Solutes and Water Activity

A selectively permeable plasma membrane separates microorganisms from their environment. Microorganisms can be affected by changes in the osmotic concentration of their surroundings. If a microorganism is placed in a hypotonic solution (one with a lower osmotic pressure), water will enter the cell and cause it to burst unless something is done to prevent the influx.

Most bacteria, algae, and fungi have rigid cell walls that maintain the shape and integrity of the cell. When microorganisms with rigid cell walls are placed in a hypertonic environment, water leaves and the plasma membrane shrinks away from the wall, a process known as plasmolysis. This dehydrates the cell and may damage the plasma membrane; the cell becomes metabolically inactive and ceases to grow.

Many microorganisms keep the osmotic concentration of their protoplasm somewhat above that of the habitat by the use of compatible solutes, so that the plasma membrane is always pressed firmly against their cell wall. Compatible solutes are solutes that are compatible with metabolism and growth when at high intracellular concentrations.

• Most procaryotes increase their internal osmotic concentration in a hypertonic environment through the synthesis or uptake of choline, betaine, proline, glutamic acid, and other amino acids; elevated levels of potassium ions are also involved to some extent.
• Algae and fungi employ sucrose and polyols—for example, arabitol, glycerol, and mannitol—for the same purpose. Polyols and amino acids are ideal solutes for this function because they normally do not disrupt enzyme structure and function.
• Since protozoa do not have a cell wall, they must use contractile vacuoles to eliminate excess water when living in hypotonic environments.
•  A few procaryotes like Halobacterium salinarium raise their osmotic concentration with potassium ions (sodium ions are also elevated but not as much as potassium). Halobacterium’s enzymes have been altered so that they actually require high salt concentrations for normal activity. 

Thus, the osmotic concentration of the cytoplasm is reduced by use of inclusion bodies which store solutes. Procaryotes also can contain pressure-sensitive channels that open to allow solute escape when the osmolarity of the environment becomes much lower than that of the cytoplasm.

Water activity

The amount of water available to microorganisms can be reduced by interaction with solute molecules (the osmotic effect) or by adsorption to the surfaces of solids (the matric effect). Because the osmotic concentration of a habitat has such profound effects on microorganisms, the degree of water availability is critical in growth 

We express water availability as water activity (aw) (also may be expressed as water potential, which is related to aw). It is equivalent to the ratio of the solution’s vapor pressure (Psoln) to that of pure water (Pwater).

aw =  Psoln

          Pwater

Water activity is inversely related to osmotic pressure; if a solution has high osmotic pressure, its aw is low.

Microorganisms differ greatly in their ability to adapt to habitats with low water activity. A microorganism must expend extra effort to grow in a habitat with a low aw value because it must maintain a high internal solute concentration to retain water. 

Some microorganisms can do this and are osmotolerant; they will grow over wide ranges of water activity or osmotic concentration. For example, Staphylococcus aureus can be cultured in media containing any sodium chloride concentration up to about 3 M. It is well adapted for growth on the skin. The yeast Saccharomyces rouxii will grow in sugar solutions with aw values as low as 0.6. The alga Dunaliella viridis tolerates sodium chloride concentrations from 1.7 M to a saturated solution.

Although a few microorganisms are truly osmotolerant, most only grow well at water activities around 0.98 (the approximate aw for seawater) or higher. This is why drying food or adding large quantities of salt and sugar is so effective in preventing food spoilage. Many fungi are osmotolerant and thus particularly important in the spoilage of salted or dried foods. 

Pressure

Most organisms spend their lives on land or on the surface of water, always subjected to a pressure of 1 atmosphere (atm), and are never affected significantly by pressure. Yet the deep sea (ocean of 1,000 m or more in depth) is 75% of the total ocean volume. The hydrostatic pressure can reach 600 to 1,100 atm in the deep sea, while the temperature is about 2 to 3°C. Despite these extremes, bacteria survive and adapt. Many are barotolerant: increased pressure does adversely affect them but not as much as it does nontolerant bacteria. Some bacteria in the gut of deep-sea invertebrates are barophilic—they grow more rapidly at high pressures. These gut bacteria may play an important role in nutrient recycling in the deep sea. Barophiles have been found among several bacterial genera (e.g., Photobacterium, Shewanella, Colwellia). Some members of the Archaea are thermobarophiles (e.g., Pyrococcus spp., Methanococcus jannaschii). 

