**3. Biotechnology of Diazotrophs**

Microorganisms which are the basis of biological preparations must comply with certain re‐ quirements, as well as have certain properties including virulence, activity and effective‐ ness, specificity, competitiveness, and technological properties (the ability to accumulate titer in the standard and production environments). Among the general requirements for the development of new biological preparations are the following: a high titer of active bac‐ teria cells, the desired length of shelf life, transportability, technological properties (solubili‐ ty, the ability to stay on the seeds, etc.), as well as the economic efficiency of their production.

The efficiency of symbiotic systems, "plant - microorganism" is defined by the virulence and level of activirty of microsymbionts. Virulence of nodule bacteria for example is the ability to penetrate into the soybean root through root hairs and lead to the formation of nodules by complex morphophysiological changes. The first stage of virulence is tumorgenic activity which is the ability to form tumors on the roots. Truly virulent strains are characterized by nodulating activity (nodulation), the ability to form complete nodules. A nodule is a com‐ plex organ of the plant, the main structure of which is made up of: tissue infected with bac‐ teria where molecular nitrogen is fixated; conductive tissue through which photosynthates are delivered and products of nitrogen fixation are taken out; and the meristem, due to which the growth of nodules occurs [1, 5, 18].

At certain stages of the formation of associative rhizocenosis and legume-rhizobial symbio‐ sis, other important properties of bacteria develop such as: nitrogen-fixing activity - the re‐ covery rate of which is N2 in NH3, and symbiotic effectiveness - the ability of plants to develop intensively, using symbiotrophic feeding on nitrogen. Symbiotic efficiency is to a great extent determined by the nitrogen-fixing activity of nodules, especially in conditions where there is a shortage of fixed nitrogen, but the efficiency of the symbiosis also depends on a number of factors not directly linked to nitrogen fixation. An important role in deter‐ mining the productivity of plant-bacterial interactions can be played by the compatibility of the metabolic systems of the partners (for example, ways of transporting nitrogen and car‐ bon), and the absence of active defense reactions in plants in response to the presence or the penetration of microorganisms. Bacteria found in the rhizosphere or nodules can synthesize substances that stimulate (phytohormones, vitamins) or inhibit (rhizobiotoxins) the develop‐ ment of the host plant. It has been found that the effective and ineffective strains of nitro‐ gen-fixing bacteria differ on a number of biochemical parameters. Effective strains most probably have a far richer metabolic fund, and their acidifying- restoring processes occur more actively [1, 3, 6, 18].

Specificity is the ability of bacteria to selectively interact with a particular species or group of plants. Specificity is one of the important systematic characteristics of nodule bacteria and is closely linked to their activity. *Rhizobium* for example is divided into: active, low-active, and non-active. It should be noted that the virulence of the bacteria and the activity may de‐ pend on the certain strain, species, varietal specificity of the plant, soil, climatic conditions and other factors [5, 6, 18].

Different races of nitrogen-fixing bacteria compete with each other. The more virulent strains will more actively colonize or inoculate relative to other strains, the root system of plants specific to them. Researchers have different interpretations of the concept of bacteria competitiveness. Some consider it to be the ability to compete with the strains which sponta‐ neously inoculate the plants; while others consider it to be resistance to the local saprophyte microflora and to displace the local strains [60, 61].

There are several methods for application of microorganisms to plants: a liquid culture, preparations on gel substrates (bacterial exopolysaccharides, silica gel, highly dispersed ma‐ terials) and applications on solid carriers (vermiculite, lignin, perlite, peat) [6, 62]. Obtaining the production-strains of nitrogen-fixing bacteria and the creation of biological preparations based on them - is a long process of research and production (Fig. 1), in which we can distin‐ guish the following stages:

1. Research:

**3. Biotechnology of Diazotrophs**

which the growth of nodules occurs [1, 5, 18].

more actively [1, 3, 6, 18].

and other factors [5, 6, 18].

production.

