Agricultural microbiology

Microbiology

Microbiology is the branch of science that deals with the study of microscopic organisms, also known as microorganisms.

Sub - disciplines:

The main sub-disciplines of microbiology include:

●       Bacteriology

●       Virology

●       Mycology

●       Parasitology

●       Phycology (Algology)

●       Microbial Ecology

●       Immunology

●       Industrial Microbiology

●       Medical Microbiology

●       Food Microbiology

●       Environmental Microbiology

●       Agricultural Microbiology

Scope :

The scope of microbiology is vast and spans across medicine, agriculture, industry, and environmental science. It helps in the diagnosis and treatment of diseases, development of vaccines, and antibiotic production. In agriculture, microbes are used for biofertilizers and pest control.

History

●          Invention of Microscope (1665–1676):

●          Robert Hooke (1665): coined the term "cell".

●          Antonie van Leeuwenhoek (1676): First to observe and describe living microorganisms (“animalcules”) using a single-lens microscope.

●   Spontaneous Generation Debate (1700s–1800s):

●   John Needham (1745): Supported spontaneous generation.

●   Lazzaro Spallanzani (1765): Disproved it by boiling broth and sealing it.

●   Louis Pasteur (1861): Definitively disproved spontaneous generation with swan-neck flask experiment.

●   Golden Age of Microbiology (1857–1914):

●   Louis Pasteur: Developed pasteurization, germ theory of disease, and vaccines (rabies, anthrax).

●   Robert Koch: Identified bacteria causing TB, cholera, anthrax; developed Koch’s postulates.

●   Discovery of major pathogens and development of sterile techniques.

●   Development of Medical Microbiology (Late 1800s):

●   Joseph Lister (1867): Introduced antiseptic surgery using carbolic acid.

●   Edward Jenner (1796): Developed smallpox vaccine (foundation of immunology).

●   Modern Microbiology(1900s–Present):

●   Alexander Fleming (1928): Discovered penicillin.

●   Discovery of viruses, antibiotics, DNA structure (Watson & Crick, 1953).

●   Rise of molecular biology, genetic engineering, biotechnolog

CLASSIFICATION OF MICROBES

SOIL MICRO-ORGANISMS:

Definition:

Soil microorganisms are microscopic living organisms found in the soil that contribute to soil health, fertility, and ecosystem functioning.

Types of Soil Microorganisms:

Bacteria:

●   Most abundant

●   Decompose organic matter

●   Involved in nitrogen fixation (e.g., Rhizobium)

Fungi:

●   Break down tough organic materials (e.g.,cellulose, lignin)

●   Form symbiotic relationships (mycorrhizae) with plant roots

Actinomycetes:

●   Filamentous bacteria

●   Decompose complex organic substances

●   Produce antibiotics (e.g., Streptomyces)

Protozoa:

●   Feed on bacteria

●   Help regulate microbial populations

●   Release nutrients in plant-available forms

Algae:

●   Perform photosynthesis

●   Contribute to soil structure and nutrient content

●   Nematodes & Microarthropods (sometimes included)

BIOFERTILIZER

DEFINITION

Biofertilizers are natural substances that contain living microorganisms, which enhance plant growth by increasing the availability of nutrients in the soil. They are eco-friendly alternatives to chemical fertilizers.

Examples: Rhizobium, Azotobacter, Azospirillum, Blue Green Algae (BGA), Mycorrhizae, Phosphate Solubilizing Bacteria (PSB).

Advantages of Biofertilizers:

●   Eco-friendly

●   Cost-effective

●   Improve Soil Health

●   Promote Plant Growth

●   Sustainable Agriculture

●   Non-toxic

Disadvantages of Biofertilizers:

●   Slow Action

●   Specificity

●   Short Shelf Life

●   Environmental Sensitivity

●   Requires Technical Knowledge.

CLASSIFICATION

1. Rhizobium

Rhizobia are soil bacteria, live freely in soil and in the root region of both leguminous and non-leguminous plants. However they enter into symbiosis only with leguminous plants,by infesting their roots and forming nodules on them. Non legume nodulated by Rhizobia isTrema or Parasponia sp.

2. Azospirillam:

 Description:

Azotobacter is a free-living, aerobic, nitrogen-fixing bacteria found in neutral to alkaline soils.

It belongs to the class Gammaproteobacteria and helps improve soil fertility by converting atmospheric nitrogen (N₂) into a usable form for plants.

It also produces growth-promoting substances like vitamins, hormones (IAA, gibberellins), and antifungal compounds.