Radiation

Many forms of electromagnetic radiation are very harmful to microorganisms. This is particularly true of ionizing radiation, radiation of very short wavelength or high energy, which can cause atoms to lose electrons or ionize. Two major forms of ionizing radiation are (1) X rays, which are artificially produced, and (2) gamma rays, which are emitted during radioisotope decay.

Low levels of ionizing radiation will produce mutations and may indirectly result in death, whereas higher levels are directly lethal. Although microorganisms are more resistant to ionizing radiation than larger organisms, they will be destroyed by a sufficiently large dose. A variety of changes in cells are due to ionizing radiation; it breaks hydrogen bonds, oxidizes double bonds, destroys ring structures, and polymerizes some molecules. Ionizing radiation can be used to sterilize items.

Some procaryotes (e.g., Deinococcus radiodurans) and bacterial endospores can survive large doses of ionizing radiation.

Extremophiles

The ability of some microorganisms to adapt to extreme and inhospitable environments is exceptional. Procaryotes are present anywhere life can exist. Many habitats in which procaryotes thrive would kill most other organisms. Procaryotes such as Bacillus infernus are  able to live over 1.5 miles below the Earth’s surface, without oxygen and at temperatures above 60°C.

Microorganisms that grow in such harsh conditions are often called extremophiles,

Thus, the various environmental factors have a great effect on the growth and distribution of microorganisms. Microorganisms must be able to respond to variations in nutrient levels, and chemical and physical nature of their surroundings. An understanding of environmental influences aids in the control of microbial growth and the study of the ecological distribution of microorganisms.

BIOFERTILIZERS - Microbial Inoculants

    Agricultural land gets poor after long term cultivation. To supplement the soil nutrient content under conventional farming system, we apply fertilizers. But this in turn pollute the ecosystem. These chemicals enter our food chain sometimes, and can be detrimental to us. In order to make agriculture sustainable, we need a balanced and responsible organic agriculture. Organic farming excludes the use of any chemical. The soil health and biodiversity is built up to sustain the plant growth for longer term. Use of biofertilizers in crop production help build up soil biological properties under organic farming. 

Bio-fertilizers include selective organisms like bacteria, fungi and algae. These are capable of fixing atmospheric nitrogen and solubilization of native and added nutrients like phosphorus, in the soil and make them available for plants. The nutrients fixed by the soil microbes are more effective than outside application.  

Biofertilizer can be defined as a ready-to-use live preparation/formulation of beneficial microorganisms, such as nitrogen fixers, phosphorus solubilizers, sulphur oxidizers or organic matter decomposers which on application, increase the availability of nutrients by their biological activity. When applied to seed, plant surfaces, or soil, they colonize the rhizosphere or the interior of the plant and promotes growth by increasing nutrients uptake.

 Bio-fertilizers are ecofriendly, cost effective and renewable source of plant nutrients. They can play a vital role in maintaining long term soil fertility and sustainability. They enhance the rate of mineralization of insoluble nutrients in the soil through numerous natural process such as nitrogen fixation, phosphorous fixation etc. They build up the soil micro-flora and there by the soil health.

 Advantages

There are many advantages of using the biofertilizers. They form an important association with other soil microbes and help in nutrient supply. Some basic advantages as listed below:

• ·         Fixes atmospheric nitrogen.
• ·         Increase nutrient availability in soil
• ·         Accelerates mineral uptake by plants through solubilization or increased absorption.
• ·         Stimulate plant growth through hormonal or antibiotics action or by decomposing organic waste.
• ·         They are cost-effective, hence, reduced cost of cultivation.
• ·         Improves soil properties and sustaining soil fertility. Lead to soil enrichment in the long term.
• ·         Are compatible with long term sustainability.
• ·         Increases crop yield.
• ·         Provide resistance against drought and soil-borne diseases
• ·         They are eco-friendly and pose no damage to the environment.

 Disadvantages 

As such there is no harmful impact of biofertilizers if it is used properly. Some constraints include,

• ·         Specific to the plants.
• ·      Low acceptability of biofertilizers because they do not produce quick and spectacular responses. Require skill in production and application.
• ·         Difficult to store.