Relationships

68

Microorganisms which are the basis of biological preparations must comply with certain re‐ quirements, as well as have certain properties including virulence, activity and effective‐ ness, specificity, competitiveness, and technological properties (the ability to accumulate titer in the standard and production environments). Among the general requirements for the development of new biological preparations are the following: a high titer of active bac‐ teria cells, the desired length of shelf life, transportability, technological properties (solubili‐ ty, the ability to stay on the seeds, etc.), as well as the economic efficiency of their

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

The efficiency of symbiotic systems, "plant - microorganism" is defined by the virulence and level of activirty of microsymbionts. Virulence of nodule bacteria for example is the ability to penetrate into the soybean root through root hairs and lead to the formation of nodules by complex morphophysiological changes. The first stage of virulence is tumorgenic activity which is the ability to form tumors on the roots. Truly virulent strains are characterized by nodulating activity (nodulation), the ability to form complete nodules. A nodule is a com‐ plex organ of the plant, the main structure of which is made up of: tissue infected with bac‐ teria where molecular nitrogen is fixated; conductive tissue through which photosynthates are delivered and products of nitrogen fixation are taken out; and the meristem, due to

At certain stages of the formation of associative rhizocenosis and legume-rhizobial symbio‐ sis, other important properties of bacteria develop such as: nitrogen-fixing activity - the re‐ covery rate of which is N2 in NH3, and symbiotic effectiveness - the ability of plants to develop intensively, using symbiotrophic feeding on nitrogen. Symbiotic efficiency is to a great extent determined by the nitrogen-fixing activity of nodules, especially in conditions where there is a shortage of fixed nitrogen, but the efficiency of the symbiosis also depends on a number of factors not directly linked to nitrogen fixation. An important role in deter‐ mining the productivity of plant-bacterial interactions can be played by the compatibility of the metabolic systems of the partners (for example, ways of transporting nitrogen and car‐ bon), and the absence of active defense reactions in plants in response to the presence or the penetration of microorganisms. Bacteria found in the rhizosphere or nodules can synthesize substances that stimulate (phytohormones, vitamins) or inhibit (rhizobiotoxins) the develop‐ ment of the host plant. It has been found that the effective and ineffective strains of nitro‐ gen-fixing bacteria differ on a number of biochemical parameters. Effective strains most probably have a far richer metabolic fund, and their acidifying- restoring processes occur

Specificity is the ability of bacteria to selectively interact with a particular species or group of plants. Specificity is one of the important systematic characteristics of nodule bacteria and is closely linked to their activity. *Rhizobium* for example is divided into: active, low-active, and non-active. It should be noted that the virulence of the bacteria and the activity may de‐ pend on the certain strain, species, varietal specificity of the plant, soil, climatic conditions - reisolation of the nitrogen-fixing microorganism from its natural habitat;






2. The preparatory stage of production:




3. Production process:





4. Storage or incubation of the biological preparations under certain temperature conditions.

5. Quality control (titer of bacterial cells / 1g of the preparative form, the presence of foreign microflora).

6. Treatment of wastewater and gas emissions, recycling waste.

The general scheme of production (see figure 1) includes the above mentioned stages; how‐ ever each case has its peculiarities. This is due to the degree of complexity of each separately organized biotechnological process (laboratory conditions, the use of shakers, industrial rockers, different fermenters), and the technological requirements of cultured organisms and choice of the preparative form of the final product (liquid culture, different carriers). For example, a kit for the fermentation of microorganisms in liquid media may consist of inocu‐ lation and production fermentors, air purification systems for fermentation, a set of connect‐ ing tubes, as well as the compressor and the gas meter.

