# An Instructional Material On ASS-307- Biopesticides and Biofertilizers Production (Elective course)

Ajeet Kumar, Sanjay Kumar Singh

<figure><img src="image_1.png" /></figure>

## To cite this version:

Ajeet Kumar, Sanjay Kumar Singh. An Instructional Material On ASS-307- Biopesticides and Biofertilizers Production (Elective course). 2025, pp.1-113. hal-05160138

HAL Id: hal-05160138

https://hal.science/hal-05160138v1

Submitted on 13 Jul 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
An Instructional Material
On
ASS-307- Biopesticides and Biofertilizers
Production
(Elective course)

<figure><img src="image_3.png" /></figure>

Authored by

Dr. AJEET KUMAR

Assistant Professor-cum-Scientist
(Soil Science-Soil Microbiology/Soil Fertility/Soil Chemistry)
Department of Microbiology
Collage of Basic Sciences and Humanities (CBS&H)

Dr. SANJAY KUMAR SINGH

Assistant Professor-cum-Scientist
Department of Soil Science
Tirhut Collage of Agriculture (TCA, Dholi), Muzaffarpur

Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur) - 848125, Bihar

June 2025
CONTENTS

**Objective of the course:** The aim of the course contents is to provide a comprehensive understanding of the Biopesticides and Biofertilizers Production.

Lecture outlines

<table><thead><tr><td>Sl. No.</td><td>Topic covered</td><td>Pg. No.</td></tr></thead><tbody><tr><td>1.</td><td>Aim of the course and learning out of the course contents Biopesticides and Biofertilizers</td><td>4-6</td></tr><tr><td>2.</td><td>History and concept of biopesticides, Importance, scope and potential of biopesticide</td><td>7-8</td></tr><tr><td>3.</td><td>Definitions, concepts and classification of biopesticides viz. pathogen, botanical pesticides, and biorationales</td><td>9-11</td></tr><tr><td>4.</td><td>Virulence, pathogenicity and symptoms of entomopathogenic pathogens and nematodes.</td><td>12-13</td></tr><tr><td>5.</td><td>Mass production technology of bio-pesticides</td><td>14-15</td></tr><tr><td>6.</td><td>Methods of application of biopesticides.</td><td>16-17</td></tr><tr><td>7.</td><td>Methods of quality control and techniques of biopesticides</td><td>18-19</td></tr><tr><td>8.</td><td>Impediments and limitation in production and use of biopesticide</td><td>20-21</td></tr><tr><td>9.</td><td>Bio-Pesticides-a special attention</td><td>22-31</td></tr><tr><td>10.</td><td>Biofertilizers - Introduction, status and scope</td><td>32-36</td></tr><tr><td>11.</td><td>Instruments Used in Microbiology Lab with Principle and Uses</td><td>37-49</td></tr><tr><td>12.</td><td>Structure and characteristic features of different types of biofertilizers like <b>bacterial biofertilizers</b>- Azospirillum, Azotobacter, Bacillus, Pseudomonas, Rhizobium and Frankia</td><td>50-55</td></tr><tr><td>13.</td><td>Structure and characteristic features of - <b>Cynobacterial biofertilizers</b>- Anabaena, Nostoc, Hapalosiphon</td><td>56-59</td></tr><tr><td>14.</td><td>Structure and characteristic features of - <b>Fungal biofertilizers</b>- AM mycorrhiza and ectomycorrhiza, endomycorrhiza and ectendomycorrhiza</td><td>60-63</td></tr><tr><td>15.</td><td>Biochemistry of biological nitrogen fixation- <b>Nitrogen fixation</b> -Free living and symbiotic nitrogen fixation.</td><td>64-86</td></tr><tr><td>16.</td><td>Mechanism of phosphate solubilization and phosphate mobilization</td><td>87-91</td></tr><tr><td>17.</td><td>Mechanism of K solubilization by Potassium Solubilizing Bacteria (KSB)</td><td>92-96</td></tr><tr><td>18.</td><td>FCO (Fertilizer Control Order) Specifications of Biofertilizers</td><td>97-99</td></tr><tr><td>19.</td><td>Quality Control of Biofertilizers</td><td>100-101</td></tr><tr><td>20.</td><td>Mass Production of Biofertilizers carrier based and liquid based biofertilizers</td><td>102-104</td></tr><tr><td>21.</td><td>Biofertilizers-Storage, shelf life and marketing</td><td>105-107</td></tr><tr><td>22.</td><td>Application technology of biofertilizers for seeds, seedlings, tubers, sets etc.</td><td>108-110</td></tr><tr><td>23.</td><td>Factors influencing the efficacy of biofertilizers</td><td>111-113</td></tr></tbody></table>

<figure><img src="image_4.png" /></figure>
**Reference Books**

1. Biofertilizers in Agriculture and Forestry- N S Subba Rao, 3rd Ed. (1993) Oxford & IBH

2. Soil Microbiology- N S Subba Rao, 1999, Oxford & IBH

3. Microbial-based biopesticides: Methods and Protocols -T. R. Glare and M. E. Moran-Diez.

4. Biopesticides Handbook- Leo M.L. Nollet, Hamir Singh Rathore, 2015, CRC Press
# Aim of the Course Contents

The aim of the course contents is to provide a comprehensive understanding of the following key aspects related to Biopesticides and Biofertilizers:

## Biopesticides

1. History and Concept of Biopesticides:

<figure><img src="image_5.png" /></figure>

Explore the historical development and conceptual foundations of biopesticides.

<figure><img src="image_6.png" /></figure>

Understand the significance, scope, and potential applications of biopesticides in modern agriculture.

<figure><img src="image_7.png" /></figure>

Definitions, Concepts, and Classification of Biopesticides:

2. Define and classify Biopesticides

<figure><img src="image_8.png" /></figure>

including pathogen-based, botanical, and biorational categories.

<figure><img src="image_9.png" /></figure>

Examine the various concepts associated with biopesticides and their classification.

3. Botanical Pesticides and Their Uses:

<figure><img src="image_10.png" /></figure>

Investigate the types and applications of botanical pesticides.

<figure><img src="image_11.png" /></figure>

Explore the uses of botanicals in biopesticides and their effectiveness.

4. Entomopathogenic Pathogens and Nematodes:

<figure><img src="image_12.png" /></figure>

Understand virulence, pathogenicity, and symptoms associated with entomopathogenic pathogens and nematodes.

<figure><img src="image_13.png" /></figure>

Explore the potential of these biopesticides in pest management.

5. Mass Production Technology of Biopesticides:

<figure><img src="image_14.png" /></figure>

Learn the techniques and technologies involved in the large-scale production of biopesticides.

<figure><img src="image_15.png" /></figure>

Understand the factors influencing the mass production of biopesticides.

6. Methods of Application of Biopesticides:

<figure><img src="image_16.png" /></figure>

Explore different methods for the effective application of biopesticides in agricultural systems.

7. Quality Control and Techniques of Biopesticides:

<figure><img src="image_17.png" /></figure>

Examine the methods and techniques used for quality control in biopesticide production.

<figure><img src="image_18.png" /></figure>

Understand the importance of maintaining high-quality standards.

8. Impediments and Limitations in Production and Use of Biopesticides:

<figure><img src="image_19.png" /></figure>

o Identify and analyze the challenges and limitations associated with the production and utilization of biopesticides.

## Biofertilizers

1. Introduction, Status, and Scope of Biofertilizers:

<figure><img src="image_20.png" /></figure>

o Provide an overview of biofertilizers, their current status, and potential scope in agriculture.

2. Structure and Characteristics of Bacterial Biofertilizers:

<figure><img src="image_21.png" /></figure>

o Explore bacterial biofertilizers such as Azospirillum, Azotobacter, Bacillus, Pseudomonas, Rhizobium, and Frankia.

<figure><img src="image_22.png" /></figure>

o Understand the structural and characteristic features of these biofertilizers.
**3. Cyanobacterial and Fungal Biofertilizers:**

* Explore the role and characteristics of cyanobacterial biofertilizers (Anabaena, Nostoc, Hapalosiphon) and fungal biofertilizers (AM and ectomycorrhiza).

**4. Nitrogen Fixation and Biochemistry:**

* Understand the mechanisms of free-living and symbiotic nitrogen fixation.

* Explore the biochemistry involved in biological nitrogen fixation.

**5. Phosphate and Potassium Solubilization:**

* Investigate the mechanisms of phosphate and potassium solubilization by biofertilizers.

**6. Production Technology and Quality Control of Biofertilizers:**

* Learn the production technology, strain selection, and quality control measures for biofertilizers.

**7. Application Technology and Factors Influencing Efficacy:**

* Explore various application technologies for biofertilizers on seeds, seedlings, tubers, and sets.

* Understand the factors influencing the efficacy of biofertilizers.

**8. Storage, Shelf Life, and Marketing of Biofertilizers:**

* Examine the storage conditions, shelf life, and marketing strategies for biofertilizers.

By covering these topics, the course aims to equip participants with a comprehensive understanding of biopesticides and biofertilizers, fostering knowledge and skills applicable to sustainable and environmentally friendly agricultural practices.

## Learning outcomes

Upon completing the course on the history, concepts, and applications of Biopesticides and Biofertilizers, participants should achieve the following learning outcomes:

### Learning outcomes of Biopesticides

**1. Understanding of Biopesticides:**

* Explain the historical development and foundational concepts of biopesticides.

* Evaluate the significance, scope, and potential applications of biopesticides in modern agriculture.

**2. Classification and Application of Biopesticides:**

* Classify biopesticides into pathogen-based, botanical, and biorational categories.

* Apply different methods of biopesticide application for effective pest management.

**3. Botanical Pesticides Knowledge:**

* Identify various botanical pesticides and describe their uses in biopesticides.

* Evaluate the effectiveness of botanicals in pest control.

**4. Knowledge of Entomopathogenic Pathogens and Nematodes:**

* Analyze the virulence, pathogenicity, and symptoms associated with entomopathogenic pathogens and nematodes.

* Assess the potential of these biopesticides in pest management strategies.

**5. Mass Production Technology:**
* Apply techniques and technologies for the large-scale production of biopesticides.

* Identify and overcome challenges associated with mass production.

## 6. Application Methods and Quality Control:

* Implement various methods for the application of biopesticides in agricultural systems.

* Employ quality control measures to ensure the efficacy and safety of biopesticides.

## 7. Awareness of Impediments and Limitations:

* Identify and analyze impediments and limitations in the production and use of biopesticides.

* Propose potential solutions to address challenges in the biopesticide industry.

## Learning outcomes of Biofertilizers

### 1. Biofertilizers Understanding:

* Summarize the introduction, status, and scope of biofertilizers in agriculture.

* Differentiate between various bacterial, cyanobacterial, and fungal biofertilizers based on their structure and characteristics.

### 2. Nitrogen Fixation and Biochemistry Knowledge:

* Explain the mechanisms of free-living and symbiotic nitrogen fixation.

* Understand the biochemistry involved in biological nitrogen fixation.

### 3. Phosphate and Potassium Solubilization:

* Describe the mechanisms of phosphate and potassium solubilization by biofertilizers.

* Apply knowledge to enhance nutrient availability in soil through biofertilizers.

### 4. Production Technology and Quality Control of Biofertilizers:

* Apply production technology, strain selection, and quality control measures for bacterial, cyanobacterial, and fungal biofertilizers.

* Ensure the production of high-quality biofertilizers for agricultural use.

### 5. Application Technology and Efficacy Factors:

* Implement various application technologies for biofertilizers on seeds, seedlings, tubers, and sets. Analyze and understand the factors influencing the efficacy of biofertilizers.

### 6. Storage, Shelf Life, and Marketing:

* Determine appropriate storage conditions and shelf life for biofertilizers.

* Develop marketing strategies for biofertilizers, considering factors such as product efficacy and market demand.

### 7. Integration of Knowledge for Sustainable Agriculture:

* Synthesize knowledge from both biopesticides and biofertilizers to promote sustainable and environmentally friendly agricultural practices.

* Formulate comprehensive strategies for integrated pest and nutrient management.

By achieving these learning outcomes, participants will be well-equipped to contribute to the adoption and implementation of bio-based solutions in agriculture, promoting sustainable and eco-friendly practices in the field.
# History and Concept of Biopesticides: Importance, Scope, and Potential

Dr. AJEET KUMAR<fnref n="1" />

## 1. Introduction

Biopesticides are biologically-based agents used to control pests, encompassing microorganisms (bacteria, fungi, viruses), biochemical substances (plant extracts, pheromones), and beneficial insects. They offer an eco-friendly and sustainable alternative to synthetic chemical pesticides, aligning with the principles of integrated pest management (IPM) and organic agriculture.

## 2. History of Biopesticides

### 2.1 Early Use

* **Ancient Practices**: Use of ash, plant extracts (e.g., neem, pyrethrum), and oil emulsions for pest control.

* **China (1100 AD)**: Use of predatory ants to control pests in citrus orchards.

### 2.2 Emergence of Microbial Biopesticides

* **1835**: Discovery of *Beauveria bassiana* as a silkworm pathogen by Agostino Bassi - foundational to microbial insecticides.

* **1874**: Bacillus thuringiensis (Bt) first isolated from infected silkworms.

* **1940s-50s**: First commercial production of Bt formulations.

* **1980s onwards**: Regulatory approvals and increased commercial development of microbial and biochemical pesticides.

## 3. Concept of Biopesticides

### 3.1 Definition

Biopesticides are natural-based pest management agents derived from living organisms or their by-products. According to the **EPA (USA)**, biopesticides are classified into three major types:

### 3.2 Types of Biopesticides

* **Microbial Biopesticides**: Contain microorganisms (e.g., *Bacillus thuringiensis*, *Trichoderma*, *Metarhizium*).

* **Biochemical Biopesticides**: Naturally occurring substances (e.g., neem oil, pheromones, plant volatiles) that control pests by non-toxic mechanisms.

* **Plant-Incorporated Protectants (PIPs)**: Genetically engineered plants expressing pesticidal properties (e.g., Bt cotton).

## 4. Importance of Biopesticides

* **Environmental Safety**: Biodegradable, non-persistent, and target-specific with minimal off-target impacts.

* **Human and Animal Health**: Reduced toxicity compared to synthetic pesticides.

<footnote n="1">¹ 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>
* **Resistance Management:** Lower chances of resistance development due to complex modes of action.

* **Organic Farming:** Essential components of certified organic crop protection.

* **Sustainability:** Promotes biodiversity and ecosystem balance.

* **Regulatory and Trade Compliance:** Safer residues align with stringent Maximum Residue Limits (MRLs).

# 5. Scope and Application

## 5.1 Agricultural Crops

* Used against pests in cereals, pulses, vegetables, fruits, and plantation crops.

* Effective in seed treatment, foliar sprays, and soil applications.

## 5.2 Horticulture and Greenhouse Cultivation

* Widely used due to enclosed conditions that support microbial efficacy.

## 5.3 Post-Harvest and Stored Grain Protection

* Entomopathogenic fungi and botanical extracts used to manage storage pests.

## 5.4 Urban and Public Health

* Larvicidal formulations (e.g., *Bacillus sphaericus*) used in mosquito control programs.

# 6. Potential and Future Prospects

## 6.1 Market Growth

* The global biopesticide market is growing at >10% CAGR, driven by demand for residue-free food and sustainability.

## 6.2 Innovations and Advancements

* **Genetic Engineering:** Development of transgenic crops with biopesticide traits.

* **Nano-formulations:** Enhanced stability and targeted delivery of bioagents.

* **AI and IoT:** Precision application and pest monitoring systems.

* **Microbial Consortia:** Synergistic blends for broader spectrum activity.

## 6.3 Challenges

* **Shelf-life and Storage:** Limited compared to synthetic pesticides.

* **Field Efficacy:** Variable results under different environmental conditions.

* **Awareness and Adoption:** Limited knowledge among farmers.

* **Regulatory Delays:** Lengthy registration processes in many countries.

# 7. Conclusion

Biopesticides represent a critical tool for achieving sustainable agriculture and ecological pest management. Their importance lies in minimizing environmental impact, promoting biodiversity and ensuring food safety. With advancing research, favourable policies and increasing consumer awareness, the scope and potential of biopesticides are poised to expand significantly.
# Definitions, Concepts, and Classification of Biopesticides: Pathogens, Botanical Pesticides, and Biorationals

Dr. AJEET KUMAR<fnref n="2" />

## 1. Introduction to Biopesticides

Biopesticides are a class of pest management agents derived from natural materials such as animals, plants, bacteria, and certain minerals. They offer environmentally friendly alternatives to conventional chemical pesticides and are often target-specific, biodegradable, and less harmful to beneficial organisms.

## 2. Definitions

* **Biopesticide:**

A formulation made from natural sources (microorganisms, botanicals, or other biologically active substances) used to control pests through non-toxic or biologically based mechanisms.

* **Pathogenic Biopesticides:**

These are formulations containing living organisms (bacteria, fungi, viruses, or protozoa) that cause disease or inhibit the development of pest organisms.

* **Botanical Pesticides:**

Plant-derived substances that exhibit pesticidal properties, such as extracts, essential oils, and secondary metabolites (e.g., neem, pyrethrin, rotenone).

* **Biorational Pesticides:**

Pesticides that are biologically based and cause minimal ecological disruption. This includes insect growth regulators, sex pheromones, and natural enzyme inhibitors.

## 3. Key Concepts

* **Eco-friendliness:**

Biopesticides are typically non-toxic to non-target organisms and decompose quickly without leaving harmful residues.

* **Specificity:**

Most biopesticides are highly specific, targeting only a particular pest or group of pests.

* **Resistance Management:**

Biopesticides play a crucial role in integrated pest management (IPM) programs by delaying resistance development in pests.

* **Mode of Action:**

Varies from microbial infection, growth disruption, repellent effects, to interference with reproductive behaviours.

<footnote n="2">² 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>
# 4. Classification of Biopesticides

## A. Microbial Biopesticides (Pathogens)

<table><thead><tr><td>Type</td><td>Example</td><td>Target Pests</td></tr></thead><tbody><tr><td>Bacteria</td><td>Bacillus thuringiensis (Bt)</td><td>Lepidopteran larvae</td></tr><tr><td>Fungi</td><td>Beauveria bassiana, Metarhizium anisopliae</td><td>Aphids, beetles</td></tr><tr><td>Viruses</td><td>Nucleopolyhedrovirus (NPV), Granulovirus (GV)</td><td>Caterpillars</td></tr><tr><td>Protozoa</td><td>Nosema locustae</td><td>Grasshoppers</td></tr></tbody></table>

## B. Botanical Biopesticides

<table><thead><tr><td>Source Plant</td><td>Active Component</td><td>Pest Type</td></tr></thead><tbody><tr><td>Neem (Azadirachta indica)</td><td>Azadirachtin</td><td>Insects (all stages)</td></tr><tr><td>Chrysanthemum spp.</td><td>Pyrethrins</td><td>Mosquitoes, flies</td></tr><tr><td>Derris spp.</td><td>Rotenone</td><td>Insects, fish</td></tr><tr><td>Garlic</td><td>Sulfur compounds</td><td>Insects, fungi</td></tr></tbody></table>

## C. Biorational Pesticides

<table><thead><tr><td>Type</td><td>Description</td><td>Mode of Action</td></tr></thead><tbody><tr><td>Insect Growth Regulators (IGRs)</td><td>Disrupt development</td><td>Juvenile hormone analogs</td></tr><tr><td>Sex Pheromones</td><td>Disrupt mating</td><td>Confusion/trapping</td></tr><tr><td>Repellents</td><td>Deter pest approach</td><td>Olfactory stimulation</td></tr><tr><td>Enzyme Inhibitors</td><td>Interfere with digestion/metabolism</td><td>Botanical alkaloids</td></tr></tbody></table>

# 5. Advantages of Biopesticides

* Environmentally safe

* Decomposes quickly

* Specific to pests

* Reduces chemical pesticide use

* Promotes biodiversity
**6. Limitations**

* Short shelf life

* Sensitivity to UV light and temperature

* Slow action compared to chemicals

* Requires frequent application

* Narrow target range

**7. Conclusion**

The classification of biopesticides into microbial (pathogens), botanical, and biorational categories provides a structured approach to their use in sustainable agriculture. They form an integral part of IPM and future pest management strategies due to their eco-compatibility and specificity.
# Virulence, Pathogenicity, and Symptoms of Entomopathogenic Pathogens and Nematodes

Dr. AJEET KUMAR<fnref n="3" />

## 1. Definitions

* **Virulence:** The degree of damage caused by a pathogen to its host. In the context of entomopathogens, it refers to the severity of disease or mortality induced in insects.

* **Pathogenicity:** The ability of a microorganism or nematode to cause disease in an insect host. It is a qualitative trait (presence or absence of pathogenic effect).

## 2. Major Groups of Entomopathogenic Organisms

<table><thead><tr><td>Type</td><td>Examples</td><td>Mode of Action</td></tr></thead><tbody><tr><td>Fungi</td><td>Beauveria bassiana, Metarhizium anisopliae</td><td>Penetrate cuticle, colonize hemocoel</td></tr><tr><td>Bacteria</td><td>Bacillus thuringiensis (Bt), Serratia spp.</td><td>Produce toxins, disrupt midgut epithelium</td></tr><tr><td>Viruses</td><td>Nuclear Polyhedrosis Virus (NPV), Granulosis Virus (GV)</td><td>Replicate inside host cells, lysis</td></tr><tr><td>Nematodes</td><td>Steinernema spp., Heterorhabditis spp.</td><td>Release symbiotic bacteria in hemocoel</td></tr></tbody></table>

## 3. Virulence Mechanisms

### A. Fungi

* Produce cuticle-degrading enzymes (proteases, chitinases).

* Hyphal bodies proliferate in the hemocoel, producing toxins.

* Rapid colonization leads to insect death within 3–7 days.

### B. Bacteria

* Bt produces crystal (Cry) toxins → ingestion by larva → pore formation in midgut → septicemia.

* Other bacteria release endotoxins and hemolysins disrupting tissue integrity.

### C. Viruses

* Infect midgut epithelial cells after ingestion.

* Viral replication → cell lysis → systemic spread → death.

### D. Nematodes

* Infective juveniles (IJs) enter insect body via natural openings.

* Release symbiotic bacteria (Photorhabdus, Xenorhabdus).

* Insect killed within 24–72 hours by toxemia/sepsis.