 Some common biofertilizer organisms are classified as follows:

 

Nitrogen fixing	Phosphorous solubilizing	Phosphorous mobilizing	Biofertilizers for Micronutrients	Plant Growth Promoting Rhizobacteria (PGPR)
Free-living: Clostridium, Azotobacter, Nostoc, Anabaena etc.	Bacteria: Bacillus subtilis, Pseudomonas striata, Bacillus circulans etc.	Arbuscular mycorrhiza: Glomus sp., Acaulospora sp, Gigaspora sp & Sclerocystis sp	Silicate and Zinc solubilizers: Bacillus sp	Pseudomonas: Pseudomonas fluorescens
Symbiotic: Rhizobium, Anabaena azollae,  Frankia etc.	Fungi: Aspergillus awamorii, Penicillium sp	Ectomycorrhiza: Laccaria sp., Boletus sp., Amanita sp
Associative Symbiotic: Azospirillum		Ericoid mycorrhizae: Pezizella ericae
		Orchid mycorrhiza: Rhizoctonia solani

  However, biofertilizers are not popular because of many difficulties. Some of them are as follows:

• ·       Inadequate popularity is due to that they cannot show instant and dramatic response like fertilizers. Inadequate awareness about its use and benefits.
• ·         Lack of promotion, extension and insufficient publicity.
• ·         Lack of availability of quality products in time to the farmers in rural areas.

 Nitrogen Fixing Biofertilizers

 The nitrogen fixing bacteria work under two conditions, symbiotically and as free living bacteria (non-symbiotic). The symbiotic bacteria make an association with crop plants through forming nodules in their roots. The free living bacteria do not form any association but live freely and fix atmospheric nitrogen.

Symbiotic Nitrogen fixers

1.     Rhizobium

The maximum utilized among all the biofertilizers, Rhizobium a symbiotic nitogen fixing bacterium growing in association with legumes. About 90% of legumes can become nodulated. In the soil the bacteria are free living and motile, feeding on the remains of dead organisms. Free living rhizobia cannot fix nitrogen and appear as straight rods. The bacteria found in root nodules exists as irregular cells called bacteroids which are often club and Y-shaped.

Several microbial genera are able to form nitrogen-fixing nodules with legumes. These include Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium, and Rhizobium - collectively called the rhizobia. They have host specific associations with legumes. 

Nitrogen fixing bacterium Rhizobium leguminosarum was isolated by Beijerinck (1888).

Ø  Rhizobium meliloti, forms nodules on alfalfa

Ø  Bradyrhizobium japonicum forms a symbiosis with soybean plants

Ø  Sinorhizobium meliloti nitrogen-fixing nodules on roots of white sweet clover

• Symbiotic nitrogen fixation by Rhizobia

Rhizobia live freely in the soil. When they approach the plant root, the plant responds by producing chemicals such as superoxide radicals, hydrogen peroxide, and N₂O. The rhizobia which survive with their antioxidant defenses can continue the infection process.

Only rhizobia and related genera with sufficient antioxidant abilities are able to proceed to the next steps in the infection process.

The plant roots release flavonoid inducer molecules that stimulate rhizobial colonization of the root surfaces. In response to this, rhizobia produce their own signaling compounds called Nod factors. Once Nod factors are produced, the outer (epidermal) cells of the roots gets altered and root hairs become deformed. In some cases, the root hairs will curl to resemble a shepherd’s crook, entrapping bacteria. In these regions, the plant cell wall is locally modified leading to the development of a bacteria-filled, tubelike structure called the infection thread.

Attachment of Rhizobium to root hairs involves specific bacterial proteins called rhicadhesins and host plant lectins that affect the pattern of attachment and nod gene expression.

 The infection thread grows toward the base of the root hair cell, division of these root cells results in the formation of a nodule. Bacteria are released from the infection thread into the nodule and each bacterial cell differentiates into the nitrogen-fixing form called a bacteroid. Bacteroids are terminally differentiated—they can neither divide nor revert back to the non-differentiated state. Further growth and differentiation lead to the development of a structure called a symbiosome.

Root Nodule Formation by Rhizobium.