**Figure 1.** The scheme of creation and production biopreparations, based on nitrogen-fixing bacteria (Sytnikov, 2012).

After establishing a positive impact of the soil microflora on plant productivity, the question arose about the practical application of micro-organisms, in particular those fixing atmos‐ pheric nitrogen. The creation of biological products on the basis of nitrogen-fixing bacteria was dictated by the need to preserve their life and functional activity in a specific prepara‐ tive form (nutrient medium or substrate) aiming to widen its practical application.

The first biological preparation based on the nitrogen-fixing nodule bacteria - Nitragin, was produced in Germany in 1896 [63]. In the Soviet Union the preparation Rizotorfin was creat‐ ed and widely used. It is a peat substrate with nutrient supplements containing highly ac‐ tive and competitive strains of rhizobia for a specific type of legume plants. Bacterial fertilizers for legume plants based on symbiotrophic nitrogen fixers are the most commonly used biological preparation of diazotrophs. For example, in the United States Nitragin and Double-noktin were produced and used on hundreds of thousands of hectares of crops. In other countries the following compounds were used: Nitrosoil and Nitrum in Argentina and Uruguay, Rhizoctonia in New Zealand, Tropical-inoculum, Nodulite and Nitrogerm in Aus‐ tralia, Ariss Agro in India, and Okadin in Egypt [64]. In Ukraine, up to 20% of crop cultures (predominantly soybean) are inoculated by biological preparations [3].

5. Quality control (titer of bacterial cells / 1g of the preparative form, the presence of foreign

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

The general scheme of production (see figure 1) includes the above mentioned stages; how‐ ever each case has its peculiarities. This is due to the degree of complexity of each separately organized biotechnological process (laboratory conditions, the use of shakers, industrial rockers, different fermenters), and the technological requirements of cultured organisms and choice of the preparative form of the final product (liquid culture, different carriers). For example, a kit for the fermentation of microorganisms in liquid media may consist of inocu‐ lation and production fermentors, air purification systems for fermentation, a set of connect‐

**Figure 1.** The scheme of creation and production biopreparations, based on nitrogen-fixing bacteria (Sytnikov, 2012).

After establishing a positive impact of the soil microflora on plant productivity, the question arose about the practical application of micro-organisms, in particular those fixing atmos‐ pheric nitrogen. The creation of biological products on the basis of nitrogen-fixing bacteria was dictated by the need to preserve their life and functional activity in a specific prepara‐

The first biological preparation based on the nitrogen-fixing nodule bacteria - Nitragin, was produced in Germany in 1896 [63]. In the Soviet Union the preparation Rizotorfin was creat‐ ed and widely used. It is a peat substrate with nutrient supplements containing highly ac‐ tive and competitive strains of rhizobia for a specific type of legume plants. Bacterial fertilizers for legume plants based on symbiotrophic nitrogen fixers are the most commonly

tive form (nutrient medium or substrate) aiming to widen its practical application.

6. Treatment of wastewater and gas emissions, recycling waste.

ing tubes, as well as the compressor and the gas meter.

microflora).

Relationships

70

To inoculate the seeds the most commonly used preparations are based on nodule bacteria from the families of *Rhizobium, Bradyrhizobium, Sinorhizobium* for the legume plants on the basis of vermiculite or perlite under the general name of Rizobofit. Symbiotrophic nodule bacteria provide nitrogen fixation of up to 350 kg / ha in lucerne during the second year of vegetation [65], as well as up to 280 kg / ha for soybean [66] and 70 kg / ha in pea [9]. As a result of many years of research it has been found that the inoculation of plants with high efficiency strains of rhizobia increases the productivity of legumes by an average of 10-25% [67]. On the basis of associative nitrogen-fixing bacteria, there has been developed a technol‐ ogy of production of a whole line of biological preparations. The list of biotechnological products and microbial preparations for crop production, has increased considerably over the past decade and includes products that are based on free-living, associative, symbiotro‐ phic nitrogen-fixing bacteria, and phosphate-mobilizing bacteria, as well as products of bi‐ nary action obtained from a combination of various microorganisms [6, 62, 64].