<footnote n="3">³ 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>
## 4. Pathogenicity Traits

<table><thead><tr><td>Parameter</td><td>Importance for Pathogenicity</td></tr></thead><tbody><tr><td>Host range</td><td>Determines spectrum of affected insects</td></tr><tr><td>Toxin production</td><td>Key for killing or weakening hosts</td></tr><tr><td>Invasion &amp; replication rate</td><td>Influences speed of mortality</td></tr><tr><td>Immune evasion ability</td><td>Ensures survival within host</td></tr></tbody></table>

## 5. Symptoms in Infected Insects

<table><thead><tr><td>Pathogen Type</td><td>Symptoms</td></tr></thead><tbody><tr><td>Fungi</td><td>White, green or red mold on cadaver, shriveling, mummification</td></tr><tr><td>Bacteria</td><td>Cessation of feeding, flaccid body, liquefied internal tissue</td></tr><tr><td>Viruses</td><td>Milky white hemolymph, body disintegration post-mortem</td></tr><tr><td>Nematodes</td><td>Rigid body, brown/red discoloration, bacterial ooze</td></tr></tbody></table>

## 6. Diagram: Infection Cycle of Entomopathogenic Nematodes

[Infective Juvenile] → [Entry into Insect Host] → [Release of Symbiotic Bacteria] → [Toxin Production & Host Death] → [Nematode Reproduction] → [Emergence of IJs]

## 7. Factors Affecting Virulence and Pathogenicity

* Environmental conditions (temperature, humidity)

* Host species and developmental stage

* Genetic variability among pathogen strains

* Dose and mode of application

## 8. Applications in IPM (Integrated Pest Management)

* Eco-friendly alternatives to chemical pesticides.

* Useful against soil-dwelling and foliage-feeding insect pests.

* Compatible with many biocontrol and cultural practices
# Mass Production Technology of Biopesticides

Dr. AJEET KUMAR<fnref n="4" />

## 1. Introduction

Biopesticides are formulations of living organisms or their by-products that are used to manage pests through natural mechanisms. Mass production of biopesticides involves the cultivation of large quantities of entomopathogenic fungi, bacteria, viruses, or beneficial nematodes under controlled conditions to ensure their viability, virulence, and field effectiveness.

## 2. Steps in Mass Production

### A. Selection of Potent Strain

* High virulence, broad spectrum activity.

* Stable under environmental stress.

* Non-toxic to humans and non-target organisms.

### B. Optimization of Culture Media

* Use of cost-effective substrates like agricultural by-products.

* Formulated for high spore/toxin yield and viability.

### C. Fermentation Process

<table><thead><tr><td>Type</td><td>Description</td><td>Suitable for</td></tr></thead><tbody><tr><td>Submerged (SmF)</td><td>Growth in liquid nutrient broth</td><td>Bacteria (Bt), Fungi (toxins)</td></tr><tr><td>Solid State (SSF)</td><td>Growth on solid substrate (e.g., rice, wheat bran)</td><td>Fungi (Metarhizium, Beauveria)</td></tr><tr><td>Surface Culture</td><td>Culture grown on the surface of solid/liquid medium</td><td>Virus production (NPV, GV)</td></tr></tbody></table>

## 3. Mass Production of Different Biopesticide Types

### A. Bacterial Biopesticides (e.g., *Bacillus thuringiensis*)

* Inoculation into nutrient-rich broth (e.g., nutrient yeast extract medium).

* Incubated at 28–30°C for 48–72 hours under agitation.

* Harvesting by centrifugation or filtration.

* Formulated as wettable powder, suspension concentrate, or granules.

### B. Fungal Biopesticides (e.g., *Metarhizium anisopliae*, *Beauveria bassiana*)

* Grown on solid substrates (e.g., broken rice, sorghum).

* Incubated for 10–15 days in sterile trays at 25–28°C.

* Spores harvested by sieving or scraping.

* Dried and mixed with talc/lignite for formulation.

<footnote n="4">⁴ 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. Viral Biopesticides (e.g., NPV)

* In vivo production using host larvae (e.g., *Helicoverpa armigera*).

* Infected larvae reared under controlled conditions.

* Virus harvested from larval cadavers.

* Purified and formulated as aqueous suspension or powder.

### D. Entomopathogenic Nematodes (e.g., *Steinernema*, *Heterorhabditis*)

* In vivo production in *Galleria mellonella* larvae or in vitro liquid fermentation.

* Harvesting of infective juveniles (IJs).

* Stored in aqueous suspensions or alginate formulations.

### 4. Formulation and Shelf Life

<table><thead><tr><td>Component</td><td>Role</td></tr></thead><tbody><tr><td>Carriers</td><td>Talc, peat, lignite, rice bran for ease of handling</td></tr><tr><td>Additives</td><td>Stabilizers, UV protectants, stickers</td></tr><tr><td>Packaging</td><td>Moisture-proof, breathable pouches</td></tr><tr><td>Storage</td><td>Refrigerated or ambient depending on shelf life</td></tr></tbody></table>

* Target shelf life: 6–12 months

* Viability tests: CFU count, spore count, IJ survival

### 5. Quality Control Parameters

<table><thead><tr><td>Parameter</td><td>Standard Requirement</td></tr></thead><tbody><tr><td>Viable count (CFU/IJ)</td><td>Minimum 1 × 10<sup>7</sup> per gram/ml</td></tr><tr><td>Contamination</td><td>Must be <0.1%</td></tr><tr><td>Moisture content</td><td><10% in dry formulations</td></tr><tr><td>pH and Toxin Profile</td><td>Within effective bioactive range</td></tr></tbody></table>

### 6. Flow Diagram: Fungal Biopesticide Production

[Mother Culture] → [Inoculum Development] → [Mass Production on Substrate]
→ [Incubation & Sporulation] → [Harvesting] → [Formulation] → [Packaging & Storage]

### 7. Challenges in Mass Production

* Contamination risk during large-scale fermentation.

* Lack of consistent quality carriers.

* Temperature and humidity control during drying and storage.

* Cost-effectiveness for smallholder farmers.

### 8. Future Innovations

* Bioreactors with automated control systems.

* Use of agro-industrial waste as media.

* Nano-formulations for increased shelf life and stability.

* AI-based process optimization for yield enhancement.
# Methods of Application of Biopesticides

Dr. AJEET KUMAR<fnref n="5" />

## 1. Introduction

Biopesticides are biological formulations containing living organisms or their derivatives used for pest management. Their success depends not only on the formulation and viability but also on **proper application techniques** to reach the target pest in the most effective manner.

## 2. Criteria for Choosing Application Method

* Target pest and life stage

* Site of infection (foliar, soil, stored grain)

* Nature of biopesticide (fungal, bacterial, viral, or nematode-based)

* Formulation type (liquid, powder, granule, etc.)

* Environmental factors (UV exposure, humidity, rainfall)

## 3. Major Application Methods

### A. Foliar Application (Spraying)

* **Target:** Leaf-feeding insects and pathogens (e.g., Bt, fungal spores)

* **Formulation:** Liquid suspensions or wettable powders

* **Equipment:** Hand sprayer, mist blower, drone-based sprayer

* **Advantages:** Direct contact with insect; easy to apply

* **Limitations:** Sensitivity to UV and wash-off by rain

### B. Soil Application

* **Target:** Soil-dwelling larvae, termites, root grubs

* **Organisms:** Fungi (Metarhizium, Beauveria), nematodes (Steinernema, Heterorhabditis)

* **Formulation:** Granules, talc-based powders, IJs in aqueous suspension

* **Method:** Mixing with compost/FYM or irrigation water

* **Advantages:** Long-lasting effect; safe from UV exposure

### C. Seed Treatment

* **Target:** Seed and seedling-borne pests

* **Agents:** Pseudomonas fluorescens, Trichoderma, Bt

* **Method:** Coating seeds with slurry before sowing

* **Formulation:** Wettable powder or liquid formulations

* **Benefits:** Early-stage protection; low dose requirement

<footnote n="5">⁵ 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>
## D. Trunk Injection / Wound Dressing

* **Target:** Borers and wood-infesting pests

* **Organisms:** Fungal or bacterial formulations

* **Method:** Injecting suspension into tree trunk or wound dressing

* **Use:** Orchards, plantations

## E. Bait Application

* **Target:** Fruit flies, cockroaches, ants, termites

* **Organisms:** *Metarhizium*, *Beauveria*, or bacterial toxins

* **Method:** Mixing biopesticide with attractants and bait substrate

* **Application:** Field edges, bait stations

## F. Drip Irrigation / Drenching

* **Target:** Nematodes, soil-borne insects, and pathogens

* **Method:** Injecting liquid formulation into irrigation lines

* **Suitable For:** High-value crops under drip systems

## 4. Formulation Types and Compatibility

<table><thead><tr><td>Method</td><td>Common Formulations</td><td>Compatible Biopesticides</td></tr></thead><tbody><tr><td>Foliar spray</td><td>WP, SC, SL</td><td><em>Bt, Beauveria, Metarhizium</em></td></tr><tr><td>Soil drenching</td><td>Granules, suspension</td><td><em>Trichoderma</em>, nematodes</td></tr><tr><td>Seed treatment</td><td>WP, DS, LS</td><td><em>Pseudomonas, Bacillus, Trichoderma</em></td></tr><tr><td>Bait</td><td>Granules, paste</td><td><em>Metarhizium</em>, <em>Bt</em> toxin</td></tr></tbody></table>

## 5. Diagram: Matching Method to Pest Type

<table><tr><td>[Seed Treatment]</td><td>→ Seed-borne pests</td></tr><tr><td>[Foliar Spray]</td><td>→ Caterpillars, aphids</td></tr><tr><td>[Soil Application]</td><td>→ Root grubs, white grubs</td></tr><tr><td>[Drip Irrigation]</td><td>→ Nematodes</td></tr><tr><td>[Bait]</td><td>→ Fruit flies, termites</td></tr></table>

## 6. Considerations for Effective Use

* Apply during **early pest stage** for best results

* Avoid **UV exposure** and apply during **cooler times of day**

* Maintain **adequate moisture** for spore/nematode survival

* Avoid mixing with **incompatible agrochemicals**

## 7. Future Trends

* Drone-assisted precision spraying

* Nano-carriers for foliar delivery

* Encapsulation for slow release

* AI-guided pest detection and smart application
# Methods of Quality Control and Techniques of Biopesticides

Dr. AJEET KUMAR<fnref n="6" />

## 1. Introduction

Quality control (QC) in biopesticide production ensures **efficacy**, **safety**, and **shelf-life** of microbial agents before commercial use. Standardized QC techniques are essential to comply with **regulatory guidelines** and maintain **bioefficacy** across production batches.

## 2. Importance of Quality Control in Biopesticides

* Ensures **purity**, **viability**, and **correct strain identity**

* Prevents contamination with unwanted microorganisms

* Assures **consistency** in field performance

* Necessary for **registration** under Insecticide Act (1968), CIB & RC (India)

## 3. Quality Parameters for Common Biopesticides

<table><thead><tr><td>Biopesticide Type</td><td>Minimum Viable Count</td><td>Purity (Contaminants)</td><td>Moisture (%)</td><td>pH Range</td></tr></thead><tbody><tr><td>Bacillus thuringiensis</td><td>1 × 10<sup>9</sup> CFU/g (solid) or ml (liquid)</td><td>&lt; 0.1%</td><td>&lt; 10%</td><td>6.5-7.5</td></tr><tr><td>Trichoderma spp.</td><td>2 × 10<sup>6</sup> CFU/g</td><td>&lt; 0.1%</td><td>&lt; 20%</td><td>6.0-7.5</td></tr><tr><td>Beauveria bassiana</td><td>1 × 10<sup>7</sup> spores/g</td><td>&lt; 0.1%</td><td>&lt; 10%</td><td>6.0-7.5</td></tr><tr><td>NPV (HaNPV)</td><td>1 × 10<sup>9</sup> PIB/ml</td><td>No bacterial/fungal growth</td><td>Liquid</td><td>5.5-6.5</td></tr></tbody></table>

## 4. Quality Control Techniques

### A. Viability Testing

* **Bacteria**: Serial dilution and plate count method on Nutrient Agar

* **Fungi**: Spore germination on PDA plates (viable if ≥ 90% spores germinate)

* **Nematodes**: Visual movement under microscope in distilled water

* **Viruses**: Occlusion bodies counted using a hemocytometer

### B. Purity Testing

* Check for contamination using general growth media:

* **Nutrient Agar**, **Potato Dextrose Agar** (PDA)

* Observe colony morphology and identify contaminating species

* Should not exceed 0.1% contamination

<footnote n="6">⁶ 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. Moisture Content Analysis

* **Gravimetric Method:** Dry 1g sample at 105°C for 6 hrs

* Moisture content = (Initial weight – Dry weight) / Initial weight × 100

* Lower moisture ensures longer shelf-life

## D. pH Testing

* Use digital pH meter or indicator strips

* Adjusted with buffer solutions or stabilizers

## E. Pathogen Identification & Authentication

* **Microscopy:** Spore or bacterial morphology

* **Molecular Techniques:**

* PCR for strain-specific genes; DNA barcoding or sequencing

* RAPD or AFLP fingerprinting

## F. Bioassay for Efficacy (Potency Test)

* Example: *Bt* tested against 3rd instar *Helicoverpa armigera* larvae

* Parameters:

* LC<sub>50</sub> (lethal concentration)

* LT<sub>50</sub> (lethal time)

* Compare with reference strain

## 5. Packaging and Labeling Standards

* Must be moisture-proof, UV-protective, and properly labeled with:

* Active ingredient

* Viable cell/spore count

* Expiry date

* Batch number

* Application method

## 6. Regulatory Guidelines in India

* **CIB & RC** – Central Insecticides Board and Registration Committee

* Must meet BIS (Bureau of Indian Standards) specifications

* QA/QC labs should be accredited under **ISO/IEC 17025**

## 7. Diagram: Quality Control Workflow

* [Raw Strain] → [Media Optimization] → [Fermentation] → [Harvesting] → [Viability Test] →
* [Contamination Check] → [Packaging] → [Labeling] → [Release]

## 8. Challenges in Quality Control

* Variation in raw materials; Lack of trained personnel in bioassay

* Inadequate storage conditions; Field inconsistency despite lab QC

## 9. Future Technologies in QC

* **Automated colony counters** for viable counts

* **Real-time PCR** for rapid identity testing

* **Digital imaging** for nematode movement

* **Portable bioassay kits** for field evaluation
# Impediments and Limitations in the Production and Use of Biopesticides

Dr. AJEET KUMAR<fnref n="7" />

## 1. Introduction

Biopesticides offer eco-friendly alternatives to chemical pesticides. However, despite their proven benefits, **production bottlenecks** and **field-level limitations** hinder their widespread adoption. These challenges must be addressed to ensure scalability and success in Integrated Pest Management (IPM) programs.

## 2. Major Impediments in Production

### A. Technical Challenges

* **Strain instability:** Genetic or phenotypic drift over subcultures

* **Low shelf-life:** Limited viability under ambient storage

* **Contamination risks:** Especially in solid-state and nematode production

* **Requirement of specific substrates:** Cost and availability issues

### B. Infrastructure Limitations

* Lack of **automated fermentation units** or **bioreactors** in small-scale setups

* Inadequate **cold storage** for live formulations

* Poor quality control in unregulated labs

### C. Regulatory Hurdles

* Delays in **registration and licensing**

* Lack of **clear, crop-specific guidelines** for use

* Absence of **standardized protocols** for QA/QC across different organisms

## 3. Field-Level Limitations

### A. Environmental Sensitivity

* Susceptibility to **UV radiation, high temperatures, and desiccation**

* Need for **moist microclimates** for successful infection (e.g., fungal spores)

### B. Limited Spectrum and Speed

* **Narrow host range** in many biopesticides (e.g., NPV for *Helicoverpa*)

* **Slower action** than chemical pesticides; not suitable for emergency control

<footnote n="7">⁷ 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. Application Difficulties

* Lack of awareness about **correct timing, dosage, and methods**

* Incompatibility with common **chemical pesticides or adjuvants**

* Limited availability of **drone or drip delivery systems**

### 4. Market and Socio-Economic Limitations

* **Higher unit cost** than synthetic pesticides in many cases

* **Weak supply chains and distribution networks**

* **Farmer skepticism** due to past inconsistent performance

* Lack of **government subsidies** or insurance coverage

### 5. Limitations in R&D and Extension

* **Inadequate funding** for basic and applied biopesticide research

* Absence of **location-specific field validation trials**

* **Limited integration** with mainstream extension services and IPM programs

### 6. Summary Table: Impediments at Different Stages

<table><thead><tr><td>Category</td><td>Key Limitation</td></tr></thead><tbody><tr><td>Production</td><td>Strain instability, contamination, shelf life</td></tr><tr><td>Infrastructure</td><td>Lack of cold storage, automation</td></tr><tr><td>Regulatory</td><td>Delayed approvals, no crop-specific protocols</td></tr><tr><td>Environmental</td><td>UV sensitivity, need for humidity</td></tr><tr><td>Application</td><td>Poor farmer training, slow action</td></tr><tr><td>Market</td><td>High cost, poor supply chain, low adoption</td></tr><tr><td>R&D/Extension</td><td>Low field support, poor awareness programs</td></tr></tbody></table>

### 7. Suggestions for Overcoming Limitations

* Develop **stress-tolerant strains and formulations**

* Provide **financial incentives** and subsidies to producers and farmers

* Strengthen **extension services** and demonstration programs

* Promote **PPP models** for biopesticide manufacturing

* Integrate with **digital tools** (apps, IoT, AI) for targeted delivery and pest tracking
# Bio-Pesticides-a special attention

Dr. AJEET KUMAR<fnref n="8" />

A fascinating and economically important area of applied microbial ecology is the biological control of pests and disease-causing agents using microorganisms. Biological control using microorganisms to initiate disease in pest populations has the potential of reducing agricultural reliance on chemical pesticides. The negative interactions (amensalism, predation and parasitism) among microbial populations and between microbes and higher organisms have always formed a natural basis for the biological control of pests and pathogens, and biological control methods take advantage of those relationships. Biological methods to control populations of disease causing organisms and pests are based on modification of host and or vector population, modification of reservoirs of pathogens, and the direct use of microbial pathogens and predators. Biotechnology may improve approaches for controlling pest and disease causing populations.

## Biological Control

Biological control is the reduction of inoculum density or disease producing activities of a pathogen or parasite in its active or dormant state by one or more organisms. This is accomplished naturally or through manipulation of the environment/host/antagonist or by mass introduction of one or more antagonists.

## Principles of Biocontrol

**Biocontrol** is based on antagonism. Which is further based on competition (for nutrients and space), parasitism (by production of volatile non-volatile antibiotics) and hyperparasitism. Competition In the soil, the dormant propagules of fungi are subjected to intense microbial competition when nutrients become available, e.g. Ammonium sulphate solution improves the biocontrol of Heterobasidium annosum by *Trichoderma viridae*.

**Hyperparasitism** It is the attack of a secondary parasite on primary parasite (parasitism of one microbe by another) observed among airborne pathogens. Rusts are often parasitised by Enderluca carices and powdery mildews by Ampelomyces quisqualis. Cladosporium species parasitise both rust and powdery mildews.

**Antibiosis** It depends on competitive saprophytic ability of a fungus. Sterile soil does not produce antibiotic substances. Several phylloplane fungi such as Aureobasidium, Alternaria, Botrytis and Helminthosporium have been shown to produce antibiotics.

## Microbial Insecticides:

Pests and diseases can be effectively controlled without chemical pesticides by good crop management practices.

## Integrated Pest Management (IPM):

It is a law that emphasises the need to restrict insect population rather than to eradicate them so that the pest population is kept at a non-injurious level. IPM is a pest management system

<footnote n="8">⁸ 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>
that in the context of the associated environment and the population dynamics of the pest species
utilises all suitable techniques and methods in as compatible a manner as possible to maintain the
pest population at levels below those causing economic injury.

Population of pathogenic or predatory microorganisms that are antagonistic towards a
particular pest population provide a natural means of controlling pest population and preparations
of such antagonistic microbial populations are called microbial pesticides or microbial
insecticides. An effective microbial pesticide possesses the following characteristics:

1. The microbial pathogen must be virulent and cause disease in the pest population when properly applied at the recommended concentration.

2. Pathogen should not be susceptible to environmental conditions. After application, it should survive long until the infection is established within the pest population.

3. Resistant stages of the pathogen like spores or cysts are best for use.

4. The pathogen should be rather specific for the pest population and must not cause disease in the non-pest population. It should rapidly establish disease in the pest population so as to minimise destruction caused by the pest.

5. The microbial pesticide should be harmless to humans and other valued plant and animal population.

The various types of microbial insecticides include

(a) Bacterial pesticides

(b) Viral pesticides and

(c) Fungal pesticides.

(a) Bacterial Pesticides

Several bacterial pathogens that have been used as insecticides include

a) Endospore forming: *Bacillus* and *Clostridium* species

b) Non-endospore forming: species of *Pseudomonas*, *Enterobacter*, *Proteus*, *Serratia*, *Xenorhabdus*.

Of the potential bacterial pesticides, *Bacillus thuringiensis* has been most extensively studied. *Bacillus thuringiensis* has been successfully tested against more than 140 insect species (Lepidoptera, Hymenoptera, Diptera and Coleoptera). At present, there are 12 groups of *B. thuringiensis*. All strains produce protein crystal inclusion bodies which act as endotoxin. They are toxic factors and are called parasporal bodies. These crystals dissolve under alkaline condition. They are not soluble in water under neutral or acidic condition. The midgut contents of the caterpillar larvae (pests) are alkaline. On ingestion, the crystal dissolves in the midgut fluid and gets digested particularly by the proteolytic enzyme present in the midgut fluid. This digested protein crystal attacks the cementing substances which are present in the gut wall, thus loosening the epithelial gut wall which helps continuous diffusion of liquid from the gut into the blood making the blood of the insect highly alkaline and leading to gut paralysis. The parasporal bodies are highly toxic for caterpillars with an LD50 value of < 0.9 mg/ g of larvae. The process of crystal synthesis and spore formation proceed simultaneously. The toxin production by a culture can be
enhanced by controlling many factors. *Bacillus thuringiensis* toxin genes have been introduced via recombinant DNA technology into the genome of plants or plant associated microorganisms.

# Method of Production Submerged fermentation

## Preparation of inoculum

The inoculum is prepared using media containing beet molasses, corn steep solids and calcium carbonate.