• The plant root releases flavonoids that stimulate the production of various Nod metabolites by Rhizobium. There are many different Nod factors that control infection specificity.
• Attachment of Rhizobium to root hairs involves specific bacterial proteins called rhicadhesins and host plant lectins.
•  A plant root hair covered with Rhizobium and undergoing curling.
• Initiation of bacterial penetration into the root hair cell and infection thread growth.
• Cell-to-cell spread of Rhizobium through infection threads followed by release of rhizobia and infection of host cells.
• Formation of bacteroids surrounded by plant-derived membranes and differentiation of bacteroids into nitrogen-fixing symbiosomes. The bacteria change morphologically and enlarge around 7 to 10 times in volume. 
• The symbiosome contains the nitrogen-fixing bacteroid inside  a peribacteroid membrane.

The symbiosomes within mature root nodules are the site of nitrogen fixation. Within these nodules, the differentiated bacteriods reduce atmospheric N₂ to ammonium. In return they receive carbon and energy from their host legume. This creates an interdependent relationship.

The nitrogenase enzyme is very sensitive to oxygen and to protect the nitrogenase, a protein called leghemoglobin is present in the nodule. Leghemoglobin binds to oxygen and helps maintain microaerobic conditions within the mature nodule. This protein is similar in structure to myo- and hemoglobins found in animals; however, it has a higher affinity for oxygen.

The genes essential for nodulation (nod, nol, and noe genes) and nitrogen fixation (nif and fix genes) show homology among rhizobia.

The molecular mechanisms by which both the legume host and the rhizobial symbionts establish productive nitrogen-fixing bacteriods within nodules continues to be an intense area of research. A major goal of biotechnology is to introduce nitrogen fixation genes into plants that do not normally form such associations.

Nitrogen fixation by rhizobia is of great importance in agriculture in several ways. The supply of fixed nitrogen enables the growth of host plants in soils that would otherwise be nitrogen limiting. Legumes are the most successful plants maybe due to their symbiotic relationships with nitrogen-fixing bacteria on Earth. Legumes (peas, beans, lentils, soybeans, alfalfa and clover) help to feed the meat-producing animals as well as humans. Crop yields are greatly improved in nodulated plants; legumes can also grow well in poor soils where there is not enough fixed nitrogen to support other types of plants. After harvest legume roots left in the soil decay, releasing organic nitrogen compounds for uptake by the next generation of plants. Farmers take advantage of this natural fertilization by rotating a leguminous crop with a non leguminous one.

Nitrogen fixation by natural means cuts down on the use of artificial fertilizers. This not only saves money but helps to prevent the many problems brought about by excessive use of commercial nitrogen and ammonia fertilizers such as eutrophication of rivers and lakes, generation of acid rain, and overgrowth of agricultural land by non-food crops. 

  2. Frankia

 Frankia is a gram-positive nitrogen-fixing filamentous free-living actinomycete found in soil. It can form a symbiotic association with actinorhizal plants by forming root nodules in some non-leguminous trees such as Casuarina and Alnus. The bacteria can supply the nitrogen requirements of the host plant. As a result, actinorhizal plants colonise and often thrive in soils that are low in plant nutrients. The waste land soil fertility can be improved by growing Casuarina.

Frankia can fix the molecular nitrogen under free living conditions and in symbiotic association with plants. The atmospheric nitrogen is fixed in the root nodules or vesicles, where nitrogen fixing enzyme, nitrogenase, is localized. The vesicles have several layers of hopanoids (a bacterial fatty ester) that create a lower oxygen concentration, which is favourable for the oxygen-sensitive nitrogenase.

Frankia inoculation can be advantageous in arid environments, disturbed sites and areas where native actinorhizal plants are absent. Frankia combination should be selected and customized for each target area and for each target species. Inoculation and nodulation before seedling transplanting improves plant survival and performance. Frankia spores can also be used for Casuarina inoculation.

 

 

Frankia nodules in roots of alder

 Non symbiotic Nitrogen fixer

 1. Azotobacter

Azotobacter is a heterotrophic free living aerobic nitrogen fixing bacteria present in alkaline and neutral soils. Azotobacter was discovered in 1901 by Dutch microbiologist and botanist Martinus Beijerinck. Azotobacter chroococcum, is the first aerobic, free-living nitrogen fixer to be identified. Azotobacter chroococcum is the most commonly occurring species in arable soils of India. Azotobacter agilis, Azotobacter beijerinckii, Azotobacter nigricans, Azotobacter tropicalis, Azotobacter vinelandii are some other common examples.