 Common Species:

●   Azotobacter chroococcum (most studied and widely used)

●   Azotobacter vinelandii

●   Azotobacter beijerinckii

●   Azotobacter paspali

●   Azotobacter insignis

 Nitrogen-Fixing Capacity:

Fixes around 20–40 kg of nitrogen per hectare per season in the soil.

Does not form nodules like Rhizobium but fixes nitrogen freely in the rhizosphere (root zone).

 Where It Is Applicable?

Used in non-leguminous crops like:

●   Wheat

●   Maize (corn)

●   Cotton

●   Potato

●   Rice

●   Vegetables

Effective in neutral to alkaline soils.

Commonly applied in biofertilizer formulations for enhancing crop yield and soil fertility in sustainable agriculture.

3.Azopirillam:

Description:

Azospirillum is a microaerophilic, associative nitrogen-fixing bacterium that lives in close association with plant roots (especially grasses).

It enhances plant growth by fixing nitrogen and producing growth-promoting substances like IAA (Indole Acetic Acid), gibberellins, and cytokinins.

Found mainly in the rhizosphere (root zone) of plants but does not form nodules. 

4. Phosphate solubilising Microorganisms

Description:

PSMs are beneficial microorganisms that convert insoluble forms of phosphorus (P) in the soil into forms that plants can absorb.

They secrete organic acids (like citric, gluconic acid) and enzymes that dissolve bound phosphates, increasing phosphorus availability.

VAM (Vesicular-Arbuscular Mycorrhiza)

AM (Arbuscular Mycorrhiza)

Both VAM and AM refer to the same type of beneficial fungi, but AM is the more modern and scientifically accurate term.

Description:

Arbuscular Mycorrhiza (AM) are a type of endophytic fungi that form symbiotic associations with the roots of most terrestrial plants.

The fungi colonize the root cortex, forming:

Arbuscules – tree-like structures for nutrient exchange

Vesicles – storage structures (in VAM specifically)

Key Features:

●   Improve phosphorus uptake significantly.

●   Also help in absorption of zinc, copper, water.

●   Increase plant tolerance to drought, disease, and stress.

●   Do not penetrate the root cells directly but form structures inside the root cortex cells

BIOPESTICIDE

Bio pesticides are living organisms which can intervene the life cycle of insect pests in such a way that the crop damage is minimized. The agents employed as biopesticides are parasites, predetors, fungi, bacteria and viruses which are natural enemies of pests. These bio agents can be conserved, preserved and multiplied under laboratory condition for field release.

Mechanism of Action of Bio-pesticides:

·  Microbial Bio-pesticides:

Contain living microorganisms like bacteria, fungi, viruses, or protozoa.

Mechanism:Infect and kill target pests by producing toxins or replicating within the host.Example: Bacillus thuringiensis (Bt): Produces crystal (Cry) toxins that damage the gut of insect larvae, leading to death.

·  Biochemical Bio-pesticides:

Naturally occurring substances that interfere with the mating, feeding, or development of pests.

Mechanism:Work as repellents, growth regulators, or pheromones disrupting pest behavior.Example:Neem oil (Azadirachtin): Acts as an insect repellent and growth inhibitor.

· Plant-Incorporated Protectants (PIPs):

Genes from pesticidal organisms are introduced into plants via genetic engineering.

MechanismPlant produces pesticidal protein, offering built-in pest resistance.

Example:Bt cotton: Produces Cry proteins to kill bollworms.

MICROBIAL BIOREMEDIATION

Bioremediation

Bioremediation is the process of using living organisms, primarily microbes like bacteria, fungi, and plants, to remove or neutralize pollutants from a contaminated site (soil, water, or air).

Microbial bioremediation

Microbial bioremediation is the use of microorganisms (mainly bacteria, fungi, or algae) to detoxify, degrade, or transform environmental pollutants into non-toxic forms.

Mechanism:

· Detection of Pollutants:

Microbes detect pollutants (like hydrocarbons or heavy metals) as potential energy or nutrient sources.Attachment and Colonization:

Microorganisms attach to the contaminated surface (soil, water, etc.) and form colonies (biofilms).

. Enzymatic Degradation:

Microbes produce specific enzymes that break down complex pollutants into simpler, less toxic compounds.Aerobic degradation (with oxygen): Organic pollutants → CO₂ + H₂O + biomass

Anaerobic degradation (without oxygen): Pollutants → Methane, CO₂, organic acids

·  Assimilation:

The byproducts are used by microbes for growth and energy.

·  Detoxification:

The final metabolites are non-toxic or less harmful than the original pollutants.