## Production medium

Production medium enhances the production of toxins within the cell which happens during sporulation. The fermentable carbohydrate and nitrogen available for growth are exhausted at the same time after the commencement of sporulation. Submerged fermentations have been mainly used to produce flowable formulations of spore crystal complex to be used as sprays.

## Semisolid fermentation

Medium consists of dextrose, yeast autolysate and a potassium source. This medium is used for propagating the culture in flasks. The production medium to produce spores and toxins contain wheat bran and solid substrate along with other nutrients. This is an aerobic fermentation with an incubation period of 36-48 hours. This fermentation gives a wettable powder or dust form which is got by just drying and grinding the bran cake. This product is more stable than flowable one but it cannot be used in the wet form since bran begins to swell when wetted.

## Bacillus thuringiensis in the Field

Insecticides based on *B. thuringiensis* are used for short-term control. They do not persist from year to year. During crop spraying, it is essential to make sufficient coverage on a leaf surface. But the *B. thuringiensis* spray must be protected from inactivation by sunlight since it is sensitive to ultra-violet light. In order to avoid this, *emulsifying agents*, stickers or binders and UV protectants can be used in the formulation. Some commercial names of *B.thuringiensis* are **DIPEL, DOOM**.

*Bacillus popilliae* and *B. lentimorbus* are potent insecticides causing Milky disease in Japanese beetle. Following the ingestion of viable spores by larvae, germination takes place in the gut and the vegetative cells penetrate the gut wall and enter the body cavity where they multiply. During this time, they remove all the nutrients from the blood and lead to the death of the larva.

## (b) Viruses As Insect Pests

Insect viruses are encased in protein coats which are insoluble in water. These protein crystals serve to protect the virus particles and due to the presence of these protein crystals, the infective viruses remain active even under normal storage condition for many years. Specificity of the virus-host relationship makes viruses an ideal candidate for use in the control of specific pest population. There are two major groups of insect viruses:

1. **Polyhedrosis virus:** where many virus particles are embedded in each protein crystal. When they occur in the host cell nucleus, they are called nuclear polyhedrosis virus (NPV), and when they occur in the host cytoplasm they are called cytoplasmic polyhedrosis virus (CPV).
2. **Granulosis virus:** where only one virus is contained in each protein crystal. They either develop in the nucleus or cytoplasm of the host cells (NGV or CGV). NPV and CGV come under the group of **Baculoviruses** and they are the most extensively studied insect viruses.

Infection is caused by ingestion of contaminated food containing NPV / GV followed by cell invasion beginning in the midgut. The GV are sprayed over the leaves and they enter the larvae feeding on the foliage. The viruses multiply fast in the larvae and make them lethargic. Affected larvae become sluggish and stop feeding and die. For example, NPV controls the pest Helicoverpa armigera. The affected larvae hang upside down from the leaves and twigs in a characteristic way and a brownish fluid oozes from them. This is a highly infective fluid and is readily disseminated amongst the healthy insect population. The spread takes place by wind, rain, etc. **TAIVIRIDAE** is a commercial NPV pesticide used to control **cotton pest bollworm** and **budworm**.

**Production:**

The specificity and obligate parasitism of insect viruses makes their mass production possible only on live insects, e.g. production of bollworm-budworm NPV (Baculovirus helothis). Bollworm larvae are raised on a semi-synthetic diet containing a water-based mixture of casein, sucrose, wheat germ, growth factors, etc. Agar is used to solidify the diet. Chemicals like formalin, sorbic acid, aureomycin are used to inhibit bacteria, yeasts and fungi. The diet is dispersed hot from a large tank to plastic trays and sealed. These are inoculated by caterpillars from an insectary and incubated under controlled conditions to produce a mass of larvae. A known volume of virus is sprayed on the diet which replicates on the caterpillars to the extent of 5000-10000 times in 5-7 days. The infected caterpillars are suctioned and then treated with water, filtered, centrifuged, precipitated and spray dried. After quality control testing, the preparation is packaged and sold.

**(c) Fungal Pesticides**

Insect mycoses are caused by Phycomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes, e.g. *Beauvaria*, *Metarrhizium*, *Entomophthora*, *Hirsutella*.

**Biological Control Agents**

*Trichoderma* is a cosmopolitan biological control agent which is a saprophyte.

* It has rapid growth rate.

* It sporulates abundantly.

* It produces antibiotics (**gliotoxin** and **viridian**).

* It is a well-known antagonist.

Commercial product **BINABTSEEPIC** can be easily applied in soil in the form of granules for control of soil-borne diseases.

Fungi used as insecticides include the following:

1. **Beauveria bassiana:** This common soil fungus has a broad host range that includes many beetles and fire ants. It infects both larvae and adults of many species. One or more companies currently are developing preparations of this fungus for EPA registration. Understanding the interactions between *Beauveria bassiana* and other soil microorganisms may be the key to successful use of this fungus.
2. *Nomuraea rileyi*: In soybeans, naturally occurring epidemic infections of *Nomuraea rileyi* cause dramatic reductions in populations of foliage-feeding caterpillars.

3. *Vericillium lecanii*: This fungus (once sold under the trade name Vertelec®) has been used in greenhouses to control aphids and whiteflies.

4. *Lagenidium giganteum*: This aquatic fungus is highly infectious to larvae of several mosquito genera. It cycles effectively in the aquatic environment (spores produced in infected larvae persist and infect larvae of subsequent generations), even when mosquito density is low.

5. *Hirsutella thompsonii*: Although preparations of this pathogen were once registered by the US EPA and marketed under the trade name Mycar®, it is no longer available commercially. *Hirsutella thompsonii* is a pathogen of the citrus rust mite.

### Some examples of fungal agents

*Trichoderma harzianum* is successfully used as a bio-control agent for diseases caused by *Rhizoctonia solanii* on beans and tomato. *T. harzianum* is the most active antagonistic agent in the case of *Fusarium* species.

### Control of Airborne Diseases

*Botrytis cinerea* was controlled when treated with *Trichoderma*. Their colonisation prevented the saprophytic establishment of *B. cinerea* on the vine (hyperparasitism). Their establishment on the floral parts limit the development of disease on the branches. Spore suspension of *Trichoderma* decrease the number of rust pustules caused by *Puccinia*.

### Seed-borne diseases

*T. harzianum* and *T. koningii* when applied to pea seeds resulted in reduction in the incidence of pre-emergence of damping off of the pea.

### Fruit diseases

Botrytis rot of strawberry was controlled with *Trichoderma*. *Anthracnose* by *Colletotrichum* was also effectively controlled by *Trichoderma* sp.

### Mechanism of Control

Antibiotics may play a crucial role in microbial antagonism. Lytic enzymes like chitinase and β -1-3, glucanase also help in controlling the diseases.

### Mode of Application

Mycelial preparation of *T. harzianum* is more effective in comparison to spore preparation of biological control caused by *Sclerotinia* spp. Wheat bran/ peat is colonised by *Trichoderma* sp. A liquid medium is used to inoculate the bran/ corn meal along with other nutrient ingredients like glucose, sodium chloride, calcium carbonate and soybean meal.

Semi-solid fermentation is preferable for most of the fungi. Since the dry conidia bran mixture is relatively stable. Embedding propagules of the bio-control agents in a matrix formed by sodium alginate (binder) and clay appear to be an effective method of delivery of *T. viridae*. It can also be applied as a seed coating with adhesives. **commercial products** available are Antagon, Biocure-F, Bioderma, Trichocan, etc. Those commercial formulations can be introduced into organic manures, composts, dry cow dung, farmyard manure, neem cake etc.
Fill in the Blank Questions based on the above contents:

1. Biological control involves reducing inoculum density or disease-producing activities of a pathogen by one or more ________.

2. Biocontrol is based on antagonism, including competition, parasitism, and ________.

3. ________ is the law that emphasizes restricting insect populations rather than eradicating them.

4. Microbial insecticides can be categorized into (a) bacterial pesticides, (b) viral pesticides, and (c) ________ pesticides.

5. An effective microbial pesticide should be specific to the pest population, harmless to humans and other valued organisms, and possess resistant stages such as ________ or cysts.

6. Bacillus thuringiensis produces protein crystal inclusion bodies known as ________ bodies, which act as endotoxins.

7. In insect viruses, Polyhedrosis viruses embed many virus particles in each protein crystal, while Granulosis viruses contain ________ virus in each crystal.

8. In the context of Integrated Pest Management (IPM), what does IPM stand for? ________ Pest Management.

9. Trichoderma harzianum is a biological control agent used for diseases caused by ________ solanii on beans and tomatoes.

10. The mode of application for mycelial preparation of *Trichoderma harzianum* is more effective than spore preparation in controlling diseases caused by ________ spp.

Answer:

1. Organisms

2. Hyperparasitism

3. Integrated pest management (ipm)

4. Fungal

5. Spores

6. Parasporal

7. One

8. Integrated

9. Rhizoctonia

10. Sclerotinia
**Multiple Choice Questions:**

1. Biological control is based on antagonism, which includes:

a) Symbiosis

b) Competition, parasitism, and hyperparasitism

c) Mutualism

d) Commensalism

2. What is the primary characteristic of an effective microbial pesticide?

a) High cost

b) Broad-spectrum toxicity

c) Harmful to non-pest populations

d) Specificity to the pest population and harmlessness to humans and valued organisms

3. Which bacterial pathogen has been most extensively studied as an insecticide?

a) *Bacillus cereus*

b) *Clostridium botulinum*

c) *Bacillus thuringiensis*

d) *Pseudomonas aeruginosa*

4. Insect viruses are categorized into:

a) Polyhedrosis and Granulosis viruses

b) Retroviruses and Coronaviruses

c) Adenoviruses and Herpesviruses

d) Lytic and Lysogenic viruses

5. What is the law that emphasizes restricting insect populations rather than eradicating them?

a) Eradication Law

b) Pest Control Law

c) Integrated Pest Management (IPM)

d) Insect Limitation Law

6. Which fungal pesticide is used as a bio-control agent for diseases caused by Rhizoctonia solanii on beans and tomatoes?

a) Beauvaria

b) Metarrhizium

c) Entomophthora

d) Trichoderma harzianum

7. How do Polyhedrosis viruses differ from Granulosis viruses?

a) Polyhedrosis viruses contain one virus in each crystal, while Granulosis viruses embed many virus particles.

b) Granulosis viruses contain one virus in each crystal, while Polyhedrosis viruses embed many virus particles.

c) Both Polyhedrosis and Granulosis viruses contain many virus particles in each crystal.

d) Both Polyhedrosis and Granulosis viruses contain one virus in each crystal.

8. What is the primary mode of application for mycelial preparation of Trichoderma harzianum?
a) Submerged fermentation

b) Spray application

c) Seed coating

d) Semisolid fermentation

9. Which commercial product is an example of a mycelial preparation of Trichoderma harzianum?

a) DIPEL

b) DOOM

c) ANTAGON

d) TAIVIRIDAE

10. What is the primary advantage of using fungal pesticides in biological control?

a) Broad-spectrum toxicity

b) Rapid growth rate

c) Harmfulness to humans

d) Specificity and obligate parasitism

Answers:

1) Competition, parasitism, and hyperparasitism

2) Specificity to the pest population and harmlessness to humans and valued organisms

3) c) Bacillus thuringiensis

4) Polyhedrosis and Granulosis viruses

5) Integrated Pest Management (IPM)

6) Trichoderma harzianum

7) b) Granulosis viruses contain one virus in each crystal, while Polyhedrosis viruses embed many virus particles.

8) Semisolid fermentation

9) c) ANTAGON

10) d) Specificity and obligate parasitism
True or False type Question:

<table><thead><tr><td>Sl. No.</td><td>True or False statement</td><td>Answer</td></tr></thead><tbody><tr><td>1)</td><td>Biological control involves the use of chemicals to eliminate pests</td><td>False.</td></tr><tr><td>2)</td><td>The negative interactions among microbial populations are not relevant to biological control</td><td>False</td></tr><tr><td>3)</td><td>Bacillus thuringiensis is a bacterial pathogen extensively studied as an insecticide, primarily due to its resistance to environmental conditions</td><td>False</td></tr><tr><td>4)</td><td>Insect viruses, specifically Polyhedrosis and Granulosis viruses, are encased in protein coats that are soluble in water</td><td>False.</td></tr><tr><td>5)</td><td>Trichoderma harzianum is a fungal biological control agent known for its slow growth rate and inability to produce antibiotics</td><td>False</td></tr><tr><td>6)</td><td>Integrated Pest Management (IPM) aims to eradicate insect populations completely</td><td>False</td></tr><tr><td>7)</td><td>The specificity and obligate parasitism of insect viruses make their mass production feasible only on live insects</td><td>True</td></tr><tr><td>8)</td><td>Antibiotics play a crucial role in microbial antagonism, aiding in the control of diseases</td><td>True</td></tr><tr><td>9)</td><td>Microbial control in agriculture is significant for its potential harm to humans and other valued plant and animal populations</td><td>False</td></tr><tr><td>10)</td><td>Biological control relies on negative interactions, such as amensalism, predation, and parasitism, for managing pests and diseases.</td><td>True</td></tr><tr><td>11)</td><td>Bacillus thuringiensis produces parasporal bodies that act as exotoxins, causing gut paralysis in pests.</td><td>False (endotoxins)</td></tr><tr><td>12)</td><td>Integrated Pest Management (IPM) focuses on eradicating insect populations to ensure agricultural productivity.</td><td>False (restricting insect populations)</td></tr><tr><td>13)</td><td>Trichoderma harzianum is a bacterial biological control agent effective against diseases caused by Rhizoctonia</td><td>False (fungus)</td></tr><tr><td>14)</td><td>Insect viruses, specifically Polyhedrosis and Granulosis viruses, are encapsulated in protein crystals that protect them and remain active for many years.</td><td>True</td></tr><tr><td>15)</td><td>The specificity of viruses makes them ideal candidates for the control of specific pest populations.</td><td>True</td></tr><tr><td>16)</td><td>Microbial insecticides should not be specific for the pest population, allowing a broader range of control.</td><td>False (specific for the pest population)</td></tr><tr><td>17)</td><td>Azolla pinnata is a bacterium commonly found in ponds, ditches, and rice fields, used as a biofertilizer for rice.</td><td>False (water fern)</td></tr><tr><td>18)</td><td>The role of Trichoderma harzianum as a bio-control agent includes rapid growth, abundant sporulation, and production of antibiotics like gliotoxin and viridian.</td><td>True</td></tr><tr><td>19)</td><td>The microbial pesticide Bacillus thuringiensis remains active from year to year and persists in the environment.</td><td>False</td></tr></tbody></table>
**Short Type Questions:**

1. Define Biological Control.

2. What are the three negative interactions that form the basis for biological control?

3. Explain the principles of Biocontrol.

4. Name the three categories of microbial insecticides.

5. What are the characteristics of an effective microbial pesticide?

6. Describe the role of parasporal bodies in Bacillus thuringiensis.

7. Differentiate between Polyhedrosis and Granulosis viruses.

8. What does IPM stand for, and what is its main emphasis?

9. Which bacterial pathogen has been extensively studied as an insecticide, and what is its primary toxic factor?

10. How does *Trichoderma harzianum* act as a bio-control agent?

**Long Type Question:**

1. Explain the role of microbial agents in biological control, detailing the categories of microbial insecticides, their characteristics, and the principles underlying their use. Provide examples and discuss the significance of microbial control in agriculture.

**Answer:**

Biological control is a method of managing pests and diseases using microorganisms to initiate disease in pest populations, thereby reducing the reliance on chemical pesticides. The negative interactions among microbial populations and between microbes and higher organisms serve as the basis for biological control. There are three main categories of microbial insecticides: bacterial pesticides, viral pesticides, and fungal pesticides.

An effective microbial pesticide should be specific to the pest population, survive environmental conditions, possess resistant stages like spores, and be harmless to humans and valued organisms. *Bacillus thuringiensis*, a bacterium, has been extensively studied. Its parasporal bodies act as endotoxins, causing gut paralysis in pests.

Insect viruses, categorized into Polyhedrosis and Granulosis viruses, are encased in protein crystals that protect them. They cause infections when ingested by pests, leading to lethargy and death. Integrated Pest Management (IPM) emphasizes restricting insect populations rather than eradication. *Trichoderma harzianum*, a fungal biological control agent, is effective against diseases caused by *Rhizoctonia solanii*. It competes, produces antibiotics, and acts as an antagonist, providing a sustainable approach to disease management.

Microbial control in agriculture is significant as it reduces the need for chemical pesticides, minimizes environmental impact, and helps maintain pest populations below economically damaging levels. This approach aligns with sustainable and environmentally friendly agricultural practices.
# Biofertilizers - Introduction, status and scope

Dr. AJEET KUMAR<fnref n="9" />

## Introduction

Indian economy is an agrarian economy, a reason that makes biofertilizers an essential need for Indian farmers. Though most of our agricultural activities are dependent on monsoon, the use of a good biofertilizer can yield a better crop to the farmers and increase the fertility of the soil. According to an estimate 240 million tons of food grains will be required to feed about one billion expected populations by 2000 AD in India and to achieve this milestone, a sizable quantity of mineral fertilizer will be required. The total fertilizer requirements of our country would be 23 million tonnes as against the present consumption level of 13 million tonnes, which is beyond any single type of nutrient source to accept the challenge of appropriate nutrient supply. Integrated use of all the sources such as mineral fertilizers, organic manures, biofertilizers etc. is the only alternate for improving soil fertility. It has now become possible to meet a large part of our total nitrogen demand through proper husbandry of biological nitrogen fixation by micro-organism in crop production system. Biofertilizers are capable of providing an economically liable level for achieving the ultimate goal of enhanced productivity. The crop microbial soil ecosystem can, therefore, be energized in sustainable agriculture.

India is fourth largest user of chemical fertilizers (12.5million tones of NPK nutrients) in the world even its soils are still being depleted of their inherent nutrients reserve as a result of wide gap between additions (12.5 million tonnes) and removal (18.5 million tonnes). One tone of produce removes an average of 32 kg nitrogen, 12 kg phosphorus and 58 kg potash. The strategy for sustaining satisfactory yield levels envisages nutrient balance and efficient nutrient cycling. This can be successfully achieved through integrated use of mineral fertilizer, bulky organic manures, compost, green manure, biological inoculant etc. At present, the nutrient use in India is much less compared to that of other countries. Against world average consumption of 95 kg ha⁻¹, and we are using 74 kg ha⁻¹ with national productivity level of merely 1.1 metric ton per hectare. At present India produces about 117 million metric tonnes of food grain for its growing population of more than 800 million people. Nitrogen fertilizer plays prominent role in increasing this food requirement. At present the vast gap cannot be filled up merely through the production of synthetic nitrogen fertilizers due to scarcity of high cost of raw materials such as fossil fuels. Biological nitrogen fixation is the key to sustain agriculture productivity application of biofertilizer in the field is the liable alternative. In fertilizer manufacturing factories, nitrogen is fixed industrially, by means of the Haber Bosch process requiring H₂ gas, very high temperature and enormous energy. Industrially fixed nitrogen has been used precipitously. It was produced 4.0 lakh metric tonnes in 1905 and this was increased to 3.5 million tonnes of nitrogen fertilizer. However, above every

<footnote n="9">⁹ 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>
hectare of land, there are 78,000 tonnes of inert nitrogen gas ($N_2$). As nitrogen is the most limiting nutrient for increasing crop productivity. Only a few prokaryotic organisms are able to fix nitrogen directly through a biological process. Annual biological nitrogen fixation (BNF) is estimated to be around 175 million tonnes of which close to 79 % is accounted for by terrestrial fixation

The term biofertilizers or which can be more appropriately called ‘microbial inoculants’ can be generally defined as a preparation containing live or latent cells of efficient strains of nitrogen fixing, phosphate solubilizing or cellulytic microorganisms used for application of seed, soil or composting areas with the objective of increasing the number of such microorganisms and accelerate certain microbial process to argument the extent of the availability of nutrients in a form which can be easily assimilated by plant. In large sense, the term may be used to include all organic resources for plant growth, which are rendered in an available form for plant absorption through microorganisms or plant associations or interactions. Soil harbours a range of microorganisms, which bring about a number of biochemical reactions in soil. The soil organisms are classified into two broad groups i.e. soil flora and soil fauna, they are again subdivided, depending upon their size such as micro and macro flora. Soil microflora includes bacteria, fungi, actinomycetes, algae etc. of these groups, bacteria are the most abundant followed by actinomycetes and fungi; algae are found under specific situation. The biomass and population of these micro-organisms in soil have been given in Table 1.

<table><thead><tr><td>Sr. No</td><td>Microorganism</td><td>Average number per g of soil (in lakhs)</td><td>Average biomass (in kg per ha)</td></tr></thead><tbody><tr><td>1.</td><td>Bacteria</td><td>1000</td><td>500</td></tr><tr><td>2.</td><td>Fungi</td><td>10</td><td>1000</td></tr><tr><td>3.</td><td>Actinomycetes</td><td>100</td><td>750</td></tr><tr><td>4.</td><td>Algae</td><td>0.01</td><td>150</td></tr></tbody></table>

The presence of these microorganisms make the soil a living and active system. These microorganisms play a significant role in the life cycle of plants and animals through a number of process such as decomposition, solubilization, nutrient fixation and supply of plants. At present, in India there is a gap of about 10 million tonnes of plant nutrients between removal by crops and replenishment through fertilizers. Supply of nutrients from organic manure has not so far been able to fill up this gap and also in years to come nutrient supply from the source is unlikely to improve due to competitive demand for alternate uses like fuel and fodder. Of late, biofertilizer are being promoted as an important component in supplementing plant nutrient need of the country. India is one of the important countries in biofertilizer production and consumption in the world. The present production capacity of different production units in the country is about 4500 tonnes per annum. The maximum production capacity is in Agro Industries Corporation followed by State Agriculture Departments, National Biofertilizer Development Centre, State Agriculture Universities and private sector. Among the different states, the maximum production capacity is in Tamil Nadu followed by M.P. U.P., Gujarat and Maharashtra.
**Thrust in Research and Development:**

The rising cost of chemically fixed nitrogen fertilizer and the massive inputs of non-renewable resource will be devastating. As an alternative there is urgency of major research effort in biological nitrogen fixation. Nitrogen in the form of ammonia is used by plants and microbes as a building block for the synthesis of amino acids and of other nitrogenous compounds. The conversion of atmospheric Nitrogen into ammonia by the nitrogen fixing microbes, mostly certain bacteria and blue green algae is called biological nitrogen fixation. Although leguminous plants have been used extensively in agriculture for centuries, it is possible to exploit these nitrogen fixing plants meaningfully. The use of new leguminous crops as well as the breeding of more vigorous and effective traditional leguminous crops can be helpful in food production. As the bacteria are the agents of fixation in bacterial plant Rhizobial technology and soil microbiology are the neglected areas. It would be a right type technology that all farmers could use legume - Rhizobial combinations constitute, a built-in nitrogen source for food crop production. Approximately 75 per cent of the nitrogen utilized by legume comes from the soil, with only 25 per cent from BNF. It is necessary to increase the rate of nitrogen fixation achieved, which would have a multiplier effect on the enhancement of crop productivity. Chemical fertilizers have temporary effect while biofertilizers have permanent effect without any production problem. Use of composite biofertilizers has permanent effect without any production problem. Use of composite biofertilizers can increase soil fertility. The cost is too much low for the biofertilizers and its proper use dose not only include a correct application of the inoculant to the seed soil but also good. Considering the prospects of biofertilizers in the country, the biofertilizers development centres are being established both in government and private sectors and It is only possible by establishing joint venture in biofertilizer agro based industry.