Apart from its ability to fix atmospheric nitrogen in soils, it can also synthesize growth promoting substances viz., auxins, and gibberellins and also to some extent the vitamins. They also facilitate the mobility of heavy metals in the soil, thus enhancing bioremediation of soil from heavy metals, such as cadmium, mercury and lead. Many strains of Azotobactor also exhibit fungicidal properties against certain species of fungus.

It is used as a bio-fertilizer for all non-leguminous plants especially rice, cotton, maize, sugarcane, pearl millet, vegetable and some plantation crops. etc. Azotobacter cells are abundant in the rhizosphere region. Its population is very low in uncultivated lands. Presence of organic matter in the soil promotes its multiplication and nitrogen fixing capacity. Azotobacter produces slime which aids in soil binding.

Field experiments on Azotobacter indicated that this is suitable when inoculated with seed or seedling of crop plants like onion, brinjal, tomato and cabbage under different ago-climatic conditions. Azotobacter inoculation decreases the requirement for nitrogenous fertilizers by 10 to 20% under normal field conditions.

Associative Symbiotic Nitrogen fixer

 1. Azospirillum

Azospirillum is a dominant soil microbe identified by Beijerinck in 1925. This Nitrogen fixing bacteria can form associative symbiosis on a large variety of plants. It. colonizes the rhizosphere of non-leguminous graminaceous plants and the intercellular spaces of root cortex.

 So far only four species of Azospirillum have been identified. They are A. lipoferum, A. brasilense, A. amazonense, A. iraquense. In Indian soils A. brasilense and A. lipoferum are very common. They reduce nitrate, perform denitrification etc.

 It is a free living or associative-symbiotic bacteria (does not form nodules but makes association by living in the rhizosphere). Azospirillurn also forms a close associative symbiosis with the higher plants. The bacteria live on root surface, sometimes also penetrates into the root tissues but do not produce any visible nodule or out growth on the root tissue. Azospirillum species establish an association with many plants particularly with non-leguminous plants such as cereals, millets, oil seeds, cotton, maize, sorghum, sugarcane etc

 Azospirillum is inoculated through seed, seedling root dip and soil application methods. The Azospirillum inoculation helps better vegetative growth of the plants, saving nitrogenous fertilizers by 25-30%. They fix nitrogen from 10 to 40 kg/ha.

 Blue Green Algae (BGA) /Cyanobacteria as Bio-fertilizers

 Blue-green algae is another important class of biofertilizer. These are small organisms appearing as a single cell or large accumulation of cells(colonies) or strings of cells (trichomes). Blue-green algae are also known as cyanophytes, cyanobacteria and cyanoprokaryotes. Cyanobacteria fix atmospheric nitrogen by forms, i.e., free-living and symbiotic associations with partners such as water fern Azolla, cycads, Gunnera, etc.

Some cyanobacterial members are endowed with the specialized cells known as heterocysts, which are thick-walled modified cells, which are considered site of nitrogen fixation by nitrogenase enzyme. The heterocysts are modified vegetative cells, which because of their thick walls and absence of photosynthesis, act as ideal sites for nitrogen fixation under aerobic conditions. Although the nitrogenase is present in vegetative cells, it remains inactive because of the presence of oxygenic photosynthesis. The enzyme is a complex, catalyzes the conversion of the molecular nitrogen into reduced form like ammonia. The fixed nitrogen may be released in the form of ammonia, polypeptides, free amino acids, vitamins, and auxin-like substances; either by secretion or by microbial degradation after the cell death.

 Nitrogen-fixing ability has not only been shown by heterocystous cyanobacteria but also by several non-heterocystous unicellular and filamentous genera. The species of BGA, known to fix atmospheric nitrogen are grouped as 3 groups. 

(i) Heterocystous –aerobic forms (ii) Aerobic unicellular forms (iii) Non-heterocystous, filamentous, micro aerophilic forms.