Factors Affecting Bioremediation

Temperature – Optimal temperatures increase microbial activity; too hot or cold can inhibit degradation.

 pH – Most microbes prefer neutral pH (6.5–7.5); extreme pH affects enzyme function.

Oxygen Availability – Aerobic microbes need oxygen; anaerobic conditions favor different pathways.Nutrient Availability – Nitrogen, phosphorus, and trace elements are essential for microbial growth.

Pollutant Concentration and Type – High toxicity or complex compounds may slow or inhibit degradation.

FERMENTATION TECHNOLOGY

Fermentation Technology :

The term fermentation is used to indicate microbial cell propagation and

generation of products under either aerobic, microaerobic, or anaerobic conditions.

Aerobic indicates condition where air is intentionally mixed with the medium;

microaerobic refers to air that is initially present, but is then used up or displaced as microbial growth occurs; while anaerobic indicates a condition where oxygen isremoved and intentionally excluded from the fermentation media since it is toxic to the cells.

Types of Fermentation Processes

Submerged Cultivation

Submerged cultivation of microbial cells in bioreactors guarantees a controlled environment for the efficient production of high-quality end products and to achieve optimum productivity and yield. Industrial bioreactors operated in batch, fed-batch, or continuous mode are utilized to culture different types of microorganisms producing a wide range of products.

Continuous Cultivation

Continuous culture represents an open system in which nutrients are aseptically and continuously added to the bioreactor, and the culture broth (containing cellsand metabolites) is removed at the same time , the volume of the culture broth is constant due to a constant feed-in and feed-out rate.Continuous culture is used for a chemostat, represented by a constant specific growth rate of cells, which is equal to the dilution rate and is controlled by the availability of the limiting nutrient,

MICROBIAL ENZYMES

A microbial enzyme refers to an enzyme produced by microorganisms like bacteria, which aids in biochemical reactions within the host cells. These enzymes play a crucial role in breaking down complex compounds in human food, enhancing digestion, and improving the utilization of nutrients.

1. Amylase

Starch hydrolyzing enzyme amylase is constituted by α-amylase and β-amylase. The α-amylasesare synthesized by plants, animals, and microorganisms, whereas, β -amylase is synthesized mainly by plants.

2. Aryl sulphatases

Arylsulphatases are typically widespread in nature, as well as in soils. They are responsible for the hydrolysis of sulphate esters in the soil and are secreted by bacteria into the external environment as a response to sulphur limitation.

3. β-Glucosidase

β-glucosidase is a common and predominant enzyme in soils.It is named according to the type of bond that it hydrolyses. This enzyme plays an important role in soils by catalyzing the hydrolysis and biodegradation of various β-glucosidespresent in plant debris decomposing in the ecosystem into glucose,which is a source of nutrient for many number of soil microorganisms.β-glucosidase is characteristically useful as a

soil quality indicator, and may give a reflection of past biological activity, the

capacity of soil to stabilize the soil organic matter, and can be used to detect

management effect on soils.

4. Cellulases

          Cellulases are a group of enzymes that catalyze the degradation of

cellulose. It has been also reported a significantly more stimulatory effect of

cellulases in black soil than reld soil.

5. Chitinases

Chitinase or chitinolytic enzymes are key enzymes responsible for the degradation and hydrolysis of chitin.Chitin is the major structural component of many fungal cell walls. They cause the degradation of cell walls of pathogenic fungi.These enzymes are effective in the control of soil-borne diseases such as Sclerotiumrolfsiiand Rhizoctoniasolani in beans and cotton, respectively.

6. Dehydrogenases

A dehydrogenase is an enzyme belonging to the group of oxidoreductases that oxidizes a substrate by reducing an electron acceptor, usually NAD⁺/NADP⁺ or a flavin coenzyme such as FAD or FMN.

7. Phosphatases

Phosphatases are the group of enzymes,hydrolyse the P from organic bound state into inorganic available forms as orthophosphates and thus play critical role in P cycle in soil.

8. Proteases

Proteases in the soil play a significant role in N mineralization,an important process regulating the amount of plant available N and plant growth.Upon death, plant and animals undergo microbial decay in the soil and the nitrogen contained in their proteins is released. Microorganisms causes breakdown of proteins with the help of proteolyticenzymes is known as ―proteolysis".Proteolysis leads to the formation ofamino acids,which served as substrates for nitrification process.This ultimately shows the N availability in soil

9. Ureases

Urease enzyme is responsible for the hydrolysis of urea fertilizers applied to the soil into NH3 and CO2 with the concomitant rise in soil pH. This, in turn, results in a rapid N loss to the atmosphere through NH3 volatilization, a process considered vital in the regulation of N supply to plants after urea fertilization.Soil urease originates mainly from plants and microorganisms found as both intra- and extra-cellular enzymes and are rapidly degraded in soil by proteolytic enzymes.