Biofertilizers are products that contain microorganisms essential for soil fertility and plant growth when added to the soil. A biofertilizer is a chemical that contains living microorganisms that colonize the rhizosphere or the interior of the plant when given to seeds, plant surfaces, or soil, and encourage growth by controlling the quantity or availability of primary nutrients to the plant host. Biofertilizers supply nutrients to plants through natural processes such as nitrogen fixation, phosphorus solubilization, and the creation of growth-promoting chemicals. They help restore the soil's natural nutrient cycle and increase soil organic matter. Healthy plants can be developed with the application of biofertilizers while also improving the soil's sustainability and health. Biofertilizers will likely decrease the need for synthetic fertilizers and pesticides, but they will not be able to completely replace them.

**Importance of Biofertilizers**

There are various uses of biofertilizers that prove their importance. They include – improving the soil quality, protecting the plants from pathogens, avoiding environmental pollution, destruction of harmful substances presents in the soil, etc. Thus, biofertilizers are very important.
# Types of Biofertilizers

## 1. Symbiotic Nitrogen-Fixing Bacteria

The symbiotic nitrogen-fixing bacteria like Rhizobium get food and shelter from the plants and provide them with fixed nitrogen in return. One of the most important symbiotic nitrogen-fixing bacteria is Rhizobium. Bacteria seek shelter and food from plants here. In exchange, they assist the plants by delivering free nitrogen.

## 2. A Loose Association of Nitrogen-Fixing Bacteria

Some bacteria aren't related directly with the plants but live around them For example, A nitrogen-fixing bacterium called Azospirillum lives near the roots of higher plants but does not form a close bond with them. This is known as rhizosphere association because these bacteria collect plant exudate and use it as food. Associative mutualism is the name given to this phenomenon.

## 3. Symbiotic Nitrogen-Fixing Cyanobacteria

There are many symbiotic nitrogen-fixing cyanobacteria like liverworts, cycad roots, the bacteria released by fern plant decay, etc. Cyanobacteria or blue-green algae from a symbiotic relationship with numerous plants. Anabaena can be found in the fern's leaf cavities. It's in charge of nitrogen fixation. The fern plants decompose and release nutrients for the rice plants to use. Azolla pinnata is a fern that grows in rice fields.

## 4. Free-Living Nitrogen-Fixing Bacteria

Free-living bacteria are found in soil, and they also perform nitrogen fixation. These include clostridium, Azotobacter, and bacillus polymyxin. They are nitrogen-fixing bacteria that live in free-living soil. *Clostridium beijerinckii*, *Azotobacter*, and other saprotrophic anaerobes are among them. Rhizobium and Azospirillum are the most extensively utilized forms of biofertilizers.

# Components of Biofertilizers

The components of a biofertilizer are:

1. **Bio Compost:**

It is eco-friendly and is produced from waste products coming from sugar industry. It also includes bacteria, fungi, and some plants.

2. **Tricho-Card:**

This eco-friendly non-pathogenic product is useful for many crops and plants, as it plays the role of a productive destroyer against the items that are harmful to the crop.

3. **Azotobacter:**

It plays an important role in atmospheric nitrogen fixation and protects the plant roots from pathogens in the soil.

4. **Phosphorus:**

To settle the exact level of need for nitrogen for a plant, and to determine the nitrogen level of the soil, phosphorus fertilizers are very helpful.
### 5. Vermicompost:

Known for quickly improving soil fertility, these are probably the most eco-friendly fertilizers that contain vitamins, sulphur, hormones, organic carbon, and antibiotics required for the growth of the plant.

## Applications of Biofertilizers

These are some important applications of biofertilizers:

1. **Seedling Root Dip:** Used for rice crops, the seedlings are planted in a waterbed for 8 to 10 hours, in this method.

2. **Seed Treatment:** In this process, the seeds are dipped in nitrogen-phosphorus mixed fertilizers. After drying them, they are planted as early as possible.

3. **Soil Treatment:** The mixture of biofertilizers and compost fertilizers is kept overnight and spread over the soil the next day. This treatment takes place before sowing the seeds.

## Disadvantages of Fertilisers

* Chemical fertilizers are supplemented by biofertilizers, not substituted for them.

* Biofertilizers only improve crop productivity by 20 to 30 percent. Unlike chemical fertilizers, they do not result in a significant improvement in productivity.

* For specific crops, specific fertilizers are necessary. This is more applicable to microorganisms that live in a symbiotic relationship. If non-specific Rhizobium is applied as a fertilizer, root nodulation, and crop production will not rise.

* Strict aseptic precautions are required during the manufacture of microbial fertilizer. During microbial mass manufacturing, contamination is a common problem.

* Microbes are killed when exposed to sunlight for an extended period of time because they are light-sensitive.

* When stored at room temperature, microbial fertilizers must be used within six months, and when stored at chilling temperature, it must be used within two years.
# Instruments Used in Microbiology Lab with Principle and Uses

Dr. AJEET KUMAR<fnref n="10" />

## List of Instruments used in Microbiology Lab

1. Analytical Balance

2. Autoclave

3. Bunsen burner

4. Centrifuge

5. Colony Counter

6. Deep Freezer

7. Homogenizer

8. Hot plate

9. Hot air oven

10. Incubator

11. Laminar Air Flow/ Laminar Hood

12. Magnetic Stirrer

13. Microscope

14. pH Meter

15. Spectrophotometer

16. Vortex Mixture/ Vortexer

17. Water Bath

18. Water Distiller

19. Culture tube

20. Test tube

21. Inoculation needle/rod/loop

22. Laboratory Heat Sealer

23. Vacuum pump

24. Microwave ovens

25. Lab incubators

26. Sand bath

27. Oil bath

28. Fermenter

<footnote n="10">¹⁰ 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>
# 1. Analytical Balance

An analytical balance is a type of balance that is commonly used for the measurement of mass in the sub-milligram range.

## Working Principle

<figure><img src="image_23.png" /></figure>

These types of balances are made with a measuring pan enclosed in a transparent covering that prevents small particles or air currents from getting collected on the pan.

<figure><img src="image_24.png" /></figure>

An electric analytical balance uses the force necessary to counteract the mass rather than measuring the mass itself.

<figure><img src="image_25.png" /></figure>

An electromagnet is used to create a force required to achieve a balance with the mass of the substance and the resulting force is displayed.

## Uses

* As they are highly precise and based on advanced technology, analytical balances are explicitly used in laboratories for the effective completion of tasks like weighing test materials and sampling amounts, formulation, density determination, purity analysis, quality control testing, and material and conformance testing

# 2. Autoclave

An autoclave is a pressurized chamber used for the process of sterilization and disinfection by combining three factors: time, pressure, and steam

## Working Principle

<figure><img src="image_26.png" /></figure>

Autoclaves use steam as their sterilization agent. The basic principle of an autoclave is that all the items within the autoclave come in direct contact with the steam for a particular period irrespective of the nature of the material- whether it is liquid, plastic ware, or glassware.

<figure><img src="image_27.png" /></figure>

The amount of time and the temperature depends on the type of material being sterilized and the increase in temperature of the cycle allows for shorter periods.

## Uses

* Autoclaves are mostly used for the sterilization of medical or laboratory equipment with the capacity of sterilizing a large number of materials at once.

* They are commonly used for the preparation of culture media during laboratory applications.

<figure><img src="image_28.png" /></figure>
## 3. Bunsen burner

Bunsen burner is a standard tool used in laboratories, named after Robert Bunsen. It is a gas-fueled single open flame.

### Working Principle

<figure><img src="image_29.png" /></figure>

This burner is made with a metal tube on a flat base with a gas inlet at the bottom of the tube, which may have an adjustable valve. On the sides of the tube are openings that can be adjusted with a collar to control the amount of air that can enter.

<figure><img src="image_30.png" /></figure>

Once the burner is connected to a gas source, the gas is forced by the gas pressure so that the gas reaches the top where the flame is ignited with a match or a lighter.

### Uses

* It is commonly used for processes like sterilization, combustion, and heating. In medical or microbiology laboratories, it is commonly used for micro-loop sterilization.

## 4. Centrifuge

A centrifuge is a device that allows the rotation of an object about a single axis, where an outward force is applied perpendicularly to the axis. A laboratory centrifuge is motor-based and allows the rotation of a liquid sample resulting in the separation of the components of the mixture.

### Working Principle

<figure><img src="image_31.png" /></figure>

A centrifuge works on the principle of sedimentation, where the high speed of the rotation causes the denser particles to move away from the center while smaller, less dense particles are forced towards the center.

<figure><img src="image_32.png" /></figure>

Thus, the denser particles settle at the bottom while the lighter particles are collected at the top.

<figure><img src="image_33.png" /></figure>

In a laboratory tabletop centrifuge, the sample tubes are aligned at an angle so that the particles have to travel a shorter distance before they hit the bottom.

### Uses

* The primary application of a centrifuge is the separation of particles suspended in a suspension. It can be used for the separation of cell organelles, nucleic acid, blood components, and separation of isotopes.

## 5. Colony Counter

A colony counter is used to estimate the density of a liquid culture by counting the number of CFU (colony forming units) on an agar or culture plates. It is used for counting microorganism which is not visible by naked eye. The magnifying glass (convex lense) is fitted in this equipment.

### Working Principle

<figure><img src="image_34.png" /></figure>

This instrument can accommodate different sizes of plates which are scanned on top with UV, white light and/or fluorescent illumination.

<figure><img src="image_35.png" /></figure>

One can accomplish the counting either manually with the touch pressure or with a digital counter.
## Uses

* A colony counter is primarily used for counting the number of colonies present on a culture plate to estimate the concentration of microorganisms in liquid culture.

# 6. Deep Freezer

## Working Principle

<figure><img src="image_36.png" /></figure>

Deep freezers are based on the principle that under extremely low temperatures, there is **minimum microbial growth** which allows for the protection and preservation.

<figure><img src="image_37.png" /></figure>

Based on this principle, we can even preserve cultures over a long period of time without any change in the concentration of the microorganisms.

## Uses

* A deep freeze can be used for the preservation of different things used in the laboratories for a very long period of time. Deep freezers are used in laboratories to store and preserve isolated microbes for a more extended period of time.

# 7. Homogenizer

Homogenizer is a device used in laboratories for the mixing of various liquids and materials like tissue, plant, food, soil, and many others.

## Working Principle

<figure><img src="image_38.png" /></figure>

This instrument is based on the principle that when large globules in coarse emulsion are passed under high pressure through a narrow orifice, they break down into smaller particles giving a more uniform and stable mixture.

<figure><img src="image_39.png" /></figure>

A homogenizer has a metal rod with narrow parallel openings in the form of a comb at the end which acts as the orifice for the homogenization process.

## Uses

* A homogenizer is primarily used to disrupt cells to acquire cell organelles for different microbiological processes.

* It is used in the preparation step before the extraction and purification of different macromolecules like proteins, nucleic acids, and lipids.

# 8. Hot plate

A hot plate is a stand-alone appliance used in microbiology laboratories as a table-top heating system.

## Working Principle

<figure><img src="image_40.png" /></figure>

Unlike the traditional ways of producing heat through fire, a hot plate produces heat by the flow of electricity.

<figure><img src="image_41.png" /></figure>

On a hot plate, electricity runs through the coils which have a high level of electrical resistance. The resistance in the coils converts the electrical energy into heat energy which causes the coils to release heat.
## Uses

* In a laboratory, hot plates are used to heat glassware and its components.

* They are used over water baths as water baths might be hazardous in case of any spills or overheating.

# 9. Hot air oven

A hot air oven is an electrical device that is used for sterilization of equipment, dried glassware or samples using dry heat.

## Working Principle

<figure><img src="image_42.png" /></figure>

A hot air oven is a type of dry heat sterilization which is performed on dry materials and on substances that do not melt or catch fire under high temperatures.

<figure><img src="image_43.png" /></figure>

There are **two types of hot air ovens** based on the working principle

<figure><img src="image_44.png" /></figure>

**Forced air hot air oven:** In this type of hot air oven, the heated air inside the oven is distributed throughout the oven with a fan. This prevents the rising of hot air towards the top while keeping the cold air at the bottom. This allows for the adequate heating of materials inside the oven.

<figure><img src="image_45.png" /></figure>

**Static air hot air oven:** In this type of oven, the heat is produced by coils present at the bottom of the oven with no fan. The hot air rises and doesn't allow the effective sterilization of the materials.

<figure><img src="image_46.png" /></figure>

The equipment inside the oven acquires heat and passes the heat towards the center, one layer at a time which allows for effective dry heat sterilization.

## Uses

* A hot air oven can be used to sterilize materials like glassware, metal equipment, powders, etc.

* It allows for the destruction of microorganisms as well as bacterial spores.

<figure><img src="image_47.png" /></figure>
# 10. Incubator

An incubator is a device used in laboratories for the growth and maintenance of microorganisms and cultures. Incubator provides an optimal temperature, pressure, moisture, among other things required for the growth of microorganisms.

## Working Principle

- The incubator is based on the principle of maintaining a proper atmosphere for the growth of microorganisms.

- Incubators have a heating system that allows for the temperature within the incubator to be adjusted according to the type of organism allowed to grow inside.

- Similarly, they are provided with adjustments for maintaining the concentration of CO₂ to balance the pH and humidity required for the growth of the organisms.

- Variation of the incubator like a **shaking incubator**, which allows for the continuous movement of the culture required for cell aeration and solubility studies.

## Uses

- Incubators have a wide range of applications including cell culture, pharmaceutical studies, hematological studies, and biochemical studies.

- Incubators can also be used in the steam cell research area.

# 11. Laminar Air Flow/ Laminar Hood

Laminar Hood is a closed device primarily for processes or instruments sensitive to microbial contamination.

## Working Principle

- A Laminar Hood is made up of stainless steel, avoiding joints and corners to prevent the accumulation of bacterial spores.

- This device creates a sterile environment with the flow of sterile air through a **High-Efficiency Particulate Air (HEPA) filter** and shortwave **ultraviolet germicidal lamp** that sterilizes the workstation.

- Laminar Air Flow has to **turn on 15 minutes before to ensure complete sterilization** and the workstation should be **cleaned with ethanol** before and after use.

## Uses

- Laminar Hood is used to conduct processes that are sensitive to contamination.

- It is used for experiments related to isolation of microorganism, plant tissue culture and for the experiments of genetic transformation.
<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>

## 12. Magnetic Stirrer/culture tube stirrer

Magnetic Stirrer is a device used in microbiology laboratories for the purpose of mixing the liquids/ or the material which is present in culture tube.

### Working Principle

<figure><img src="image_50.png" /></figure>

This device consists of a rotating magnetic or an electromagnet creating a rotating magnetic field that allows the stir bar (a piece of heavy metal) to move around in the vessel.

<figure><img src="image_51.png" /></figure>

It is coupled with a heating system to heat the liquid while it is mixed.

### Uses

* It is usually used for mixing various liquid components in a mixture in a chemical or microbiology laboratory.

* This device is used in place of other stirrers as it is noise-free and because the size of the stir bar is so tiny, there is less chance of contamination.

<figure><img src="image_52.png" /></figure>

## 13. Microscope

Microscopes are devices that allow the observer to have an exceedingly close view of minute particles.
## Working Principle

<figure><img src="image_53.png" /></figure>

There are different types of microscopes, each of which works on its respective principles. However, there is some commonality in them.

<figure><img src="image_54.png" /></figure>

The basic principle in a microscope is magnification. Based on the relative position of the object from the lens or electromagnets, different positions, nature, and magnification of the image can be achieved.

<figure><img src="image_55.png" /></figure>

Different types of microscopes are developed to cater to the specific needs of the observation. However, the common theme is magnification.

## Uses

* Based on the type of microscopes, different microscopes are used for different purposes.

* They are primarily used for the observation of minute particles which cannot be observed with naked eyes.

## 14. pH Meter

A pH meter is a device used in laboratories that measure the H-ion concentration in water-based solutions to determine the acidity or alkalinity of the solution. A **pH meter** is **often termed** a “**potentiometric pH meter**” as it **measures the difference** in electric potential between the **reference** and a **pH electrode**.

### Working Principle

<figure><img src="image_56.png" /></figure>

In a potentiometric pH meter, single or multiple glass electrodes, connected to a bulb selective to hydrogen ions, are attached to a metal rod.

<figure><img src="image_57.png" /></figure>

When the bulb with the electrodes is dipped into a solution, hydrogen ions in the solution exchange with positive charges on the electrode generating an electrochemical potential which is displayed in terms of pH units on display.

## Uses

* A pH meter is primarily used to measure the acidity of microbial cultures, soil, and water treatment plant.

* It can be used to measure the acidity level in wine and cheese during their production.

## 15. Spectrophotometer

The spectrophotometer is an optical instrument for measuring the intensity of light in relation to wavelength. Based on the amount of light absorbed by a coloured solution, a quantitative analysis of the solution can be done.

### Working Principle

<figure><img src="image_58.png" /></figure>

Spectrophotometry is based on the **Beer-Lambert Law**, which states the absorbance of light by a solution (of a particular wavelength) is directly proportional to the concentration of the substance.

<figure><img src="image_59.png" /></figure>

Different wavelengths of light are passed through a solution as different substances have better absorbance at different wavelengths. Based on the absorbance of a particular wavelength, the quantitative analysis of a solution can be done.
## Uses

* In a microbiology laboratory, a spectrophotometer is applied for the measurement of the substance concentration of protein, nucleic acids, bacterial growth, and enzymatic reactions.

# 16. Vortex Mixture/ Vortexer

A vortex mixture is one of the basic technologies used for the **mixing of samples in glass tubes or flasks** in laboratories.

## Working Principle

<figure><img src="image_60.png" /></figure>

It is based on the simple principle of causing reactions and homogenization by agitating the mixture.

<figure><img src="image_61.png" /></figure>

Motorized draft shafts present on the mixer oscillate and transfer the movement to the sample tubes causing the sample fluids to undergo turbulent flow.

## Uses

* Vortex mixer is mostly used for the mixing of various sample fluids in the sample tubes and also allows for the homogenization of cells and cell organelles.

# 17. Water Bath

Water Bath is a conventional device that is used for chemical reactions that required a controlled environment at a constant temperature.

## Working Principle

<figure><img src="image_62.png" /></figure>

A sensor in the device transfers water temperature to a reference value which is then amplified and a control system generates a signal for the heating system which heats the water to the desired temperature.

## Uses

* Water baths are primarily used for heating samples under a controlled temperature.

* These are suitable for heating chemicals that might be flammable under direct ignition.

<figure><img src="image_63.png" /></figure>

# 18. Water Distiller

A water distiller is a device that purifies water by the process of distillation. This is commonly used in, microbiology laboratories, organic chemistry laboratories and medical laboratories.

## Working Principle

<figure><img src="image_64.png" /></figure>

A water distiller is based on the principle of distillation.
<figure><img src="image_65.png" /></figure>

According to this process, water is first brought to a boil and then condensed into liquid form to obtain pure distilled water.

## Uses

* It is used to obtain distilled water required for lab tests as well as for the preparation of culture media.

## 19. Culture Tube

Culture tubes are often used in microbiology lab for handling and culturing all kinds of live organisms, such as molds, bacteria, seedlings, plant cuttings, etc. It is also used to store samples and other fluids. The mouth of the tube is plain cut because rounded mouth causes problems in removing cotton plug of tube.

<figure><img src="image_66.png" /></figure>

## 20. Test Tube

Test tubes are widely used by chemists to handle chemicals, especially for qualitative experiments and assays. Their spherical bottom reduces mass loss when pouring, make them easier to wash out, and allow convenient monitoring of the contents.

<figure><img src="image_67.png" /></figure>

## 21. Inoculation needle/rod/loop

An inoculation needle is equipment used in the lab of microbiology. It is used in the transfer of microbial organisms from plate culture to needle by first sterilizing the needle to prevent contaminations. Inoculation needle is made up of **Nichrome alloy** {Nickel, Chromium, Manganese & Fe} which is heat resistant.

An inoculation loop, also known as a **smear loop** is a simple tool, primarily used by microbiologists to take and transfer inoculum of a microorganism culture.

## 22. Laboratory Heat Sealer

This instrument is used to seal the **pouches, plastic bags, sachets** that are used in packaging of biofertilizer. The apparatus is used to ensure the intactness or band of the plastic bags and other packaging bags that are used to pack different consumable as well as non-consumable goods.
## 23. Vacuum pump

Vacuum pumps are mechanical devices that enable the removal of air and gas molecules from a sealed area to create an area devoid of air or gas, their purpose is to clean and seal. Vacuum pumps come in wet or dry variants depending on the media being pumped through them.

## 24. Microwave ovens

Microwave ovens are used for **heating and defrosting in laboratories**. Improper use of a microwave can pose a number of hazards including: Ignition of flammable vapors. Electrical shock from ungrounded or faulty units. Microwave ovens used in laboratories for the rapid heating of material-either to dry them completely or to subject a work piece to sudden thermal stress or electric field stress. **Determinations of the moisture content in soil or leaf tissue samples**, for example, can be made **within tens of seconds** rather than hours. It is often assumed that placing a load within a microwave oven will result in it being heated evenly as well as quickly.