Form of Cyanobacteria	Cyanobacterial members
Unicellular	Chroococcidiopsis, Dermocapsa, Gloeocapsa (Gloeothece)∗, Myxosarcina, Pleurocapsa∗, Xenococcus
Filamentous heterocystous	Anabaena∗, Anabaenopsis, Aulosira, Calothrix∗, Camptylonema, Chlorogloea, Fischerella∗, Gloeotrichia, Nodularia, Nostoc∗, , Stigonema, Tolypothrix, Westiella,
Filamentous non-heterocystous	Lyngbya, Myxosarcina, Oscillatoria, Schizothrix, Trichodesmium

 ∗Some strains of these genera live symbiotically with other plants

 Cyanobacteria can contribute to about 20–30 kg N ha−1 as well as the organic matter to the soil, quite significant for the economically weak farmers unable to invest for costly chemical nitrogen fertilizer.

        Many Asian countries like China, Vietnam, India, etc. have been utilizing cyanobacteria in paddy cultivation as the alternative to nitrogen fertilizers. Nitrogen availability to plants is increased due to application of cyanobacteria in agriculture ecosystems, particularly the rice fields. Inoculation of cyanobacteria (in vitro) in wheat crops, could enhance the plant shoot/root length, dry weight, and yield. It has also been suggested that cyanobacteria can improve the bioavailability of phosphorus to the plants by solubilizing and mobilizing the insoluble organic phosphates present in the soil with the help of phosphatase enzymes.

Nostoc

 Nostoc is a filamentous blue green alga usually formed of ball-like gelatinous colonies composed of filaments called trichomes. Along the filament large, spherical or cylindrical, colourless empty cells called heterocyst are found where nitrogen fixation occurs. Terrestrial species are found abundantly in rice fields where the soil is moist, mixed with many small plants like lichens, mosses, etc., on moist rocks, bottom of lakes and springs. Nostoc sp. are found also in paddy fields and stagnant water.

 

 Nostoc

 Gloeocapsa

 Gloeocapsa (Greek: gloia (gelatinous) and Latin: capsa (case) are single or clustered cells enclosed in layers of mucilage. They are found on rocks or moist soils. Some are symbiotic with fungi, forming lichens.

 

Gloeocapsa

 

Anabaena -Azolla system

    Blue-Green alga, Anabaena azollae) forms a symbiotic relationship with Azolla (aquatic fern) and fixes atmospheric nitrogen. Azolla is a free-floating water fern that floats in water and fixes atmospheric nitrogen. Azolla is considered to be a potential bio-fertilizer in terms of nitrogen contribution to rice. Anabaena azollae is associated with the Azolla in the dorsal lobe of each vegetative leaf. Anabaena-Azolla is used in paddy fields where the blue green algae perform photosynthesis as well as fix the atmospheric nitrogen in flooded rice ecosystem. 

Anabaena-Azolla

Azolla is a fast growing water fern and can double its weight within a week. Azolla is rich organic manure also. It mineralizes the soil nitrogen rapidly which is made available to the crop in a very short period. Nitrogen release from Azolla is slow but steady, without leaching losses. It also serves as a protein rich feed to fish and poultry. The blue green algae also synthesize and liberate some growth promoting substances viz., auxin and amino compounds which stimulate the growth of rice plants. Cyanobacteria thus build up natural fertility (C, N) in soil.

 Application of Biofertilizers 

Can be as seed treatment or seed inoculation/seedling root dip.

 Rhizobium: For all legumes, Rhizobium is applied as seed inoculant.

 Azospirillum/Azotobacter: In the transplanted crops, Azospirillum is inoculated through seed, seedling root dip and soil application methods. For direct sown crops, Azospirillum is applied through seed treatment and soil application.

A typical Blue green algal composite consists of Nostoc, Anabaena, Calothrix, Tolypothrix, Plectonema, Aphanotleca, Gleocapsa, Oscillatoria, Cylindrospermum, Aulosira and Scytonema and they are applied in rice fields mainly.

 Conclusion

Biofertilizers are a vital component for the soil fertility management in sustainable organic farming. Once they are established, the soil fertility can be maintained over the years. Almost all the essential plant nutrients can be supplied through biofertilizers to the crops. They help enhance the absorption and make available the nutrient to the plants. These microorganisms may be symbiotic, associated or free living in nature. The use of these inoculants based upon effective quality control system and powerful support machinery.

 References

 Text Book of Microbiology by Michael J. Pelczar

http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.719.2177&rep=rep1&type=pdf

https://www.bio-fit.eu/q8/lo1-why-biofertilizers?start=4

http://eagri.org/eagri50/SSAC222/lec17.pdf