstrations.

* o Collaboration with **Krishi Vigyan Kendras (KVKs)** and NGOs.

#### 2. Branding & Packaging

* o Attractive, informative labels with usage instructions.

* o Highlight benefits (e.g., "Increases yield by 20%").

#### 3. Distribution Channels

* o **Agri-input dealers**, cooperatives, and online platforms.

* o Government subsidies (e.g., **Paramparagat Krishi Vikas Yojana - PKVY**).
4. Digital Marketing

o Social media, YouTube tutorials, and e-commerce (Amazon, BigHaat).

5. Certifications & Quality Assurance

o ISI, FCO (Fertilizer Control Order), or ISO certification.

o Lab testing reports for credibility.

C. Challenges in Marketing

<figure><img src="image_129.png" /></figure>

Farmer skepticism (preference for chemical fertilizers).

<figure><img src="image_130.png" /></figure>

Short shelf life (requires efficient supply chain).

<figure><img src="image_131.png" /></figure>

Competition from synthetic fertilizers.

Conclusion

o Storage: Maintain **cool, dry, and dark conditions** to prolong shelf life.

o **Shelf Life:** Ranges from 6–24 months, depending on formulation.

o **Marketing:** Requires **awareness, branding, and strong distribution networks.**

Biofertilizers have immense potential in **sustainable agriculture**, but their success depends on proper handling and effective promotion
# Application technology of biofertilizers for seeds, seedlings, tubers, sets

Dr. AJEET KUMAR<sup>21</sup>

## 1. Introduction

Biofertilizer application requires specialized techniques to ensure optimal microbial colonization. This lecture covers precision application methods for different planting materials.

**Learning Objectives:**

* ✓ Master seed treatment technologies

* ✓ Learn seedling inoculation methods

* ✓ Understand tuber/set treatment protocols

* ✓ Compare traditional vs advanced application systems

## 2. Seed Application Technologies

### A. Slurry Treatment (Most Common)

**Procedure:**

1. Prepare 10% sugar/jaggery solution (adhesive)

2. Mix biofertilizer @ 500g/kg seed

3. Coat uniformly, shade-dry for 30 mins

4. Sow within 24 hours

**Advantages:**

* o Cost-effective (<₹2/acre)

* o Suitable for small seeds (pulses, cereals)

**Limitations:**

* Short window between treatment & sowing

### B. Advanced Seed Coating Methods

<table><thead><tr><td>Technology</td><td>Specifications</td><td>Crops</td></tr></thead><tbody><tr><td>Film Coating</td><td>Polymer + microbes (5-20µm layer)</td><td>Hybrid maize, vegetables</td></tr><tr><td>Encapsulation</td><td>Alginate beads (2-3mm)</td><td>High-value seeds</td></tr><tr><td>Bio-Priming</td><td>Hydration (12h)-Dehydration with microbes</td><td>Rice, wheat</td></tr></tbody></table>

**Efficacy Data:**

* Film coating increases rhizosphere colonization by 40%

* Bio-priming enhances germination rates by 15-25%

<footnote n="21"><sup>21</sup> Assistant Professor-cum-Scientist (Soil Science-Soil Microbiology, Soil fertility, Soil Chemistry), Department of Microbiology, College of Basic Sciences & Humanities (CBS&H), RPCAU, Pusa-848125, Bihar. E-mail: ajeet.sri@rpcau.ac.in</footnote>
### 3. Seedling Treatment Methods

#### A. Root Dip Technique

**For:** Paddy, tomato, cabbage transplants

**Protocol:**

1. Prepare 10L suspension (1kg biofertilizer + water)

2. Dip roots for 30-60 minutes

3. Transplant immediately

**Modified Approaches:**

* **Gel Dip:** Carboxymethyl cellulose-based (extends viability)

* **Mycorrhizal Dip:** For perennial crops

**\*\*B. Nursery Bed Application**

**Method:**

1. Mix 5kg biofertilizer with 100kg FYM

2. Apply to 1,000m² nursery bed

3. Cover with thin soil layer

**Best For:**

* Vegetable seedlings

* Forest species propagation

### 4. Tuber & Set Treatment

#### A. Tuber Application (Potato, Turmeric)

**Step-by-Step:**

1. Prepare slurry (1kg biofertilizer + 50L water)

2. Spray on tubers (5kg/treatment for 1 acre)

3. Dry in shade before planting

**Innovation:**

1. **Wax Coating:** Beeswax + microbes extends shelf-life

#### B. Set Treatment (Sugarcane, Banana)

**Two Approaches:**

1. **Furrow Application:**

* 10kg biofertilizer + 50kg sand per acre

* Apply in planting furrows

2. **Direct Coating:**

* Biofilm application on suckers

* Uses starch-based adhesives

### 5. Equipment for Large-Scale Application

<table><thead><tr><td>Equipment</td><td>Capacity</td><td>Suitable For</td></tr></thead><tbody><tr><td>Rotary seed treater</td><td>500kg/hour</td><td>Seed cooperatives</td></tr><tr><td>Vacuum coater</td><td>Precision coating</td><td>Research stations</td></tr></tbody></table>
<table><thead><tr><td>Equipment</td><td>Capacity</td><td>Suitable For</td></tr></thead><tbody><tr><td>Dip-tank systems</td><td>10,000 seedlings/hour</td><td>Commercial nurseries</td></tr></tbody></table>

**Cost Comparison:**

* Manual treatment: ₹50/acre

* Mechanical coating: ₹120/acre (but 5× faster)

**6. Recent Advances**

* ✓ **Nano-film Coatings:** 100nm microbial layers

* ✓ **Drone Spraying:** For field-established crops (5L/ha)