## 25. BOD incubators

BOD (Biological Oxygen Demand) incubators are essential equipment in the laboratory that provides a temperature-controlled environment to support growth of microbiological cultures.

**Here the materials kept in desired temperature for desired period of time.**

## 26. Sand bath

A sand bath is a common laboratory equipment made from a container filled with heated sand. It is used **to provide even heating for another container during a chemical reaction**.

<figure><img src="image_68.png" /></figure>

## 27. Oil bath

An oil bath is a type of heated bath used in a laboratory, most commonly used **to heat up chemical reactions**. It's essentially a container of oil that is heated by a hot plate or (in rare cases) a Bunsen burner. It is used in laboratories for reactions that require heating temperatures up to **200 degrees Celsius**. Oil baths provide uniform heat compared to other heaters. An oil bath is a **heating device which uses boiling oil as the temperature regulator**. Since different oils have different boiling points, it is possible to obtain a temperature near to the desired temperature by selecting oil with a boiling point as close as possible to the desired temperature.
<figure><img src="image_69.png" /></figure>

## 28. Fermenter

A fermenter is the equipment, which provides the proper environment for the growth of a desired organism. It is generally a large vessel in which, the organism may be kept at the required temperature, pH, dissolved oxygen concentration and substrate concentration. Different models of fermenters are available depending upon the necessity. A simple version model contains steam generator, sterilization process devices and agitator. A sophisticated fermenter contains pH regulator, oxygen level regulator, anti-foam device, temperature controller, etc.

<figure><img src="image_70.png" /></figure>
**True or False type questions with Answer:**

1. **True or False:** An analytical balance measures mass by counteracting the mass with an electromagnet.

* **Answer:** True

2. **True or False:** Autoclaves use dry heat to sterilize equipment in laboratories.

* **Answer:** False (They use steam for sterilization)

3. **True or False:** A centrifuge works on the principle of sedimentation.

* **Answer:** True

4. **True or False:** A deep freezer is used to sterilize laboratory equipment.

* **Answer:** False (It is used to preserve samples by maintaining extremely low temperatures)

5. **True or False:** A homogenizer is used to heat glassware in microbiology laboratories.

* **Answer:** False (It is used to mix and homogenize materials)

6. **True or False:** A Bunsen burner is commonly used for micro-loop sterilization in microbiology laboratories.

* **Answer:** True

7. **True or False:** A hot air oven uses moist heat to sterilize equipment.

* **Answer:** False (It uses dry heat for sterilization)

8. **True or False:** The principle of a spectrophotometer is based on the Beer-Lambert Law.

* **Answer:** True

9. **True or False:** Laminar Air Flow provides a sterile environment using a HEPA filter.

* **Answer:** True

10. **True or False:** A pH meter measures the temperature of a solution.

* **Answer:** False (It measures the hydrogen-ion concentration or pH of a solution)
# Structure and Characteristic Features of Different types of Biofertilizers

Dr. AJEET KUMAR<fnref n="11" />

## Bio-Fertilizers

Bio-fertilizers are defined as preparations containing active (living cells) or latent cells of efficient strains of certain microorganisms that help crop plants to utilize the atmospheric nitrogen to increase the nitrogen content of soil, and can dissolve the insoluble phosphate of the soil to release the phosphorus in the rhizosphere when applied through seed or soil. They accelerate certain microbial processes in the soil which augment the extent of availability of nutrients in a form easily assimilated by plants.

## Processing of carrier material

The use of ideal carrier material is necessary in the production of good quality biofertilizers. Peat soil, lignite, vermiculite, charcoal, press mud, farmyard manure and soil mixture can be used as carrier materials. The following points are to be considered in the selection of carrier material; Cheaper in cost, Should be locally available, High organic matter content, No toxic chemicals, Water holding capacity of more than 50%, Easy to process, friability and vulnerability.

## Preparation of carrier material

The carrier material (peat or lignite) is powdered to a fine powder so as to pass through 212 micron sieve. The pH of the carrier material is neutralized with the help of calcium carbonate (1:10 ratio), since the peat soil / lignite are acidic in nature (pH of 4 - 5). The neutralized carrier material is sterilized in an autoclave to eliminate the contaminants

Use of biofertilizers is one of the important components of integrated nutrient management, as they are cost effective and renewable source of plant nutrients to supplement the chemical fertilizers for sustainable agriculture. Several microorganisms and their association with crop plants are being exploited in the production of biofertilizers. They can be grouped in different ways based on their nature and function.

<table><thead><tr><th>S. No.</th><th>Groups</th><th>Examples</th></tr></thead><tbody><tr><td colspan="3">N<sub>2</sub> fixing Biofertilizers</td></tr><tr><td>1.</td><td>Free-living</td><td>Azotobacter, Beijerinckia, Clostridium, Klebsiella, Acetobacter, Anabaena, Nostoc,</td></tr><tr><td>2.</td><td>Symbiotic</td><td>Rhizobium, Frankia, Anabaena azollae</td></tr><tr><td>3.</td><td>Associative Symbiotic</td><td>Azospirillum</td></tr></tbody></table>

<footnote n="11">¹¹ 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>
## P Solubilizing Biofertilizers

1. Bacteria
   *Bacillus megaterium* var. phosphaticum, *Bacillus subtilis*
   *Bacillus circulans*, *Pseudomonas striata*

2. Fungi
   *Penicillium sp*, *Aspergillus awamori*

## P Mobilizing Biofertilizers

1. Arbuscular mycorrhiza
   *Glomus sp*, *Gigaspora sp*, *Acaulospora sp*, *Scutellospora sp*.
   *& Sclerocystis sp*.

2. Ectomycorrhiza
   *Laccaria sp*, *Pisolithus sp*, *Boletus sp*, *Amanita sp*.

3. Ericoid mycorrhizae
   *Pezizella ericae*

4. Orchid mycorrhiza
   *Rhizoctonia solani*

## Biofertilizers for Micro nutrients

1. Silicate and Zinc
   *Bacillus* sp.

## Plant Growth Promoting Rhizobacteria

1. *Pseudomonas*
   *Pseudomonas fluorescens*

## Types of Bio-fertilizers:

There are following types of biofertilizers: Bacterial, fungal, algal, aquatic fern and earthworms

### Bacterial Bio-Fertilizers

#### Bacterial Bio-Fertilizers

Here, live cells of bacteria are used as fertilizers. They are the principal **nitrogen fixers** in the soil and may be either free-living or symbiotic (within the nodules of legume roots, leaf nodules of certain plants and stem nodules of *Sesbania grandiflora*). Some bacterial genera can **solubilize phosphorus** from the bound form (*Bacillus*, *Pseudomonas* sp.).

Bacteria like *Agrobacterium* sp. and *Pseudomonas fluorescence* supply growth hormones like indole acetic acid to the plants. Certain groups of bacteria like the *Pseudomonas fluorescence* living in association with the rhizosphere of most of crop plants (rhizobacteria promoting plant growth) supply all the essential nutrients required for the growth of the crop and in addition, protects the plant roots from the attack by soil-borne pathogens (saprophytic suppression). The nitrogen fixing ability of the bacteria can be studied under the following heads.

**Free-living nitrogen fixers:** Among the large number of free-living nitrogen fixers, the *Azotobacter* requires oxygen to flourish and *Klebsiella* and *Rhodospirillum* can survive both in the presence and absence of oxygen. Apart from fixing nitrogen, *Azotobacter chroococcum* has the ability to synthesize and secrete B-vitamins, growth hormones and antifungal antibiotics into its environment. *Azotobacter* has one drawback, its nitrogen fixing ability is regulated by the presence of nitrogenous compounds in its environment. Thus it cannot be used along with a nitrogenous
fertilizer. Klebsiella is another promising free-living nitrogen fixer, and it has been possible to isolate its 'nif' genes (17 of them) and to introduce them into E.coli.

## Benefits of Liquid Bio-fertilizer

The advantages of Liquid Bio-fertilizer over conventional carrier based Bio-fertilizers are listed below:

* Longer shelf life -12-24 months.

* No contamination.

* No loss of properties due to storage upto 45° c.

* Greater potentials to fight with native population.

* High populations can be maintained more than 10⁹ cells/ml upto 12 months to 24 months.

* Easy identification by typical fermented smell.

* Cost saving on carrier material, pulverization, neutralization, sterilization, packing and transport.

* Quality control protocols are easy and quick.

* Better survival on seeds and soil.

* No need of running Bio-fertilizer production units through out the year.

* Very much easy to use by the farmer.

* Dosages is 10 time less than carrier based powder Bio-fertilizers.

* High commercial revenues.

* High export potential.

* Very high enzymatic activity since contamination is nil.
# Structure and characteristic features of bacterial biofertilizers

(Azospirillum, Azotobacter, Bacillus, Pseudomonas, Rhizobium and Frankia)

## 1. Rhizobium

Rhizobium is a soil habitat bacterium, which is able to colonize the legume roots and fixes the atmospheric nitrogen symbiotically. The morphology and physiology of Rhizobium will vary from free-living condition to the bacteroid of nodules. They are the most efficient biofertilizer as per the quantity of nitrogen fixed concerned. They have seven genera and highly specific to form nodule in legumes, referred as cross inoculation group. Rhizobium inoculant was first made in USA and commercialized by private enterprise in 1930. The bacteria infect the legume root and form root nodules within which they reduce molecular nitrogen to ammonia which is in reality utilized by the plant to produce valuable proteins, vitamins and other nitrogen containing compounds. The site of symbiosis is within the root nodules. It has been estimated that 40-250 kg N / ha / year is fixed by different legume crops by the microbial activities of Rhizobium. The percentage of nodules occupied, nodules dry weight, plant dry weight and the grain yield per plant the multistrain inoculant was highly promising.
**Structure:** Rhizobia are rod shaped bacteria, 0.8 µm in diameter and 2 µm in length, often with flagellae. They assume a different shape when inside their host, being irregularly shaped or often 'Y'-shaped

### Physical features of liquid Rhizobium

* Dull white in colour

* No foam formation,

* No bad smell

* pH 6.8-7.5

## 2. Azotobacter

Of the several species of Azotobacter, A. chroococcum is the dominant inhabitant in arable soils capable of fixing N₂ (2-15 mg N₂ fixed /g of carbon source) in culture media. The bacterium produces abundant slime which helps in soil aggregation. The numbers of A. chroococcum in Indian soils rarely exceeds 10⁵/g soil due to lack of organic matter and the presence of antagonistic microorganisms in soil. It is well known free living nitrogen fixing aerobic bacterium. It is used as a Bio-Fertilizer for all non leguminous plants especially rice, cotton, vegetables etc. Azotobacter cells are not present on the rhizosplane but are abundant in the rhizosphere region.

<figure><img src="image_71.png" /></figure>

### Physical features of liquid Azotobacter

The pigmentation that is produced by Azotobacter in aged culture is melanin which is due to oxidation of tyrosine by tyrosinase enzyme. Some of the pigmentation are described below-

* A. chroococcum : Produces **brown-black** pigmentation in liquid inoculum.

* A. beijerinchii : Produces **yellow-light brown** pigmentation in liquid inoculum

* A. vinelandii : Produces **green fluorescent** pigmentation in liquid inoculum.
➤ *A. paspali* : Produces green fluorescent pigmentation in liquid inoculum.

➤ *A. macrocytogenes*: Produces, pink pigmentation in liquid inoculum.

➤ *A. insignis*: Produces grayish-blue pigmentation in liquid inoculum.

➤ *A. agilities* : Produces green-fluorescent pigmentation in liquid inoculum.

Role of liquid *Azotobacter* as a Bio-control agent

*Azotobacter* produces antifungal substance which inhibits the growth of soil fungi like
*Aspergillus, Fusarium, Curvularia, Alternaria, Helminthosporium, Fusarium* etc.

3. Acetobaceter

This is a sacharophilic bacterium and is associated with sugarcane, sweet potato and sweet sorghum plants and fixes 30 kg N/ha/year. Mainly this bacterium is commercialized for sugarcane crop. It is known to increase yield by 10-20 t/ acre and sugar content by 10-15 %.

<figure><img src="image_72.png" /><figcaption>Acetobacter colony</figcaption></figure>

<figure><img src="image_73.png" /><figcaption>Acetobacter colony</figcaption></figure>

4. Azospirillum

<figure><img src="image_74.png" /></figure>

Azospirillum lipoferum and A. brasilense are primary inhabitants of soil, the rhizosphere, and intercellular spaces of root cortex of graminaceous plants. They perform the associative symbiotic relation with the graminaceous plants. Azospirillum are N₂ fixing organisms isolated from the root and above ground parts of a variety of crop plants. They are Gram negative, Vibrio or Spirillum having abundant accumulation of polybetahydroxybutyrate (70 %) in cytoplasm.

Five species of *Azospirillum* have been described to date

➤ A. brasilense

➤ A. amazonense

➤ A. irakense

➤ A. lipoferum

➤ A. halopraeferens

The organism proliferates under both anaerobic and aerobic conditions but it is preferentially micro-aerophilic in the presence or absence of combined nitrogen in the medium. Apart from nitrogen fixation, growth promoting substance production (IAA), disease resistance and drought tolerance are some of the additional benefits due to *Azospirillum* inoculation.
Phosphatic Bio-fertilizers: Phosphate solubilizing microorganisms (PSM)

Several soil bacteria and fungi, notably species of *Pseudomonas*, *Bacillus*, *Penicillium*, *Aspergillus* etc. secrete organic acids and lower the pH in their vicinity to bring about dissolution of bound phosphates in soil. Increased yields of wheat and potato were demonstrated due to inoculation of peat based cultures of *Bacillus polymyxa* and *Pseudomonas striata*. They act in different ways. Some bacteria secrete organic acids such as lactic acid, acetic acid and citric acid which solubilize the bound phosphate to forms which are available to plants. Others produce sulphuric acid by oxidation of sulphur which acts like the organic acids in making phosphate available. Carbonic acid formed by the action of carbon dioxide released by bacteria during respiration and water acts in a similar way. Some organisms give off hydrogen sulphide which reacts with the iron salt, ferrous phosphate, to form ferrous sulphide and thus releases the phosphate. Microbes are also responsible for decay of dead animal and plant litter in the soil during which humic and fulvic acids are formed which bind the metal ions such as Fe, Al, Mn and Ca and release the phosphate ions for the plants. They are used as seed inoculants.

<figure><img src="image_75.png" /></figure>

<figure><img src="image_76.png" /></figure>
# Structure and characteristic features of Cynobacterial biofertilizers: (Algae as Bio-fertilisers)

Dr. AJEET KUMAR<fnref n="12" />

Another group of free-living nitrogen fixers are the **cyanobacteria** commonly called the **blue-green algae (BGA)**. More than a hundred species of BGA can fix nitrogen.

Nitrogen fixation takes place in specialized cells called the **heterocysts** (large, thick walled and metabolically inactive cells) which depend on vegetative cells for energy to fix nitrogen while the fixed nitrogen is utilized by the vegetative cells for growth and development. BGA are very common in the rice fields (the micro-aerophilic condition and alkalinity are conducive to the algal population).

If no chemical fertilizers are added, inoculation of the algae can result in 10-14 % increase in crop yields. Unlike *Azotobacter*, the BGA are not inhibited by the presence of chemical fertilizers. On the other hand, presence of inorganic minerals (superphosphate) accelerates their growth. They are easy to produce and usually they are mass produced in cement tanks filled with fresh-water. Since they do not require any processing, they are quite cheap. Cost of 10 kg may be only Rs.30-40. Algal effect can be seen in subsequent years without the use of fresh inoculants.

Algal bio-fertilisers or cyanobacterial biofertilizers are Anabaena, Nostoc and Oscillatoria. Algae have proved beneficial in the case of certain crops like vegetables, cotton and sugarcane when added along with chemical fertilizers.

## Aquatic Fern (Azolla) as a Bio-fertilizer:

Azolla is a tiny water fern common in ponds, ditches and rice fields. It has been used as a bio-fertilizer for rice. The nitrogen accumulated in the Azalla is made available to the rice crop when the fern decomposes. The nitrogen fixing work is accomplished by the symbiotic relationship between the fern and a BGA, *Anabaena azollae*. The alga inhabits some of the cells on the underside of the Azolla frond and fixes atmospheric nitrogen. It is dependent on the fern for photosynthesis which supply the energy for nitrogen fixation. In addition to nitrogen, the decomposed Azolla also provides K, P, Zn and Fe to the crop. It also controls aquatic weeds which would otherwise compete with the crop for nutrients.

## AZOLLA (Botanical name is Azolla pinnata)

It belongs to the family Azollaceae. Azolla is a water fern. It is considered as aquatic weed commonly found in floating in idle ponds, tanks shallow ditches and canals. Azolla is associated with rice field. A blue green algae (BGA) *Anabaena azollae* living in the epidermal cavity of lower side of the leaf of Azolla. The symbiotic association of *Azolla pinnata* and *Anabaena azollae* is termed as Azolla Anabaena complex. This algae fixes atmospheric nitrogen for Azolla and in

<footnote n="12">¹² 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>
exchange, the plant provides shelter and food to the algae. As this complex fixes atmospheric nitrogen, it has great potentiality for use, in agricultural field as biofertilizer and can be used as an alternative to nitrogen fertilizers. It is recommended as green manure because of its large biomass and high N content (4-6%N) for sub merged rice fields, with in maximum temperature of 38°C. The yield increase is 15-20% Fresh Azolla contains 90-95 water. Decomposed Azolla contains the following plant foods.

<table><thead><tr><td>S.No.</td><td>Nutrient</td><td>Per cent (%)</td></tr></thead><tbody><tr><td>1</td><td>Nitrogen</td><td>4 to 6</td></tr><tr><td>2</td><td>Phosphorus</td><td>0.5 to 0.9</td></tr><tr><td>3</td><td>Potassium</td><td>2 to 6</td></tr><tr><td>4</td><td>Calcium</td><td>0.4 to 1</td></tr><tr><td>5</td><td>Magnesium</td><td>0.5</td></tr><tr><td>6</td><td>Manganese</td><td>0.11 to 0.16</td></tr><tr><td>7</td><td>Iron</td><td>0.06 to 0.16</td></tr><tr><td>8</td><td>Total ash</td><td>9 to 10</td></tr></tbody></table>

## Ideal conditions for growth and multiplication of Azolla

1. Depth of water should be maintained at 8-10 cm in the field

2. Temperature in between 14 to 35°C. Optimum for quick growth and multiplication is 20 to 28°C.

3. Suitable soil pH is 6.0 to 8.0

4. Single Super Phosphate should be applied at the rate of 20-25 kg ha⁻¹, should be applied

5. Furadon granules @2-3 kg /week should be applied to control leaf eating insects and snails.

## Methods of growing Azolla

1. Green manuring

2. Dual cropping

3. Composting

4. Just growing

### 1. Green manuring

When there is standing water in the field, Azolla is grown by spreading 200-500 kg /ha fresh Azolla. After 25 days of the whole field will be covered by Azolla, if condition is ideal. Green Azolla is incorporated by ploughing after dewatering in the field. This will add 30-40 kg of N fertilizer /ha.

### 2. Dual cropping

1. Azolla nursery is prepared simultaneously with the paddy nursery

2. The area of Azolla nursery should be half that of paddy nursery (seed bed).

3. The Azolla nursery should be kept free from other aquatic weeds and there should be 8-10 cm standing water in the Azolla nursery.

4. Where the paddy seeds are sown in the paddy nursery fresh Azolla is spread in the Azolla nursery @40-80 grams per square meter.
5. After 25 days the Azolla will increase an extent of 60 times and super phosphate is applied @ 25 grams/Sq. Meter/week.

6. Within 25 days the paddy seedlings will be ready for transplanting. Before transplantation of paddy seedlings, there will be standing water in the main field. Azolla is taken out from the Azolla nursery and spread in the transplanted field @ 200-500 kg/ha.

7. Single super phosphate @25-50 kg /ha /week is applied for 3 weeks and water level is maintained 5-10 cm.

8. After another 25 days, the whole field may be covered by Azolla. Azolla is incorporated after dewatering the field. This will add 30-40 kg N/ha besides other nutrients.

3. **Composting:** Azolla is grown in permanent Azolla nursery. The Azolla is collected every week from the nursery and kept in layers on the side of nursery. Within 10 days, the Azolla will decompose and can be used in any crop

4. **Just growing**

Azolla can be grown along with paddy. Nitrogen will be added where it is not incorporated. Azolla when grown in one hectare of land throughout the year and ideal conditions are maintained it can fix 800 kg of nitrogen per hectare azolla is suitable for only flooded rice and fixes up to 40-80 kg N/ha. Symbiotically with *Anabaena azollae*. The yield increase is 15-20 %.

**Advantages of azolla:**

1. It contains higher Nitrogen content

2. Release of nitrogen is easy

3. There is no leaching loss

4. Releases nutrient slowly

5. Weed interference is less where grown as dual crop

**V. Blue green algae or Cyanobacteria:**

BGA is phototrophic in nature. These are photosynthetic, prokaryotic microorganisms and capable of fixing molecular nitrogen. BGA are found to grown in a paddy filed and marshland. It is used as biofertilizer for irrigated rice crop. The rice field ecosystem provides an environment favourable for the growth of BGA with respect to their requirements viz., light, water and temperature. Nitrogen fixation by BGA have been estimated to be 25-30 kg N/ha. Yield increases by 10-15 %. BGA has some species viz., *Nostoc*, *Anabaena*, *Olocia*, *Calothrix*, *Tolipothix* etc., They fix nitrogen in the presence of sunlight. They also contain growth promoting substances like auxin, Indole acetic acid and Gibberellic acid.

**Recommendation:** application of 10 kg /ha flakes of BGA. It is applied 10 days after the transplanting of rice crop.

**Algal production technology:**

Mainly three methods are followed.

1) Trough method

2) Pit method

3) Field scale
## 1. Trough method:

<figure><img src="image_77.png" /></figure>

Take 3 m x 2 m x 20 cm size (L x B x h) galvanized iron sheet trough or to the convenient size. Add about 8 to 10 kg of soil and well mixed with 200 grams of SSP.

<figure><img src="image_78.png" /></figure>

The soil pH should be about neutral. If acidic correct by lime. Add water to the trough 5-15 cm (height) to protect from insects, and add Carbofuran 3.0 G at the rate of 25 grams / tray.