PLANT MICROBE INTERACTION

a) Plant microbe interaction

It mainly constitutes the association of microorganism with plants little in a positive

way or in a negative way. The positive approach is mainly the symbiotic relationships and the negative approach constituents mainly pathogen plant interactions.

b) Plant microbe – microbe interactionAlso called tripartite symbiosis. Interaction between the macrobiont and a microbiont

Eg: Alnus – Frankia –Mycorrhiza

Casuarina – Frankia – Mycorrhiza

c) Microbe – microbe interaction

Different species or genera interact in a positive or negative way and exhibit various types of intererationship.

Interrelationship between microorganisms: Beneficial and harmful relationship Microbial ecosystem of soil is the sum of the biotic and the abiotic components of soil. Many of these organisms depend upon one another for direct and indirect nutrients.The microorganisms that inhabit the soil exhibited many different types of associations or interactions. Some of the associations are indifferent or neutral, some are beneficial type of interactions and some are detrimental or negative.

I. Beneficial / positive interactions

A. Neutralism

B. Symbiosis / mutualism

C. Protoco-operation

D. Communalism

A. Neutralism

It is a type of neutral association, in which two dissimilar organisms inhabiting the same environment without impacting each other microorganisms as entirely independent. Each could utilize different nutrients without producing metabolic end products that are inhibitory.

B. Symbiosis / Mutualism

It is an obligatory or highly specific interaction between two populations in which both of them benefit from each other. It usually requires close physical connection in which both partners can act, as if they are one, when in separate their metabolic activities are different.

Based on the partner selection it is classified as obligate symbiosis and facultative symbiosis. Based on the purpose of interaction also it is classified. Based on the purpose it is classified as service-resource type, resource-resource type and service to service type

1. Obligate symbiosis: It occurs when both microorganism live together in close proximity, and both species cannot survive without its mutualistic partner. Symbiotic association is evident in soil among several groups of organisms algae and fungi in lichens, bacteria residing within protozoan cells, bacteria and roots in the Rhizobium legume symbiosis, fungi and roots in mycorrhizae.

a. Lichens

In lichens, the algae and fungi are in intimate physical and physiological relationship. The alga benefits from the protection afforded by the fungal hyphae thatenvelop and protect it from environmental stresses. While the fungi gains benefit by making use of the CO2 fixed by its photosynthetic partner as well as the oxygen. Where ever the BGA are the participants, the fungi also benefits from the fixed N2.

b. Mycorrhizae

It is a mutualistic association among mycorrhizal fungi and plant roots, in which plants provide fungus with carbohydrates and offer it protection. In turn the fungus increases the surface area of plant roots for absorbing water, nitrogenous compounds, phosphorus, and other inorganic nutrients (e.g., phosphate) from the surrounding soil and delivers them to the

plant which improves plant growth and health.

c. Symbiotic N2 Fixation

The nitrogen-fixing bacteria provide the plants with nitrogenous compounds, while in return the plants provide the nitrogen-fixing bacteria with carbohydrates. This mutualistic  association improves plant growth and health, and it has different types which include

Rhizobium spp. with root nodules of legume plants and Frankia which is an actinomycete(nodule-forming filamentous bacteria) with the roots of Alnus and Casuarina trees which are non legumes.

2. Facultative mutualism : It occurs when one of the two partners can survive without its mutualistic partner by itself in some conditions. Membership in this association is not usually specific and one organism can be replaced by the other. It is also termed synergism.

It is a loose association

C. Proto co-operation

Synergism (protocooperation) is a relationship that occurs between two or more

populations at which both or all of them benefit. In this relationship microbial populations perform a function which may not be performed individually or produce a new product that neither each population can produce alone.

This relationship is different from mutualism because as it is not an obligatory interaction, none of the species depend on the relationship for existence, as each member can live and produce its own food individually.

D. Commensalisms

It is a relationship at which one population benefits, while the other population is unaffected (neither harmed nor benefited). It is a very common relationship between different microbial populations. It is the type of beneficial association, in which only one population benefits, while the other population is unaffected (neither harmed nor benefited). This is a very common relationship between different populations.

III. Negative / harmful / deleterious interactions

Detrimental effects of one species on its neighbours are quite common in soil, and they are demonstrated by the decreases in abundance or metabolic activities of the susceptible organisms. It consists of different relationships between different populations either two or more, at which one population at least is harmed while the other is either harmed, benefited, or not affected.