* ✓ **Smart Stickers:** pH-sensitive release on seed surface

**7. Field Validation Data**

<figure><img src="image_132.png" /></figure>

**ICAR Trials (2023):**

* Proper seed treatment gave 22% yield boost in chickpea

* Root dip increased tomato survival rate by 18%

**8. Best Practice Guidelines**

1. Always use fresh batches (<3 months old)

2. Avoid chemical fungicide contact for 48 hours

3. Morning/evening application reduces UV damage

4. Combine with organic amendments (FYM, compost)

**9. Discussion Questions**

1. Compare economics of manual vs mechanical application

2. How would you adapt these methods for arid regions?

3. Design a quality check protocol for treated seeds

**10. References**

* ✓ FAO (2022) *Microbial Inoculant Application Manual*

* ✓ ICAR Handbook (2023) *Biofertilizer Application Technologies*

* ✓ Journal of Applied Microbiology (2024) *Nano-coating Innovations*
# Factors Influencing the Efficacy of Biofertilizers

Dr. AJEET KUMAR<fnref n="22" />

## 1. Introduction

Biofertilizers are microbial inoculants that enhance soil fertility and plant growth by fixing atmospheric nitrogen, solubilizing phosphorus, or producing growth-promoting substances. However, their effectiveness depends on multiple biotic and abiotic factors.

**Learning Objectives:**

1. Understand the key factors affecting biofertilizer performance.

2. Analyze the role of environmental and management practices.

3. Discuss strategies to optimize biofertilizer efficacy.

## 2. Key Factors Influencing Biofertilizer Efficacy

### A. Microbial Strain Characteristics

1. **Strain Selection**

o Specificity of strains to host plants (e.g., *Rhizobium* for legumes).

o Competitiveness against native soil microbes.

o Genetic stability and adaptability.

2. **Viability and Shelf Life**

o Survival during storage and transportation.

o Carrier material quality (e.g., lignite, peat, liquid formulations).

### B. Soil Factors

1. **Soil pH**

✓ Most biofertilizers (e.g., *Rhizobium*, *Azotobacter*) prefer neutral pH (6.5–7.5).

✓ Acidic soils inhibit microbial activity; liming may be required.

2. **Soil Moisture**

o Optimal moisture (40–60% water-holding capacity) is crucial.

o Drought reduces microbial mobility, while waterlogging causes anaerobiosis.

3. **Soil Temperature**

o Mesophilic bacteria (25–30°C) are most common.

o Extreme temperatures (>40°C or <10°C) reduce efficiency.

4. **Soil Organic Matter**

o Enhances microbial proliferation and nutrient cycling.

o Low organic carbon limits biofertilizer survival.

5. **Soil Salinity and Heavy Metals**

* High salinity inhibits nitrogen fixation and P-solubilization.

* Heavy metals (Cd, Pb) are toxic to beneficial microbes.

<footnote n="22">²² Assistant Professor-cum-Scientist (Soil Science-Soil Microbiology, Soil fertility, Soil Chemistry), Department of Microbiology, College of Basic Sciences & Humanities (CBS&H), RPCAU, Pusa-848125, Bihar. E-mail: ajeet.sri@rpcau.ac.in</footnote>
## C. Environmental Conditions

### Climate

* Rainfall affects leaching and microbial dispersal.

* UV radiation can reduce microbial viability in surface applications.

### Seasonal Variations

* Kharif (rainy season) vs. Rabi (winter) crops may require different strains.

## D. Host Plant Factors

### 1. Plant Species and Variety

* Specificity (e.g., *Rhizobium* works best with legumes).

* Root exudates influence microbial colonization.

### 2. Plant Growth Stage

* Early application (seed treatment/seedling dip) improves colonization.

* Late-stage application may be less effective.

## E. Agricultural Practices

### 1. Inoculation Method

* Seed coating, soil application, or foliar spray.

* Liquid formulations show better adherence than carrier-based ones.

### 2. Chemical Fertilizers and Pesticides

* High N-P-K fertilizers suppress N-fixing bacteria.

* Pesticides/fungicides may be toxic to biofertilizer microbes.

### 3. Crop Rotation and Intercropping

* Legume-non-legume rotation enhances soil N content.

* Continuous monoculture reduces microbial diversity.

### 4. Tillage Practices

* Minimum tillage preserves microbial communities.

* Deep plowing may disrupt established biofertilizer networks.

## 3. Strategies to Enhance Biofertilizer Efficacy

* ✓ **Combined Inoculation:** Co-inoculation of N-fixers + P-solubilizers (e.g., *Rhizobium* + *Pseudomonas*).

* ✓ **Biochar Amendment:** Improves soil structure and microbial retention.

* ✓ **Encapsulation Techniques:** Protects microbes from environmental stress.

* ✓ **Farmer Awareness:** Proper storage, application timing, and dosage.

## 4. Case Studies/Recent Advances

* o **PGPR (Plant Growth-Promoting Rhizobacteria):** *Bacillus* and *Pseudomonas* strains enhancing drought resistance.

* o **Nano-Biofertilizers:** Improved delivery systems for better microbial survival.

## 5. Conclusion

The efficacy of biofertilizers is influenced by microbial, soil, plant, and management factors. Optimizing these parameters can maximize their benefits, reducing dependency on chemical fertilizers.
**6. Discussion Questions**

1. How does soil pH affect the performance of phosphate-solubilizing bacteria?

2. Compare the advantages of liquid vs. carrier-based biofertilizers.

3. What integrated approaches can farmers adopt to improve biofertilizer efficiency?