<figure><img src="image_79.png" /></figure>

Sprinkle the algal culture on the surface of the standing water and keeping it in air (open) and exposed to sunlight.

<figure><img src="image_80.png" /></figure>

In hot summer months, the growth of algae will be rapid in about 7 to 10 days and form a thick mat, where algal growth becomes sufficiently thick stop watering and allow the water to evaporate completely, the dry algae cracks in to flakes.

<figure><img src="image_81.png" /></figure>

Collect the dry algal flakes from the trough and store in bags for the use in the field. One trough gives 1.5 to kg mats.

## 2. Pit-method:

<figure><img src="image_82.png" /></figure>

Adopted by number of farmers in Bihar and India

<figure><img src="image_83.png" /></figure>

Demark the area in the field for algal production

<figure><img src="image_84.png" /></figure>

Flood the area with a depth of about 12.5 cm

<figure><img src="image_85.png" /></figure>

Apply SSP @12.0 kg /40 m² area

<figure><img src="image_86.png" /></figure>

Apply the algal culture

<figure><img src="image_87.png" /></figure>

In clayey soils good growth of algae takes place in about two weeks in clear sunny weather while in loamy soils it takes about 3-4 weeks

<figure><img src="image_88.png" /></figure>

Once algal growing mats are formed, they are allowed to dry up in the field and deed algal flakes are thin collected and stored in bags.

<figure><img src="image_89.png" /></figure>

BGA benefit the rice crop up to 25-30 kg N /ha

<figure><img src="image_90.png" /></figure>

Dose: Apply BGA @10-15 kg /ha after transplanting.

## Actinomycetes as Bio-fertiliser

Frankia is an actinomycete and forms nitrogen fixing nodules in trees and shrubs. The organism invades the cells of a developed lateral root and causes it to fuse into a nodule. Entry into the host changes the structure of the microbe.
# Structure and characteristic features of - Fungal biofertilizers

Dr. AJEET KUMAR<fnref n="13" />

## Fungi as Bio-fertiliser

Some nonpathogenic fungi help in plant growth by forming associations with the host plant roots called mycorrhizae (myca- fungi, rhiza -root). examples are Trichoderma, Gigaspora, Glomus, etc. One group of mycorrhizae forms a sheath around the fine lateral roots and replaces the root hairs by dichotomous branching of the fungal hyphae. They are called ectomycorrhizae because they do not traverse intracellularly. The ectomycorrhizae help the plant by solubilizing nutrients near the plant roots and making it easy for the plants to feed. They also prevent the roots from being attacked by nematodes (by entangling them). Another group called the endomycorrhizae penetrate the roots and establish symbiotic relation with the plants.

The fungi helps the roots in obtaining inorganic nutrients while obtaining essential organic nutrients from the host. There is yet another group called ect-endomycorrhiza or vesicular-arbuscular mycorrhiza (VAM fungi) wherein they are partly outside the host roots and partly intracellular.

The mycorrhizae are of particular significance as they assist plants to absorb phosphates from the soil. Phosphate ions are not mobile in the soil, thus when a plant uses up all the available phosphate near its roots, it starves for want of phosphates. The mycorrhizal mycelia spread far and wide, away from the depleted zone and translocate the phosphates directly to the host. Plants with restricted root system, having short stubby roots and few root hairs are particularly benefited by association with mycorrhiza. Absorption of elements which are required in minute quantities by plants is also facilitated by mycorrhizae. Mycorrhizae are valuable particularly in times of drought. They enable plants to survive and grow in the driest and poorest of soils, even in leached soils that have been mined by deforestation and overcropping.

## Cultivation of mycorrhizal fungi

It is easy to mass cultivate ectomycorrhizal fungi since they can grow in an artificial fungal medium. They are mass produced in liquid media, dried and pressed into cakes or cultivated in the form of spores and packaged. The seed inoculation is performed just as that of Rhizobium. Cultivation of endo mycorrhizal fungi is quite difficult since they need a live host for survival. Hence the host plants harbouring such mycorrhizal fungi are specially grown in laboratory conditions, their roots excised, powdered and the mixture used as a fertilizer.

<footnote n="13">¹³ 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>
## MYCORRHIZA

The term 'mycorrhiza' (literally, fungus root) was first used by A. B. Frank to characterise the association between higher plants and fungi. The symbiotic association between the roots of some plants and some fungi is called mycorrhizal association. The fungus is highly habitat limited and is usually found in the immediate vicinity of or within the roots.

### Occurrence of Mycorrhiza

They occur on almost all terrestrial plants though not as specific as the nitrogen fixing symbiosis. Thus a plant may have several mycorrhizae that can form symbiosis with its roots. Extent of symbiosis depends on fertility. High soil fertility leads to low mycorrhizal infection and poor symbiosis and vice versa. Roots supply carbohydrates to the fungi which absorb nutrients from the soil and supply them to the crop.

### Types of mycorrhizae

Mycorrhizae are of two kinds:

**Ectomycorrhiza** Ectomycorrhizal symbiosis is a mutually beneficial union between fungi and the roots of vascular and non-vascular plants. The host of an ecomycorrhizal fungus is usually a gymnosperm (pine). The typical ectomycorrhizal fungi are basidiomycetes (Agaricus), ascomycetes or phycomycetes members. Usually more common in the temperate regions, they are capable of growing apart from the host on media containing simple sugars and vitamins. Ectomycorrhizae have poor competitive saprophytic ability hence they have a tough time competing with other microbes in the soil.

<figure><img src="image_91.png" /></figure>

During the infection process, ectomycorrhizal fungi in soil are stimulated by the root metabolites to grow toward the root. The hyphae aggregate around the root and penetrate between
the root epidermis and the cortex. A structure called Hartig net is formed which is a fungal sheath surrounding the root in which fungal hyphae penetrate between the root cells.

Eventually, the root gets surrounded by a fungal mantle. The fungal hyphae replace the fine lateral root hairs of the host root system thus modifying it structurally. Thus the host root system that is infected with ectomycorrhiza looks stunted and dichotomously branched. The fungal hyphae on the exterior of the roots usually serve as an extension of roots and store large amounts carbohydrates

**Endomycorrhiza** In this case, the fungal hyphae penetrate the host root cells. They are quite common among the Ericaceae and Orchidaceae members of higher plants as well as fruit trees like citrus, coffee, rubber, etc.

## Vam Fungi

Vesicular-arbuscular mycorrhiza (VAM) fungi shown in are geographically ubiquitous. They are commonly found in association with agricultural crops, shrubs, tropical tree species and some temperate trees. Their nutritional requirements are not specific. VAM associations are formed by non septate Zygomycetes and Phycomycetes. Some examples are *Glomus*, *Gigaspora*, *Acaulospora*, *Entrophospora* and *Scutellospora* of which *Glomus* is the most common fungus.

The fungi are obligate biotrophs and do not grow on synthetic media and hence are classified according to the morphological characteristics of the spores formed in the soil. VAM fungi produce large resting spores (0.2 mm). The spores can survive adverse conditions. The germ tube dies if it is unable to encounter and successfully penetrate the host root. The fungal hyphae traverses and ramifies in the root cortex. Branches from the intercellular hyphae enter cortical cells where further branching results in highly branched hyphal structures called arbuscles. They are short-lived and serve as the nutrient transfer

<figure><img src="image_92.png" /></figure>
Mechanism between the fungus and the host. Phosphate transfer possibly occurs across living membranes of the host and the fungus via arbuscles. When the association is well established, hyphal swellings called vesicles form on the mycelium inside and outside the root. The vesicles are sac-like terminal swellings at the tip of hyphae and contain many lipid droplets and function as storage organs. External mycelium form very thick-walled chlamydospores. These fungi are disseminated both actively and passively. Active dissemination is form one root to another by mycelial growth through soil and passive dissemination is through biotic agents like rodents, worms, insects and birds or abiotic agents like wind and water. VAM fungi interact with other soil microbes like the free-living and symbiotic nitrogen fixers and phosphate solubilises to improve their efficiency for the biochemical cycling of elements to the host plants.

## Constraints in usage of biofertilizers

1. Farmers acceptance has been for away from satisfaction

2. Difficult to demonstrate striking increase in yield due to biofertilizers unlike chemical fertilizers

3. Strains fail to establish if the soil moisture in the surface in the surface zone

4. Fungicide treatment is a problem in crops like groundnut

5. Inoculants manufacturing is for below the requirement

6. Availability of quality of inoculants is another problem

7. Seed inoculation is no more suitable when seeds are sown on dry soil under

8. When seeds are coated with toxic chemicals and pesticides

9. Soil containing higher number of ineffective native Rhizobia
# Biochemistry of Biological Nitrogen Fixation

Dr. AJEET KUMAR<fnref n="14" />

Nitrogen is an essential element for plant growth and development and a key issue of agriculture. Nitrogen fertilizers contribute to resolving the challenge the world is facing, feeding the human population. The Green Revolution was accompanied by an enormous increase in the application of nitrogen fertilizer. There is however a high heterogeneity of its distribution throughout the world. Biological Nitrogen Fixation (BNF) is known to be a key to sustain agriculture and to reduce soil fertility decline.

## Nodulation: From the Infection Process to the Functioning of the Nitrogenase

The production of nitrogen fertilizer by industrial fixation generates large quantities of carbon dioxide, contributing to earth warming. The natural process of BNF offers an economic means of reducing environmental problems and improving the internal resources. It is a process that allows microorganisms to convert atmospheric nitrogen ($N_2$) to ammonia ($NH_3$) assimilable by associated plants.

$$6 \text{ N} + 6\text{H}^+ + \text{ATP} \xrightarrow{6e^-} 2 \text{NH}_3 + \text{ADP} + \text{Pi}$$

Different types of associations are listed as bellows:

<table><thead><tr><th>Types of association</th><th>Microorganisms</th><th>Host plants</th></tr></thead><tbody><tr><td rowspan="3">Symbiotic</td><td>Bacteria(ex. <i>Rhizobium</i>)<sup>a</sup></td><td>Legumes</td></tr><tr><td>Actinomycetes (ex. <i>Frankia</i>)</td><td>Actinorhiza</td></tr><tr><td>Cyanobacteria (ex. <i>Anabaena azollaea</i>)</td><td>Fern</td></tr><tr><td>Non symbiotic</td><td>Bacteria (ex. <i>Azotobacter</i>, <i>Azospirillum</i>)</td><td>Cereals</td></tr><tr><td>Free living systems</td><td>Bacteria (ex. <i>Thiobacillus</i>, <i>Clostridium</i>)</td><td></td></tr></tbody></table>

<sup>a</sup>: Rhizobium-leguminous plants is the most studied symbiotic association. There are six main genus of rhizobia:

1. Allorhizobium (fast growing)

2. Azorhizobium (fast growing)

3. Bradyrhizobium (slow growing)

4. Mesorhizobium (intermediate growing)

5. Rhizobium (fast growing)

6. Sinorhizobium (fast growing)

<footnote n="14">¹⁴ 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>
## Nodule formation:

Leguminous plants and rhizobia communicate through the gene expression by reciprocally transmitting signals for the activation of the symbiotic genes in two partners. A type of phenolic called flavonoids, are released by host roots plants into the rhizosphere. Flavonoids act as a chemo-attractant for the bacteria to the plant roots, and eventually colonies of rhizobia attach to the root hairs. Flavonoid signal activates expression of nodulation (nod) genes.

In the rhizobial strains there are numerous nodulation genes including the nod genes nodABC and nodD (Figure 1). On the surface of the rhizobial bacteria the flavonoids are recognized by an expressed nodD protein. Then nodD binds to a promoter DNA sequence, and thereby activates transcription of nod genes of the operons. A group of nod genes encode enzymes synthesise the rhizobial nodulation signal, Nod factor (Figure 2), which triggers development of the root nodules by the plant. The plant roots recognise Nod factor, through binding to a surface protein receptor at the sub apical root tip. Perception of Nod factor induces a development inside the root, producing pronounced curling of the root hairs entrapping the rhizobia which establish infection of root. Bacteria gain access to plants cell membrane. The plasma membrane invaginates to form novel infection structure known as the infection thread, a tubular structure that extends from the root hair tip to the lower cells of the root cortex. Rhizobia enter the infection threads in which they actively multiply. At the same time the underlying root cortex cells are quickly proliferated to constitute the nodule primordia. The infection threads branch out into cells of the nodule primordia. The rhizobia are finally released into the nodule cell and enveloped in a membrane derived from the host cell plasma membrane. At this stage, rhizobial bacteria become bacteroids able to fix nitrogen.

## Importance of nitrogen fixation

The process of nitrogen fixation is carried out in microbes.

## Key Points

* Nitrogen fixation takes elemental nitrogen (N₂) and converts it into a ammonia, a format usable by biological organism.

* The fixed form of nitrogen (NH₃) is needed as an essential component of DNA and proteins. Therefore, it is needed for all life on earth.

* Nitrogen fixation is carried out by the enzyme nitrogenase, which are found in microbes.
**Key Terms**

* **Fixation:** The act of uniting chemically with a solid substance or in a solid form; reduction to a non-volatile condition; — said of gaseous elements.

* **cofactor:** A substance, especially a coenzyme or a metal, that must be present for an enzyme to function.

* **Heterometal:** Describing a complex containing two (or more) different metals

* **symbiosis:** A close, prolonged association between two or more organisms of different species, regardless of benefit to the members

* **sulfide:** Any compound of sulfur and a metal or other electropositive element or group.

* **reduction:** A reaction in which electrons are gained and valence is reduced; often by the removal of oxygen or the addition of hydrogen.

* **enthalpy:** In thermodynamics, a **measure of the heat content of a chemical** or physical system.

* **oxidation:** A reaction in which the atoms of an element lose electrons and the valence of the element increases.

* **reduction:** A reaction in which electrons are gained and valence is reduced; often by the removal of oxygen or the addition of hydrogen.

* **proteoglycan:** Any of many glycoproteins that have heteropolysaccharide side chains.

**Nitrogen fixation:**

The conversion of atmospheric nitrogen into ammonia and organic derivatives, by natural means, especially such conversion, by microorganisms in the soil, into a form that can be assimilated by plants.

Nitrogen fixation also refers to other biological conversions of nitrogen, such as its conversion to nitrogen dioxide. Nitrogen fixation is a process by which nitrogen ($N_2$) in the atmosphere is converted into ammonia ($NH_3$). Atmospheric nitrogen or elemental nitrogen ($N_2$) is relatively inert: it does not easily react with other chemicals to form new compounds. Dinitrogen is quite inert because of the strength of its $N≡N$ triple bond. To break one nitrogen atom away from another requires breaking all three of these chemical bonds. Fixation processes free up the nitrogen atoms from their diatomic form ($N_2$) to be used in other ways. Nitrogen fixation, natural and synthetic, is essential for all forms of life because nitrogen is required to biosynthesize basic building blocks of plants, animals, and other life forms, e.g., nucleotides for DNA and RNA and
amino acids for proteins. Therefore, nitrogen fixation is essential for agriculture and the manufacture of fertilizer. Microorganisms that fix nitrogen are bacteria called diazotrophs.

## Nitrogenase and Nitrogen Fixation

Some higher plants, and some animals (termites), have formed associations (symbioses) with diazotrophs. Diazotrophs are microbes. Biological nitrogen fixation was discovered by the German agronomist *Hermann Hellriegel* and Dutch microbiologist Martinus Beijerinck.

Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by an enzyme called nitrogenase. Nitrogenases are enzymes used by organisms to fix atmospheric nitrogen gas ($N_2$). There is only one known family of enzymes that accomplishes this process. All nitrogenases have an iron – and sulfur-containing cofactor that includes a heterometal complex in the active site (e.g., FeMoCo). In most species, this heterometal complex has a central molybdenum atom. However, in some species it is replaced by a vanadium or iron atom. Enzymes responsible for nitrogenase action are very susceptible to destruction by oxygen. Many bacteria cease production of the enzyme in the presence of oxygen. Many nitrogen-fixing organisms exist only in anaerobic conditions, respiring to draw down oxygen levels, or binding the oxygen with proteins.

## History of Nitrogen Fixation

For thousands of years farmers were aware that plants belonging to the legume family, promoted crop growth when planted with other non-legumes. Growing a legume crop in a field could also result in the next year's crop of non-legume plants giving a far greater yield. This led to the practice of crop rotation, a practice which can be traced back to techniques recorded in Roman literature.

<figure><img src="image_93.png" /></figure>

**Martinus Beijerinck:** Work done by Martinus Beijerinck was key to the discovery of rhizobia, symbiotic bacteria found on the roots of legumes, responsible for nitrogen fixation.

While the ancient Romans were aware of the improved results gained through crop rotation, they did not know that these benefits were brought about through the replenishment of nitrogen in the soil. Later people knew legumes did replenish nitrogen in the soil, but did not know how atmospheric ($N_2$) was converted into ammonium ($NH_3$) by legumes until research done in the 19th century.
Hermann Hellriegel (1831-1895), a noted German agricultural chemist, discovered that leguminous plants took atmospheric nitrogen and replenished the ammonium in the soil through the process now known as nitrogen fixation. He found that the nodules on the roots of legumes are the location where nitrogen fixation takes place.

## HELLRIEGEL'S AND BEIJERINCK'S DISCOVERIES

Hellriegel did not determine what factors in the root nodules carried out nitrogen fixation. Martinus Willem Beijerinck (March 16, 1851 – January 1, 1931), a Dutch microbiologist and botanist, explored the mechanism responsible, discovering that the root nodules contained microbes. He further demonstrated that these microbes were bacteria, which he named rhizobia. These rhizobia perform the chemical processes of nitrogen fixation. In addition to having discovered this biochemical reaction vital to soil fertility and agriculture, Beijerinck is responsible for the discovery of this classic example of symbiosis between plants and bacteria. The bacteria in the root nodules are needed to provide nitrogen for legume growth, while the rhizobia are dependent on the root nodules as an environment to grow.

## Nitrogen Fixation Mechanism

The conversion of N₂ to NH₃ depends on a complex reaction, essential to which are enzymes known as nitrogenases.

### Key Points

* ✓ Nitrogen fixation does result in the release of energy, but the activation of this reaction takes energy in the form of ATP hydrolysis.

* ✓ Nitrogenases are metalloenzymes, which are proteins that have metallic molecules as subunits.

* ✓ While a great deal is known about how nitrogenases reduce nitrogen, some steps are unknown.

### Biological nitrogen fixation (BNF)

It occurs when atmospheric nitrogen is converted to ammonia by an enzyme called nitrogenase. The reaction for BNF is:

$$N_2 + 8 H^+ + 8 e^- \rightarrow 2 NH_3 + H_2$$

This type of reaction results in N₂ gaining electrons and is thus termed a reduction reaction. While the equilibrium formation of ammonia from molecular hydrogen and nitrogen has an overall negative enthalpy of reaction (i.e. it gives off energy), the energy barrier to activation is very high
without the assistance of catalysis, which is done by nitrogenases. The enzymatic reduction of N₂ to ammonia therefore requires an input of chemical energy, released from ATP hydrolysis, to overcome the activation energy barrier.

**A General Catalytic Mechanism Scheme for Nitrogenase:**

a) Components I and II are dissociated; II is ready for reduction.

b) ATP binds to component II, which receives electrons from an **electron donor** (ferredoxin or flavodoxin); binding of ATP induces an allosteric conformational change which allows association of the two proteins. Electrons flow from the [4Fe-4S] cluster on II to the P cluster on I.

c) Electrons are further shuttled to the iron-molybdenum cofactor (FeMoco), and ATP is hydrolised to ADP. This step is repeated several times before a molecule of N₂ can bind to FeMoco.

d) The protein complex dissociates, and nitrogenase reduces dinitrogen to ammonia and dihydrogen.

(Legend:I: component I (dinitrogenase; MoFe protein); II: component II (dinitrogen reductase; Fe protein); ATP: adenosine triphosphate; ADP: adenosine diphosphate; Fdx: ferredoxin; Fld: flavodoxin).

Nitrogenase is made up of two soluble proteins: component I and II. Component I known as MoFe protein or nitrogenase contains 2 Mo atoms, 28 to 34 Fe atoms, and 26 to 28 acid-labile sulfides, also known as a iron-molybdenum cofactor (FeMoco). Component I is composed of two copies each of two subunits (α and β); each subunit's stability depends on the other in vivo.

Component II known as Fe protein or nitrogenase reductase is composed of two copies of a single subunit. This protein has four non-heme Fe atoms and four acid-labile sulfides (4Fe-4S). Substrate binding and reduction takes place on component I, which binds to ATP and ferredoxin or flavodoxin proteins (Fdx or Fld) (see step B).

The hydrolysis of ATP supplies the energy for the reaction while the Fdx/Fld proteins supply the electrons. Note this is a reduction reaction which means that electrons must be added to the N₂ to reduce it to NH₄. Thus, the role of component II is to supply electrons, one at a time to component I. ATP is not hydrolyzed to ADP until component II transfers an electron to component I. The association of nitrogenase component I and II and later dissociation occurs several times to allow the fixation of one N₂ molecule.
Nitrogenase ultimately bonds each atom of nitrogen to three hydrogen atoms to form ammonia (NH₃). The nitrogenase reaction additionally produces molecular hydrogen as a side product, which is of special interest for people trying to produce H₂ as an alternative energy source to fossil fuels.

## Anaerobiosis and N₂ Fixation

Nitrogen fixing bacteria have different strategies to reduce oxygen levels, which interfere with nitrogenase function.

### Key Points

* The iron (Fe) found in nitrogenases is very sensitive to oxygen, if there is too much oxygen this will in the end disrupt nitrogenase function.

* Some bacteria produce barriers which protect themselves from oxygen, while others use proteins such as leghemoglobin to bind up oxygen which may interfere with nitrogenases.

* Portions of leghemoglobin are thought to be produced by rhizobia residing in plant nodules, while other parts are produced by the plant, an elegant example of symbiosis.

Central to nitrogen fixation (N₂ to NH₃) are the enzymes that do the actual fixation, these are known as nitrogenases. Due to the oxidation carried out by oxygen, most nitrogenases, which are essential large reduction complexes are irreversibly inhibited by O₂, which degradatively oxidizes the Fe-S cofactors. In essence, O₂ binds to the iron (Fe) found in nitrogenases and blocks their ability to bind to N₂. To protect nitrogenases, there are mechanisms for nitrogen fixers to protect nitrogenase from oxygen in vivo. One known exception is the nitrogenase of Streptomyces thermoautotrophicus, which is unaffected by the presence of oxygen. This is complicated by the fact the bacteria still need the presence of oxygen for proper respiration.