This include

a) Competition

b) amensalism

c) parasitism and predation

a. Competition

It is a relation that occurs between different populations in the soil which use the same limiting resources that are insufficient to support all the individuals. The rivalry for limiting nutrients or other common needs. These resources include raw materials important for life such as water, light, nutrients, oxygen, and space for occupying or any other resources, which is essential for survival and reproduction. In this relation the best adapted microbial species will predominate or infact, eliminate other species which are dependent upon the same limited nutrient substances. Also, organisms which have the capability to grow faster are considered good competitors.

b. Amensalism (Antagonism)

It is the most common negative relationship in nature at which one microbial population suppresses or adversely influences the growth or the activities of the other population in the same environment by producing inhibitory substances either directly or indirectly. The release of products by one species is toxic to its neighbours. The population that produces the inhibitors is not affected by them and therefore gains the antagonistic edge. These inhibitors may be antibiotics, toxins, organic acids, alcohols, or other allelochemicals, lytic enzymes, as well as harmful gases like methane, ethylene, HCN, nitrite, or sulfides or other volatile sulfur compounds. The population that adversely affects the other is called antagonistic species, and it constantly has great practical importance. Antagonism is a type of ammensalism..

C. Parasitism

It is a relationship between two dissimilar organisms that is called host-parasite relationship in which one of them (parasite) lives in or on the other organism (host). The parasite lives in close contact with the host and forms metabolic association with the host and feeds on their cells, tissues, or fluids in which the parasite is profited, while the host is adversely affected. Sometimes the relation between the host and parasite could be diverged from parasitic relationship to a pathogenic relationship.This relationship is widely spread in soil communities and characterized by its long period of contact and the specialization between parasite and host. Also, parasite is usually smaller than the host (in most cases). This relationship has two sides, one is useful while the other harmful. If the parasitism is accomplished on bacteria that are considered pathogenic to plants, it is considered as a useful relationship for plant growth andhealth. While if the parasitism is accomplished on bacteria that are considered profitable to plants, it is considered as a harmful relationship for plant growth and health

d. Predation

Predation is one of the most dramatic interrelationships among the microorganic in nature, at which predator organism directly attacks a prey organism and feeds on it. This relationship has short duration, at which predators may or may not kill their prey prior to feeding on them, but the normal result is generally absorption of the prey‘s tissue through ingestion and subsequently the death of prey. Prey may be larger or smaller than predator.

QUALITY CONTROL OF BIO-INOCULANTS

Bioinoculants (also called microbial inoculants or biofertilizers) contain beneficial microorganisms that promote plant growth or soil health. Ensuring their quality is essential for effectiveness and safety. Quality control includes physical, chemical, microbiological, and functional parameter

Key Aspects:

1.  Viability Count

The number of viable (live) microorganisms is checked.

Expressed as CFU (Colony Forming Units) per gram or ml.

Standard: 10⁷ to 10⁹ CFU/g or ml for most bioinoculants.

2. Contamination Check

Must be free from harmful or unwanted microbes.

Tested using selective media to identify contamination.

3. pH and Moisture Content

Ideal pH: ~6.5 to 7.5 (neutral range).

Moisture: 20–40% for solid carriers; affects microbial survival.

4. Shelf Life

Monitored over time to ensure viability during storage.

Minimum shelf life: 6 months (depending on formulation and carrier).

5. Carrier Material Quality

Should be sterile, non-toxic, and have high water-holding capacity.

Examples: peat, lignite, charcoal, or liquid carriers.

6. Functionality Testing

Tests to ensure:

Nitrogen fixation (e.g., in Rhizobium, Azotobacter)

Phosphate solubilization (e.g., in PSM).

Mycorrhizal association (e.g., in AM/VAM fungi).

Done through lab assays or pot/field trials.

7. Labeling and Packaging

Must include:

·         Strain name

·         CFU count

·         Date of manufacture & expiry

·         Storage instructions

·         Application method

8. Adherence to National Standards

In India, bioinoculants are regulated by:

Fertilizer Control Order (FCO), 1985.

Standards provided for specific microbial strains (e.g., Rhizobium, Azospirillum, PSM).

Standard Requirement

Viable Cell Count ≥ 10⁷–10⁹ CFU/g or ml

Contamination - Absent in standard media

pH - 6.5 – 7.5

Moisture Content - 20% – 40%

Shelf Life - Minimum 6 months

Carrier Material - Sterile, non-toxic, high water-holding

Packaging & Labeling - As per FCO and BIS standards