Some microbes have a proteoglycan rich extra cellular matrix which traps a layer of water, often referred to as a **slime layer**. This **slime layer acts as a barrier for oxygen**. The ability of some nitrogen fixers such as azotobacteraceae to employ an oxygen-amendable nitrogenase under aerobic conditions has been attributed to a high metabolic rate, allowing oxygen reduction at the cell membrane.
<figure><img src="image_94.png" /></figure>

**Leghemoglobin:** Leghemoglobin, the protein which binds to oxygen, allowing nitrogenases to function in an oxygen free environment. The ribbons represent protein folds, while the conglomerate of spheres are the position of the iron containing heme group which binds the oxygen.

Many rhizobia, nitrogen fixing bacteria, live in a symbiotic relationship with plants known as legumes. They have an interesting strategy to deal with O₂. In plants infected with Rhizobium, (legumes such as alfalfa or soybeans), the presence of oxygen in the root nodules would reduce the activity of the oxygen-sensitive nitrogenase. In these situations, the roots of such plants produce a protein known as leghemoglobin (also leghaemoglobin or legoglobin). Leghemoglobin buffers the concentration of free oxygen in the cytoplasm of infected plant cells to ensure the proper function of root nodules. Leghemoglobin is a nitrogen or oxygen carrier; naturally occurring oxygen and nitrogen interact similarly with this protein. Leghemoglobin buffers the concentration of free oxygen in the cytoplasm of infected plant cells to ensure the proper function of root nodules. It has close chemical and structural similarities to hemoglobin, and, like hemoglobin, is red in colour. Leghemoglobin has a high affinity for oxygen, about ten times higher than of human hemoglobin. This allows an oxygen concentration that is low enough to allow nitrogenase to function but not so high as to bind all the O₂ in the bacteria, providing the bacteria with oxygen for respiration.

Leghemoglobin is produced by legumes in response to the roots being infected by rhizobia, as part of the symbiotic interaction between the plant and this nitrogen-fixing bacterium. Interestingly, it is widely believed that leghemoglobin is the product of both the plant and the bacterium in which a protein precursor is produced by the plant and the heme (an iron atom bound in a porphyrin ring, which binds O₂) is produced by the bacterium. The protein and heme come together to function, allowing the bacteria to fix-nitrogen, giving the plant usable nitrogen and thus the plant provides the rhizobia a home.
# Genetics and Regulation of N₂ Fixation

Through control of gene expression, nitrogen fixing bacteria can turn on and off the proteins needed for nitrogen fixation.

## Key Points

* Nitrogen fixation takes a great deal of energy. If the conditions are not favorable for nitrogen fixation or there is enough ammonia around, nitrogen-fixing bacteria turn off the production of proteins needed for nitrogen fixation.

* Nitrogen fixing protein production is regulated by the nif regulon.

* The nif regulon contains factors which both turn on and off the production of proteins needed for nitrogen-fixation.

## Key Terms

* ✓ **regulon**: A group of genes that is regulated by the same regulatory molecule. The genes of a regulon share a common regulatory element binding site or promoter. The genes comprising a regulon may be located non-contiguously in the genome.

* ✓ **operon**: A unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and structural genes that are transcribed together.
NIF REGULON

<figure><img src="image_95.png" /></figure>

**Nif Regulon:** This is a schematic representing many of the proteins in the nif regulon and where they act in the pathway needed for nitrogen fixation.

The fixation of atmospheric nitrogen ($N_2$) is a very energy intensive endeavor. If there is no need for $N_2$ fixation, the production of proteins needed for fixation are tightly controlled. The nif genes are responsible for the coding of proteins related and associated with the fixation of atmospheric nitrogen into a form of nitrogen available to plants. These genes are found in nitrogen fixing bacteria and cyanobacteria. The nif genes are found in both free living nitrogen fixing bacteria and in symbiotic bacteria in various plants.

The nif genes are genes encoding enzymes involved in the fixation of atmospheric nitrogen. The primary enzyme encoded by the nif genes is the nitrogenase complex which is in charge of converting atmospheric nitrogen to other nitrogen forms such as ammonia, which plants can use
for various purposes. Besides the nitrogenase enzyme, the nif genes also encode a number of regulatory proteins involved in nitrogen fixation. The expression of the nif genes is induced as a response to low concentrations of fixed nitrogen and oxygen concentrations (the low oxygen concentrations are actively maintained in the root environment). Nitrogen fixation is regulated by nif regulon, which is a set of seven operons which includes 17 nif genes. Nif genes have both positive and negative regulators. Some of nif genes are: Nif A, D, L,K, F,H S,U,Y,W,Z.

Activation of nif genes transcription is done by the nitrogen sensitive NifA protein. When there isn't enough fixed nitrogen factor available for the plant's use, NtrC, which is a RNA polymerase, triggers NifA's expression. NifA then activates the rest of the transcription for the nif genes. If there is a sufficient amount of reduced nitrogen or oxygen is present, another protein is activated, NifL. In turn, NifL inhibits NifA activity, which results in the inhibition of nitrogenase formation. NifL is then regulated by other proteins that are sensors for the levels of O₂ and ammonium in the surrounding environment.

The nif genes can be found on bacteria's chromosomes, but many times they are found on bacteria's plasmids with other genes related to nitrogen fixation, such as the genes needed for the bacteria to communicate with the plant host.

Nitrogen in the Biosphere

* Nitrogen is required in large amounts as an essential component of proteins, nucleic acids, and other cellular constituents.

* Nitrogen is the most abundant element in our atmosphere - nearly 79% in the form of N₂ gas.

* In order for nitrogen to be used for growth it must be "fixed" (combined) in the form of ammonium (NH₄⁺) or nitrate (NO₃⁻) ions.

* Plants are able to use nitrogen in the form of nitrate (NO₃⁻) or ammonia (NH₃⁺), but these compounds are present in limited supply in the soil.

<figure><img src="image_96.png" /></figure>
# Nitrogen Fixation

* The growth of all organisms depend on the availability of Nitrogen (e.g. amino acids)

* Nitrogen in the form of Dinitrogen ($N_2$) makes up 80% of the air we breathe but is essentially inert due to the triple bond ($N=N$)

* In order for nitrogen to be used for growth it must be "fixed" (combined) in the form of ammonium ($NH_4$) or nitrate ($NO_3$) ions.

## Nitrogen Fixation

### Nitrogen Fixation

#### Enzymatic reduction of atmospheric Nitrogen

$$ \underset{\text{(Dinitrogen)}}{N_2} \xrightarrow[2e]{2H^+} \underset{\text{(Di-imide)}}{HN = NH} \xrightarrow[2e]{2H^+} \underset{\text{(Hydrazine)}}{H_2N-NH_2} \xrightarrow[2e]{2H^+} \underset{\text{(Ammonia)}}{2NH_3} \quad [6e + 6H^+ + N_2 \rightarrow 2NH_3] $$

#### Energy intensive process :

$$ N_2 + 8H^+ + 8e^- + 16 \text{ ATP} = 2NH_3 + H_2 + 16 \text{ ADP} + 16 \text{ Pi} $$

* ☐ Performed only by selected bacteria and actinomycetes

* ☐ Performed in nitrogen fixing crops (eg: soybeans)
# Nitrification

Two step reactions that occur together :

1st step: catalyzed by Nitrosomonas (Slow RKⁿ)

$$2\text{NH}_4^+ + 3\text{O}_2 \rightarrow 2\text{NO}_2^- + 2\text{H}_2\text{O} + 4\text{H}^+$$

2nd step: catalyzed by Nitrobacter (Fast RKⁿ)

$$2\text{NO}_2^- + \text{O}_2 \rightarrow 2\text{NO}_3^-$$

# Nitrogen Fixation Process

## Energetics

- N≡N

- Haber-Bosch (100-200 atm, 400-500°C, 8,000 kcal kg⁻¹N)

- Nitrogenase (4,000 kcal kg⁻¹N)
## Properties of dinitrogen which makes it inert

* Dinitrogen - two N atoms connected by triple bond

* Breaking the N≡N bond is difficult - high dissociation energy of 942 kJ mol⁻¹

* Breaking first π bond requires 540 kJ mol⁻¹

<figure><img src="image_97.png" /></figure>

## Other important properties

<table><thead><tr><th>Property</th><th>Gas</th><th>Nitrogen</th><th>Carbon</th><th>Oxygen</th><th>Argon</th></tr></thead><tbody><tr><td>Ionization potential (eV)</td><td></td><td>14.3</td><td>11.256</td><td>13.614</td><td>15.755</td></tr><tr><td>Electron affinity (eV)</td><td></td><td>0.073</td><td>1.595</td><td>1.461</td><td>0</td></tr><tr><td>Solubility in water (mole/cm³)</td><td></td><td>0.083</td><td>Insoluble</td><td>0.153</td><td>0.140</td></tr></tbody></table>

* High ionization potential and low electron affinity - difficult to reduce and oxidize

* Solubility very less - reactions in solution phase - difficult
## Biological fixation of dinitrogen

* Enzyme nitrogenase

* Present in soil bacteria, root nodules and algae

* Two decades of research - mechanism not established

* Enzyme contains Mo and Fe

* Proposed mechanism - complexation of N₂ to metal ions

* Reduces bond strength - breaking 1<sup>st</sup> π bond easier

## Limitations with biological route:

* Nitrogenase - sensitive to O₂ - requires O₂ free environment

* Sensitive to environmental conditions - temperature, pH

* Cannot be used for large scale N₂ fixation

## Different types of associations:

<table><thead><tr><td>Types of association</td><td>Microorganisms</td><td>Host plants</td></tr></thead><tbody><tr><td rowspan="3">Symbiotic</td><td>Bacteria(ex. Rhizobium)<sup>a</sup></td><td>Legumes</td></tr><tr><td>Actinomycetes (ex. Frankia)</td><td>Actinorhiza</td></tr><tr><td>Cyanobacteria (ex. Anabaena azollaea)</td><td>Fern</td></tr><tr><td>Non symbiotic</td><td>Bacteria (ex. Azotobacter, Azospirillum)</td><td>Cereals</td></tr><tr><td>Free living systems</td><td>Bacteria (ex. Thiobacillus, Clostridium)</td><td></td></tr></tbody></table>

### aSix main genus of Rhizobia:

1. Allorhizobium (fast growing)

2. Azorhizobium (fast growing)

3. Bradyrhizobium (slow growing)

4. Mesorhizobium (intermediate growing)

5. Rhizobium (fast growing)

6. Sinorhizobium (fast growing)
## Key Terms

* **Fixation:** The act of uniting chemically with a solid substance or in a solid form; **reduction** to a non-volatile condition.

* **Cofactor:** A substance, especially a coenzyme or a metal, that must be present for an enzyme to function.

* **Heterometal:** Describing a complex containing **two or more different metals**

* **Symbiosis:** A close, prolonged association between two or more organisms of different species, regardless of benefit to the members

* **Sulfide:** Any compound of sulfur and a metal or other electropositive element or group.

* **Reduction:** A reaction in which **electrons are gained** and valence is reduced; often by the **removal of oxygen** or the **addition of hydrogen**.

* **Enthalpy:** In thermodynamics, a **measure of the heat content of a chemical** or physical system.

* **Oxidation:** A reaction in which the atoms of an element **lose electrons** and the valence of the element increases.

* **Proteoglycan:** Any of many glycoproteins that have hetero-polysaccharide side chains.

## Genetics of Nitrogenase

<table><thead><tr><td>Gene</td><td>Properties and function</td></tr></thead><tbody><tr><td>nifH</td><td>Dinitrogenase reductase</td></tr><tr><td>nifDK</td><td>Dinitrogenase</td></tr><tr><td>nifA</td><td>Regulatory, activator of most nif and fix genes</td></tr><tr><td>nifB</td><td>FeMo cofactor biosynthesis</td></tr><tr><td>nifEN</td><td>FeMo cofactor biosynthesis</td></tr><tr><td>nifS</td><td>Unknown</td></tr><tr><td>fixABCX</td><td>Electron transfer</td></tr><tr><td>fixK</td><td>Regulatory</td></tr><tr><td>fixLJ</td><td>Regulatory, two-component sensor/effector</td></tr><tr><td>fixNOQP</td><td>Electron transfer</td></tr><tr><td>fixGHIS</td><td>Transmembrane complex</td></tr></tbody></table>
# Nitrogen fixing organisms

* Free living aerobic bacteria

* Azotobacter

* Beijerinckia

* Klebsiella

* Cyanobacteria
    (lichens)

* Free living anaerobic bacteria

* Clostridium

* Desulfovibrio

* Purple sulphur bacteria

* Purple non-sulphur
    bacteria

* Green sulphur bacteria

* Free living associative bacteria

* Azospirillum

* Symbionts

* Rhizobium
    (legumes)

* Frankia (alden trees)

# Free-living N₂ Fixation

## Energy

* 20-120 g C used to fix 1 g N

## Combined Nitrogen

* *nif* genes tightly regulated

* Inhibited at low NH₄⁺ and NO₃⁻ (1 µg g⁻¹ soil, 300 µM)

## Oxygen

* ✓ Avoidance (anaerobes)

* ✓ Microaerophilic Respiratory protection

* ✓ Specialized cells (**heterocysts**, vesicles)

* ✓ Spatial/temporal separation

* ✓ Conformational protection

## Associative N₂ Fixation

* Phyllosphere or rhizosphere (tropical grasses)

* *Azospirillum*, *Acetobacter*

* 1 to 10% of rhizosphere population
* Some establish within root

* Same energy and oxygen limitations as free-living

* Acetobacter diazotrophicus lives in internal tissue of sugarcane, grows in 30% sucrose, can reach populations of $10^6$ to $10^7$ cells g$^{-1}$ tissue, and fix 100 to 150 kg N ha$^{-1}$ y$^{-1}$

## Phototrophic N$_2$-fixing Associations

* Lichens–cyanobacteria and fungi

* Mosses and liverworts–some have associated cyanobacteria

* Azolla-Anabaena (Nostoc)–cyanobacteria in stem of water fern

* Gunnera-Nostoc–cyanobacteria in stem nodule of dicot

* Cycas-Nostoc–cyanobacteria in roots of gymnosperm

## Formation of a Root Nodule

<figure><img src="image_98.png" /></figure>
**Infection Process:**

* Attachment

* Root hair curling

* Localized cell wall degradation

* Infection thread

* Cortical cell differentiation

* Rhizobia released into cytoplasm

* Bacterioid differentiation (symbiosome formation)

* Induction of nodulins

**Role of Root Exudates:**

**General**

* Amino sugars, sugars

**Specific**

* Flavones (luteolin), isoflavones (genistein),
flavanones, chalcones

* Inducers/repressors of *nod* genes

* Vary by plant species

* Responsiveness varies by rhizobia species

<figure><img src="image_99.png" /></figure>

<figure><img src="image_100.png" /><figcaption>Genistein</figcaption></figure>

**Nodule Metabolism:**

<figure><img src="image_101.png" /></figure>

**Oxygen metabolism**

* ✓ Variable diffusion barrier

* ✓ Leghemoglobin
<figure><img src="image_102.png" /></figure>

## Nitrogen metabolism

* $NH_3$ diffuses to cytosol

* Assimilation by GOGAT

* Conversion to organic-N for transport

<figure><img src="image_103.png" /></figure>

## Carbon metabolism

* ✓ Sucrose converted to dicarboxylic acids

* ✓ Functioning TCA in bacteroids

* ✓ C stored in nodules as starch

## Nitrogen Fixation

* All nitrogen fixing bacteria use highly conserved enzyme complex called **Nitrogenase**

<figure><img src="image_104.png" /></figure>

<figure><img src="image_105.png" /></figure>

<figure><img src="image_106.png" /></figure>

<figure><img src="image_107.png" /></figure>

<figure><img src="image_108.png" /></figure>

<figure><img src="image_109.png" /></figure>
* Nitrogenase is composed of two subunits: an **iron-sulfur protein** and a **molybdenum-iron-sulfur protein**

* Aerobic organisms face special challenges to nitrogen fixation because nitrogenase is inactivated when oxygen reacts with the iron component of the proteins.

<figure><img src="image_110.png" /></figure>
<figure><img src="image_111.png" /></figure>

Nitrogenase

<figure><img src="image_112.png" /></figure>

Dinitrogenase
reductase

$$
N_2 + 8H^+ + 8e^- \rightarrow 16 Mg ATP \rightarrow 2NH_3 + H_2 + 16MgADP
$$

<figure><img src="image_113.png" /></figure>

Nitrogenase proteins and cofactors. (A) Shown is one half of the nitrogenase complex, with one αβ pair of the MoFe protein α₂β₂ tetramer and one bound Fe protein dimer. The location of bound nucleotides and the three metal clusters are also shown.

Barney BM, et al., Dalton Trans. 2006, 2277-84.
<figure><img src="image_114.png" /></figure>
# Mechanism of phosphate solubilization and phosphate mobilization

Dr. AJEET KUMAR<fnref n="15" />

## INTRODUCTION

Much of the soluble phosphorus applied as fertilizer to soil is fixed into one of the many sparingly soluble forms (tricalcium phosphate, aluminium phosphate, Iron phosphate and manganese phosphate) and only 10-18 % is utilized by the standing crop. A variety of mechanism have been accounted for the increased mobilization of soil phosphorus in plant rhizosphere such as excretion of organic acids (Moghimi et al. 1978), exudation of reducing substances (Gardner, 1982), the production of mucilage and sloughed off root cells which provide an energy substrate for microbial activity in the rhizosphere which subsequently dissolve mineral and organic phosphorus (Banik and Dey, 1982), Hydrolysis of organic phosphorus by phosphatase enzymes (Helal and Sauerbeck, 1984). Many phosphates solubilizing microorganism isolated from the rhizosphere of different crops predominantly of the genera *Bacillus polymixa*, *B. megaterium*, *B. circulans* *Pseudomonas striata*, species of *Aspergillus* and *Penicillium* have shown the ability to solubilise sparingly soluble phosphate *in vitro* as well as *in vivo* condition by secreting the organic acid and inorganic acids. Kumar and Maurya (2006) observed a more rapid initial release of phosphorus from tricalcium phosphate in the presence of the fungal species of *Aspergillus sulfurious*, *Aspergillus niger*, *Aspergillus flavus*, *Aspergillus terreus*, and *Rhizopus stolonifer*. Fungi in soils are able to traverse distances more easily than bacteria thus it is more important to phosphorus solubilisation (Kucey, 1983). The solubilizing microorganisms convert insoluble inorganic phosphatic compounds into soluble form. Such microbes not only assimilate phosphorus but they cause a large portion of soluble phosphate to be released in quantities in the excess of their own requirement. With this view an attempt has been made to summarize the information's about the mechanisms of microbial phosphorus solubilisation.

## PHOSPHATE FIXATION AND RETENTION

Phosphate ions attracted to soil constituents with such a bond that they become insoluble and difficultly available to plants. This process is called phosphate fixation or phosphate retention.

### Phosphate Fixation in Acidic Soil

Acid soils usually contain significant amount of soluble and exchangeable $Al^{3+}$, $Fe^{3+}$, and $Mn^{2+}$ ions. These ions serve as bridge in between clay and phosphate ions. **Precipitation and**

<footnote n="15">¹⁵ 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>
**Adsorption:** The phosphate ion enters into a chemical reaction with these free metal ions. Al³⁺ + 3 H₂PO₄⁻ → Al(H₂PO₄)₃↓

The product formed is difficultly soluble in water and precipitates in solution. With the passage of time the Al phosphate ppt become less soluble consequently less available to plants. Phosphate is fixed through adsorption on the surface of hydrous oxide of iron and aluminium.

## Phosphate Fixation in Alkali Soils

An alkaline soil contains high amounts of soluble and exchangeable Ca²⁺ and CaCO₃. Phosphate is reported to react with the ionic and carbonate form of calcium.

$$3\text{Ca}^{2+} + 2\text{PO}_4^{3-} \rightarrow \text{Ca}_3(\text{PO}_4)_2\downarrow$$

Insoluble

$$3\text{CaCO}_3 + 2\text{H}_2\text{PO}_4^- \rightarrow \text{Ca}_3(\text{PO}_4)_2 + 2\text{H}_2\text{O} + 2\text{CO}_2\uparrow$$

Insoluble
### Bacteria (PSB)

*Bacillus polymixa*

*Bacillus subtilis*

*Bacillus megaterium*

*Bacillus circulans*

*Pseudomonas striata*

*Pseudomonas rathonia*

### Fungi (PSF)

*Aspergillus niger*

*Aspergillus flavus*

*Aspergillus sulfurious*

*Aspergillus awamori*

*Aspergillus wentii*

*Aspergillus fomitopsis*

*Penicillium fuscum*

*Penicillium digitatum*

*Penicillium bilaii*

*Penicillium lilacinum*

## PHOSPHATE SOLUBILISING MICROORGANISM (PSM)

Fungal species are more efficient solubilises than bacteria.

### MECHANISM OF PHOSPHATE SOLUBILIZATION

1. Production of organic acids

Lowering of pH

Chelating effect

2. Production of inorganic acid

3. Production of CO₂

4. Production of H₂S

5. The production of mucilage and sloughed off root cells.

#### Production of organic acids

Phosphate solubilising microorganism produces various kinds of organic acids which are the main cause of solubilisation of insoluble phosphate by lowering the pH. Amount of phosphate dissolution depends up on many factors like types of organic acids **(Monocarboxylic acid):** Formic acid, Acetic acid, Lactic acid, Gluconic acid; **(Dicarboxylic acid):** Oxalic acid, Tartaric acid, Malic acid, Succinic Acid; **(Tricarboxylic acid):** Citric acid) produced such as aliphatic, aromatic; Structures of organic acids and amount of organic acids.

### CHELATION

The nonvolatile organic acids mainly responsible for phosphate liberation in solution possibly by their chelating action. These acids were produced as a result of the decomposition of glucose in

Pikovskaya's medium (1948) by *Bacillus megaterium*, *Bacillus circulans*, *Escherichia freundii*, *Penicillium digitatum* and *Penicillium fuscum*.

#### Production of inorganic acid

Nitrifying bacteria like *Nitrosomonas* and *Nitrobacter* produces the nitric acids which reacts with the insoluble phosphate and convert into soluble form. The possible chemical reactions are as follows:

$$Ca_3(PO_4)_2 + 4 HNO_3 \rightarrow Ca(H_2PO_4)_2 + 2Ca(NO_3)_2$$
Similarly sulphur oxidizing bacteria like *Thiobacillus* spp. Oxidizes the sulphur containing compounds and produces SO₂ which react with water and form sulphuric acid which reacts with the insoluble phosphate and convert into soluble form.

$$Ca_3(PO_4)_2 + 2H_2SO_4 \rightarrow Ca(H_2PO_4)_2 + 2CaSO_4$$

## Production of CO₂

The CO₂ produced by microorganism and plant roots is responsible for solubilization of inorganic phosphate in soil.

$$CO_2 + H_2O \rightarrow H_2CO_3$$

$$Ca_3(HPO_4)_2 + 2H_2CO_3 \rightarrow Ca(H_2PO_4)_2 + 2CaCO_3$$

## Production of H₂S

The H₂S produced by sulphate reducing bacteria (*Desulfovibrio*) particularly in the anaerobic condition.

$$FePO_4 + H_2S \rightarrow FeS + H_2PO_4^-$$

## Exudation of organic compounds from plant root

A variety of organic substances available at the root region, which directly or indirectly influences the quality and quantity of microorganism.

1. Sugars : Glucose, fructose, sucrose, maltose.

2. Amino acids : glutamine, glycine, methionine.

3. **Organic acid** : Tartaric, oxalic, citric, butyric, Succinic, Fumaric, glycolic.

4. Fatty acids and sterols: Palamitic, stearic, oleic, linolenic, cholesterol, sitosterol.

5. Nucleotides, flavonones and enzymes: flavonones; Adenine, guanine, uridine, cytidine; phosphatase, invertase, proteinase.

Mucilage and sloughed off root cells provide an energy substrate for microbial activity in the rhizosphere which subsequently dissolve insoluble phosphorus.

## Conclusions

* When soluble phosphates are added to soil, it is quickly fixed into insoluble forms, which greatly reduces the efficiency of phosphatic fertilizers.

* In acidic soils, the phosphorus is fixed primarily by Fe, Al, and Mn and in alkaline soils by Ca and Mg.

* Production of organic acid in microenvironment of root zone of plant is the main cause of phosphorus solubilization.

* PSF are more effective in solubilization than PSB.
<figure><img src="image_115.png" /></figure>

Besides concentration of organic acid, types of organic acid produced by PSM is more important.

**References:**

Gardner, W.K (1982) The acquisition of phosphorus by Lupinus albus L. I. Some characteristics of the soil root interface. *Plant and Soil*. 68: 19-32.

Moghimi, A; Lewis, D.G and Oades, J.M (1978) Release of phosphate from calcium phosphates by rhizosphere products. *Soil biology and biochemistry*. 10: 277-281.

Banik, S and Dey, B.K (1982) Available phosphate content on an alluvial soil as influenced by inoculation of some isolate phosphate solubilising microorganism. *Plant Soil*. 69: 353-364.

Helal, H.M; and Sauerbeck, D. (1984) Influences of plant roots on C and P metabolism in soil. *Plant and Soil*. 76: 175-182.

Kucey, R.M.N (1983) Phosphate solubilizing bacteria and fungi in various cultivated and virgin Alberta soils. *Canadian Journal of Biological Science*. 63: 671-678.

Pikovskaya, R.I (1948) Mobilization of phosphorus in soil in connection with vital activity of some microbial species. *Mikrobiologiya*.17: 362-370.

Kumar, A and Maurya, B.R (2006) Efficacy of phosphate solubilising fungi on acidity and solubilisation of tricalcium phosphate in broth. *Plant Archive*. 6(1): 309-311.
# Mechanism of K solubilization by Potassium Solubilizing Bacteria (KSB)

Dr. AJEET KUMAR<fnref n="16" />

Potassium is one of the essential macronutrients for plant growth and development, playing a crucial role in various physiological processes such as enzyme activation, photosynthesis, and nutrient transportation. However, a significant portion of soil potassium exists in insoluble forms, making it inaccessible to plants. Enter potassium solubilizing bacteria (KSB) - a group of remarkable microorganisms capable of converting insoluble potassium into forms that plants can readily absorb. The potential of KSB to enhance soil fertility and boost crop yields has garnered significant interest in agricultural research and sustainable farming practices. By harnessing the natural capabilities of these bacteria, farmers can reduce their dependence on chemical fertilizers, leading to more eco-friendly and cost-effective farming solutions. This discovery not only promises to revolutionize traditional farming methods but also contributes to the broader goal of sustainable agriculture. In this article, we delve into the fascinating world of potassium solubilizing bacteria, exploring their mechanisms of action, methods for their screening and their potential applications in agriculture. Join us as we unlock nature's secret to more productive and sustainable farming.

**Introduction:**

Potassium (K) solubilization is done by a wide range of saprophytic bacteria, fungal strains and Actinomycetes. There are strong evidences that soil bacteria are capable of transforming soil K to the forms available to plant effectively. There is considerable population of KSB in plant rhizosphere. These include both aerobic and anaerobic isolates however the most frequently KSB in soil are aerobic in nature. A considerably higher population of KSB is commonly found in the rhizosphere in comparison with non-rhizosphere soil. Solubilization of K by KSB from insoluble and fixed forms is an important aspect regarding K availability in soils. The ability to solubilize the silicate rocks by *B. mucilaginosus*, *B. circulanscan*, *B. edaphicus*, *Burkholderia*, *A. ferrooxidans*, *Arthrobacter* sp., *Enterobacter hormaechei*, *Paenibacillus mucilaginosus*, *Cladosporium*, *Aminobacter*, *Sphingomonas*, *Burkholderia* and *Paenibacillus glucanolyticus* has been reported. Among the soil bacterial communities, *B. mucilaginosus*, *B. edaphicus* and *B. circulanscan* have been described as effective K solubilizers. KSB are usually present in all soils and have been isolated from rhizosphere soil, non-rhizosphere soil, paddy soil and saline soil. KSB are isolated by serial dilution plate method using modified Aleksandrov medium including 5.0 g glucose; 0.5 g MgSO₄·7H₂O; 0.1 g CaCO₃; 0.006 g FeCl₃; 2.0 g Ca₃(PO₄)₂; 3.0 g potassium aluminium silicate; and 20.0 g agar in 1 L of deionized sterile water. The pH of this medium is adjusted to 7.2 by

<footnote n="16">¹⁶ 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>
adding 1 N NaOH. The plates are incubated at 28 ± 2°C in biological oxygen demand incubator for 3-4 days. The colonies exhibiting clear zones are selected and diameter of the solubilization zone is calculated and the values are recorded for each sample.

Recently, a modified plate assay for rapid screening of KSB has been developed. This assay is based on improved visualization of halo zone formation around the colonies on agar plates, through inclusion of an acid-base indicator dye (bromothymol blue, BTB), to modify the Aleksandrov medium. This assay is also time saving, more sensitive and beneficial in comparison to the Aleksandrov plate assay.

<figure><img src="image_116.png" /><figcaption>K solubilization on Aleksandrov agar plate</figcaption></figure>

<figure><img src="image_117.png" /><figcaption>K solubilization on modified agar medium plate</figcaption></figure>

**Figure 1.** Comparison of Potassium Solubilization on Aleksandrov Agar Plate (a) and Modified Agar Medium Plate (b) After 72 Hours of Incubation.

Quantitative estimation of K solubilization is performed by flame photometry wherein culture broth is centrifuged and supernatant is used for precipitation of cobalt nitrite. Standard curve for quantification of K is prepared using various concentrations of KCl solution. In this assay, mica is usually used as a source of insoluble form of K, although other K sources were also used in screening KSB (e.g., insoluble magnesium trisilicate, muscovite, illite powder, montmorillonite, kaolinite, potassium-feldspar, biotite, waste mica, bentonite, wood ash and potassium aluminium silicate). In addition, the amount of K solubilization in different culture media (e.g., different pH, temperature, K source, and carbon source) is different. For example, the amount of K solubilization by *B. edaphicus* in the liquid media was more and a better growth was detected on illite than feldspar. In general, the microbial solubilization of K is strongly influenced by pH, oxygen, the bacterial strains used and kind of K bearing minerals; in fact, moderate alkalinity favours the solubilization of silicate.

**Mechanisms of K solubilization by KSB**

It is generally believed that microorganisms contribute to the release of K⁺ from K-bearing minerals by several mechanisms. Released H⁺ can directly dissolve the mineral K as a result of slow releases of exchangeable K, readily available exchangeable K. As occurs in the case of P
solubilization, the major mechanism of K mineral solubilization is by production the organic and inorganic acids and production of protons (acidolysis mechanism) which are able to convert the insoluble K (mica, muscovite, and biotite feldspar) to soluble forms of K, easily taking up by the plant.

The types of various organic acids such as oxalic acid, tartaric acids, gluconic acid, 2-ketogluconic acid, citric acid, malic acid, succinic acid, lactic acid, propionic acid, glycolic acid, malonic acid, fumaric acid, etc. have been reported in KSB, which are effective in releasing K from K-bearing minerals. It has also observed that the type of the organic acid produced by KSB is different. Among the different organic acids involved in the solubilization of insoluble K, tarteric acid, citric acid, succinic acid, α-ketogluconic acid and oxalic acid are the most prominent acids released by KSB.

Microbial decomposition of organic materials also produces ammonia and hydrogen sulfide that can be oxidized in the soil to form the strong acids such as nitric acid and sulphuric acid. Hydrogen ions displace $K^+$, $Mg^{2+}$, $Ca^{2+}$ and $Mn^{2+}$ from the cation exchange complex in a soil. In addition to decreasing soil pH, organic acids produced by KSB can release of K ions from the mineral K by chelating (complex formation) $Si^{4+}$, $Al^{3+}$, $Fe^{2+}$ and $Ca^{2+}$ ions associated with K minerals. It was reported that KSB weathered phlogopite via aluminum chelation and acidic dissolution of the crystal network. In addition, The *B. altitudinis* strain accelerates weathering of K feldspar, change mineral surface morphology and induce the formation of new mineral complex. This strain dissolved K feldspar and significantly released more Si, Al and Fe by producing more organic acids. The KSB have a considerable role in proving K to plant by storing K in their biomass, which is potentially available to plants. It has been reported that the production of various extracellular polymers (primarily proteins and polysaccharides) can also led to release of K from K bearing minerals. These substances serve as attachment structures to mineral surface. Solution containing fresh microbial exopolysaccharides increases the dissolution rate of feldspars probably by forming complexes with framework ions in solution. KSB also synthesize biofilms, which create controllable microenvironments around microbial cells for weathering. Biofilm formation on aluminosilicate increases the residence time of water as compared to the residence time at the bare mineral surface and enhances the mineral weathering.

It was accepted that the microbial biofilms not only accelerated the weathering process but also regulated denudation losses by acting as a protective layer covering the mineral-water-hyphal/root hair interface in the mycorrhizosphere and rhizosphere of vascular plants. Besides, biofilm formation on mineral surface promoted the corrosion of potassium rich shale and the release of K, Si and Al in the bacteria-mineral contact model. In addition, it is known that the release of organic acids from the plant roots can be effective in enhancing mobilization of mineral K. Therefore, it can be suggested that other PGPRs (e.g., IAA producing bacteria) can also have a role in providing K for plant by increasing root exudates. In general, the most important mechanisms known in K mineral solubilization by KSB are (i) by lowering the pH; (ii) by enhancing chelation of the cations bound to K; and (iii) acidolysis of the surrounding area of microorganism.
## Potentialities of KSB

KSB can accelerate weathering of K minerals; especially when they are in direct contact with mineral surfaces by different action mechanisms. Attempts have been made to use of K mobilizing bacteria for solubilizing K from different K bearing minerals and hence to improve plant nutrition. Although KSB could be an alternative and viable technology to solubilize insoluble K into soluble form. The application in agricultural practice is still prevented by several factors, example, (i) lack of awareness about bio-fertilizers amongst the farmers; (ii) slow influence of the K biofertilizer on crop yield; (iii) less interest in scientific community on the development of K biofertilizer technologies; (iv) culture collection banks not yet developed for KSB due to the loss of efficient strains developed by scientists; and (v) and deficiency in technology in respect to product formulations are the major limitations.

## Conclusions

K-bearing minerals significantly contribute to potassium (K) fertilization for crop plants, which can only absorb K from the soil solution. While K can be released into the soil solution from insoluble minerals, this release is typically insufficient to meet plant requirements due to the low concentration of soluble K and its relative immobility in the soil. Consequently, K-containing fertilizers are commonly applied in extensive agricultural systems to supply available K. However, these fertilizers are expensive and their long-term use leads to increased input costs, reduced agricultural profitability, and numerous environmental problems due to the accumulation of heavy metals in the soil and plant systems. The use of potassium solubilizing bacteria (KSB) offers a promising alternative to solubilize soil K reserves and make them available to plants, thereby promoting plant growth and reducing the need for K-fertilizers. K solubilization is facilitated by a variety of bacteria, including *B. mucilaginosus*, *B. edaphicus*, *B. circulans*, *Pseudomonas*, *Burkholderia* and *Acidithiobacillus ferrooxidans*. Research has demonstrated that KSB can dissolve K from insoluble K-bearing minerals by secreting organic acids. The primary mechanism through which KSB make K available to plants involves the production of organic acids, which can either directly increase dissolution through proton or ligand-mediated mechanisms or indirectly by forming complexes in solution with reaction products. Thus, applying KSB as biofertilizers not only enhances plant growth and yield but also reduces the need for agrochemicals, supporting eco-friendly crop production. These technologies are becoming increasingly vital in modern agricultural practices, especially given the changing agricultural landscape and environmental hazards associated with chemical fertilizers, which call for a greater role for biofertilizers in the future.

## Future Perspectives

To fully realize the potential of KSB, several areas of research and application need to be explored:

1. Conduct field studies to assess the potential of KSB techniques in agricultural production systems and evaluate their impacts on crop growth and soil behaviour.

2. Investigate the role of KSB in increasing the availability of other nutrients affected by pH, such as phosphorus (P), nitrogen (N), iron (Fe), and zinc (Zn).
3. Study the effects of other plant growth-promoting rhizobacteria (PGPRs) such as indole-3-acetic acid (IAA) producers, ACC deaminase producers, phosphate solubilizers, and nitrogen fixers on K availability.

4. Examine the interactions (both positive and negative) between KSB and other PGPRs concerning K availability.

5. Determine the optimal conditions for KSB activity, including the role of organic matter.

6. Evaluate plant species that are effective in K uptake and support K solubilizing microbial populations.

7. Understand the mechanisms of KSB to determine the best bacterial strains for use with specific plants in various geographic regions.

8. Study the stability of KSB inoculants in the soil and assess soil mineralogical properties before inoculation.

9. Conduct further research on the molecular biology of KSB, as there is currently limited information compared to other PGPRs.

10. Addressing these areas will enhance our understanding and application of KSB in sustainable agriculture
# FCO (Fertilizer Control Order) Specifications of Biofertilizers

Dr. AJEET KUMAR<fnref n="17" />

## 1. Introduction

The Fertilizer Control Order (FCO), 1985, regulates the quality, production, and distribution of fertilizers in India, including biofertilizers. The FCO ensures that biofertilizers meet minimum microbial and physical standards for efficacy and safety.

**Learning Objectives:**

* ✓ Understand the FCO guidelines for biofertilizers.

* ✓ Learn the microbial and carrier specifications.

* ✓ Discuss labeling, packaging, and storage requirements.

## 2. Importance of FCO Specifications

* ✓ Ensures **minimum viable microbial count** for effectiveness.

* ✓ Prevents **substandard or contaminated products** in the market.

* ✓ Standardizes **production, testing, and labeling** for farmer trust.

## 3. FCO-Approved Biofertilizers & Their Specifications

### A. Bacterial Biofertilizers

<table><thead><tr><td>Biofertilizer</td><td>Microorganism</td><td>Minimum Viable Cells</td><td>Contamination Limit</td></tr></thead><tbody><tr><td>Rhizobium</td><td>Rhizobium spp.</td><td>1 × 10<sup>9</sup> CFU/g</td><td>No contamination at 10<sup>-6</sup> dilution</td></tr><tr><td>Azotobacter</td><td>Azotobacter spp.</td><td>1 × 10<sup>8</sup> CFU/g</td><td>No contamination at 10<sup>-6</sup> dilution</td></tr><tr><td>Azospirillum</td><td>Azospirillum spp.</td><td>1 × 10<sup>8</sup> CFU/g</td><td>No contamination at 10<sup>-6</sup> dilution</td></tr></tbody></table>

<footnote n="17">¹⁷ 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>
<table><thead><tr><th>Biofertilizer</th><th>Microorganism</th><th>Minimum Viable Cells</th><th>Contamination Limit</th></tr></thead><tbody><tr><td>Phosphate-Solubilizing Bacteria (PSB)</td><td>Bacillus, Pseudomonas</td><td>1 × 10<sup>8</sup> CFU/g</td><td>No contamination at 10<sup>-6</sup> dilution</td></tr><tr><td colspan="4"><strong>B. Fungal Biofertilizers</strong></td></tr><tr><th>Biofertilizer</th><th>Microorganism</th><th>Minimum Viable Cells</th><th>Contamination Limit</th></tr><tr><td>Phosphate-Solubilizing Fungi (PSF)</td><td>Aspergillus, Penicillium</td><td>1 × 10<sup>8</sup> CFU/g</td><td>No contamination at 10<sup>-6</sup> dilution</td></tr><tr><td>Mycorrhiza (AMF)</td><td>Glomus, Gigaspora</td><td>100 propagules/g</td><td>No contamination at 10<sup>-6</sup> dilution</td></tr></tbody></table>

### C. Carrier-Based vs. Liquid Biofertilizers

* ✓ **Carrier-based (Solid):** Peat, lignite, or charcoal (pH 6.5–7.5, moisture <30%).

* ✓ **Liquid Formulations:** Must maintain 1 × 10⁸ CFU/mL for 6 months.

### 4. Physical & Chemical Specifications (FCO Guidelines)

* ✓ **Moisture Content:** ≤30–40% (carrier-based).

* ✓ **pH:** 6.5–7.5 (neutral range for microbial survival).

* ✓ **Particle Size:** 90% should pass through 150–212 µm sieve.

* ✓ **Contaminants:**

* o No pathogenic bacteria (e.g., *E. coli*, *Salmonella*).

* o Heavy metals (As, Cd, Pb) within permissible limits.

### 5. Labeling & Packaging Requirements (FCO Compliance)

**Mandatory Information:**

* Name of biofertilizer & strain (e.g., *Rhizobium leguminosarum*).

* CFU count at production & expiry date.

* Batch number, manufacturing date, and expiry (shelf life: 6–12 months).
* Storage instructions (e.g., "Store in cool, dry place").

**Prohibited Claims:**

* False promises (e.g., "100% yield increase").

* Unapproved microbial strains.

**6. Quality Control & Testing Methods**

<figure><img src="image_118.png" /></figure>

**Serial Dilution & Plate Count:** To check CFU/g.

<figure><img src="image_119.png" /></figure>

**PCR & DNA Sequencing:** For strain authentication.

<figure><img src="image_120.png" /></figure>

**Field Trials:** To validate performance under different agro-climatic conditions.

**7. Recent Amendments & Challenges**

<figure><img src="image_121.png" /></figure>

**New Additions (2023):** *Trichoderma* (biocontrol agent) included under FCO.

**Challenges:**

* Adulteration with inert materials.

* Poor shelf life due to improper storage.

**8. Case Study: Impact of FCO Regulations**

* **Before FCO:** Inconsistent biofertilizer quality → low farmer trust.

* **After FCO:** Standardized products → increased adoption in organic farming.

**9. Conclusion**

FCO specifications ensure **quality, efficacy, and safety** of biofertilizers. Compliance is essential for sustainable agriculture and reducing chemical fertilizer dependency.

**10. Discussion Questions**

* Why is a **minimum CFU count** necessary for biofertilizers?

* How does **carrier material** affect microbial survival?

* What improvements can be made in FCO regulations for better enforcement?

**11. References**

* **Fertilizer Control Order (FCO), 1985** (Amended 2023).

* **BIS Standards (IS 8268:2015)** for microbial inoculants.

* Kumar, R. (2020). *Biofertilizers: Science & Technology*. TERI Press.
# Quality Control of Biofertilizers

Dr. AJEET KUMAR<fnref n="18" />

**Formulation:**

Inoculation of the carrier with the culture broth means the mixing of broth and carrier. This operation must be done in aseptic conditions to avoid any contamination.

**Quality control of formulation:**

The quality of the carrier-based inoculum depends upon the viable cell count and the presence or absence of contaminants.

o A good culture must contain about $10^7–10^8$ viable cells or CFUs per g of culture.

o No contaminants are permissible at $10^{-5}–10^{-6}$ dilutions. These critical values differ according to the type of biofertilizer.

o As per I.S.I., in case of Rhizobium, the carrier based culture must contain at least $10^7$ cell or CFUs per g of culture and no contaminants are permissible below $10^{-6}$ dilution. In case of Azotobacter, So, the enumeration of cells density and contaminants are important task in the production of carrier based microbial biofertilizer.

**Bioactivity of Azotobacter:**

Nitrogenase is the enzyme catalyzing the reduction of nitrogen into ammonia. This enzyme can also reduce acetylene into ethylene as well. Acetylene and ethylene can easily be measured by a gas chromatograph. In a closed system, if a portion of gas is substituted by acetylene and acetylene is allowed to be reduced for a certain period, the proportion of acetylene and ethylene can be measured by passing the mixture of gas through the column of gas chromatograph and measuring the peak developed.

The nitrogenase activity (hence, the nitrogen fixation activity) of Azotobacter or other free living bacteria can be extrapolated by **Acetylene Reduction Assay (ARA)** method, but this technique can not be applied in case of Rhizobium as this bacteria can not fix nitrogen in free condition. In this case the plant containing the nodules is to be taken for assay.

**Bioefficacy of Rhizobium (Nodulation):**

The study of nodulation efficiency of Rhizobium is an important task in Biofertilizer Technology. The number of viable cells in the inoculum can easily be enumerated by dilution plating method but this technique will not show whether the viable cells have retained their nodulation efficiency during the long term laboratory procedure like preservation, production and storage. This technique will enumerate the number of viable cells, which have retained the

<footnote n="18">¹⁸ 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>