MICROBIOLOGY - M.H. Serhiichuk - 2008

Class="center">Chapter 12. HUMAN USE OF MICROORGANISMS

Agriculture

In agriculture, microorganisms are used to enhance soil fertility, control plant pests, silage fodder, and produce microbial protein.

The application of biological products in agriculture offers the following advantages:

- biological preparations are based on living microorganisms and their metabolites;

- the constituent microorganisms are indigenous to the environment;

- the action of biological products relies on natural processes (Nitrogen Fixation, converting phosphorus into plant-available forms, and natural antagonism among microorganisms);

- the substances comprising biological products readily degrade in the environment;

- biological products are harmless to humans and animals.

BACTERIAL PREparations for soil fertility enhancement.

Following the discovery of ROOT nodule Bacteria, the idea emerged to utilize them for improving crop yields by enhancing MOLECULAR NITROGEN FIXATION. This concept was first implemented by F. Nobbe and L. Hiltner in Germany in 1896.

Preparations based on root nodule bacteria include nitragin, N-germ, nitrazon, rhizotorphin, among others.

Nitragin is a preparation consisting of living Cells of a specific species of root nodule bacteria. Its primary function is the Fixation of Atmospheric nitrogen.

The application of root nodule bacterial preparations is particularly essential when new legume crops are introduced into a given area and the soil microflora lacks the appropriate bacteria. Legume crops develop intensively only in the presence of their specific strains of root nodule bacteria in sufficient quantities, which is not always observed under natural conditions. The cultivation of certain legumes is restricted to specific regions, meaning that soils may not contain all necessary strains of nodule bacteria. In Ukraine, soils are sparsely colonized by soybean root nodule bacteria. In the transitional zones between the forest-steppe and Polissya, as well as in Polissya, the Carpathian foothills, and Transcarpathia, There is a scarcity of bacteria specialized for alfalfa, vetch, sainfoin, and peas. This is because these crops were previously cultivated only sporadically or not grown at all in these areas.

How can the positive effect of inoculating legume crops with a *Rhizobium* culture be explained in cases where soils have long been cultivated and already contain nodule bacteria within their microflora? First, cross-infection may occur under natural conditions, meaning that legumes are infected by bacteria from closely related plant groups. In such instances, nodules may form, but they function suboptimally, or ineffective nodules are produced. Artificial inoculation introduces an active strain of root nodule bacteria—applied directly to the seeds—into The Root System of the legume. Second, nodule bacteria present in soil previously used for growing non-legume crops exist merely as ordinary Saprophytes. Frequently, the conditions in such soils prove unfavorable for nodule bacteria, leading to a substantial decline in their population; consequently, natural infection fails to establish an effective Symbiosis.

Sufficient soil colonization by indigenous (native) root nodule bacteria does not always satisfy agricultural demands, as these populations may lack sufficient activity. Microbial breeders therefore propose The Use of more active strains.

Industry produces two forms of nitragin: soil-based and dry.

Soil-based nitragin is a culture of root nodule bacteria grown in sterile soil. One gram of such a preparation must contain at least 3·10⁶ cells. The application rate for seed Treatment is 500 g/ha. To produce this preparation, soil (or peat) rich in organic matter is mixed with up to 30% sand and chalk to adjust the pH to 6.6-7.0. The soil is sterilized, packaged into polyethylene bags, inoculated with the appropriate strain of root nodule bacteria, and subsequently cultivated at a Temperature of 26-28 ⁰C for 4-6 days (or 10-11 days for slow-growing strains).

Dry nitragin is a powder composed of living cells of root nodule bacteria. One gram of the preparation contains 8-10·10⁹ cells. This facilitates transportation, although the preparation must be mixed with a filler prior to use.

Nodule bacterial preparations are species-specific and intended for treating seeds of a particular legume crop, as indicated on the packaging (e.g., "Soybean", "Vetch", "Sainfoin", etc.). The return on investment for using these preparations on forage grasses is 10-15 units of profit per unit of cost, and 20-30 or more for grain legumes.

Preparations based on free-living nitrogen fixers have also been developed, including azotobacterin, diazobacterin, and diazophyt.

Azotobacterin is a preparation of living *Azotobacter chroococcum* cells. It is used to increase crop yields of vegetables in both greenhouse and open-field cultivation by supplying growth-promoting substances secreted by the microorganisms (Vitamins, Hormones, Amino Acids), improving plant mineral and Water Nutrition, and enhancing Disease resistance.

The production technology is identical to that of nitragin. One gram of the soil-based preparation must contain at least 50 million cells. For seed treatment, 3-6 kg of azotobacterin is applied per hectare. One gram of the dry preparation must contain at least 10⁹ *A. chroococcum* cells.

Azotobacterin is additionally valuable because *A. chroococcum* synthesizes a range of BIOLOGICALLY ACTIVE SUBSTANCES: nicotinic and pantothenic acids, pyridoxine, biotin, heteroauxin, gibberellin, and Other Compounds that stimulate seed germination and accelerate plant growth.

The bulk of fixed nitrogen is removed with the harvest, while a certain amount remains in the soil. Nodule bacteria ensure the yield formation of most legumes without depleting soil nitrogen reserves. Only after cultivating peas and soybeans might a slight decrease in soil nitrogen content be observed. Due to this property, legumes serve as excellent predecessors in crop rotations, making it possible to reduce nitrogen fertilizer rates for subsequent crops or eliminate them altogether.

The cultures forming The basis of both nitragin and azotobacterin are asporogenic bacteria. Since the Number of viable cells decreases during storage, such preparations are manufactured shortly before use.

Preparations have been developed to mobilize sparingly soluble forms of phosphorus in the soil, including phosphobacterin, albobacterin, and polymyxobacterin.

Phosphobacterin is a preparation containing spores of Bacillus megaterium var. phosphaticum, which transform complex organophosphorus compounds and poorly accessible mineral phosphates into forms of phosphorus readily available to plants. Industrial production yields soil and dry formulations of phosphobacterin. One gram of the dry preparation must contain 8·10⁹ spores. The hectare dosage for treating cereal seeds is 5 g of the preparation, whereas for corn, potatoes, and vegetable crops, it is 15 g. The shelf life of phosphobacterin is practically unlimited. The culture comprising the preparation synthesizes A number of biologically active substances: thiamine, pyridoxine, biotin, pantothenic and nicotinic acids, and vitamin B₁₂.

Preparations with complex action, containing associations of various cultures, are also utilized.

Bactofill is a preparation designed to enrich the soil with available sources of nitrogen and phosphorus, based on the bacteria Azospirillum brasilens, Azotobacter vinelandii, Bacillus megaterium, Bacilluspolymxa, Pseudomonas fluorescens, and Streptomyces albus.

Microbial preparations against plant pests. Bacteria, Fungi, and Viruses are used as producers for the manufacture of such preparations. The main requirements for microbial preparations include high virulence toward pests, rapid action, safety for humans, flora, and fauna, ease of application, and prolonged shelf life.

Entomopathogenic preparations. Currently, preparations against 160 species of insects are produced. Bacterial entomopathogenic preparations, such as enterobacterin, insectin, alestin, toxobacterin, and bitoxibacillin, are created on the basis of the bacterium Bacillus thuringiensis. This Gram-positive spore-forming bacterium produces the following insect-toxic products:

- phospholipase, an enzyme that breaks down Phospholipids in insect Tissues;

- β-exotoxin, which disrupts RNA Synthesis in insect cells;

- δ-endotoxin, a parasporal crystalline endotoxin formed within the Bacillus thuringiensis Cell during sporulation. The toxin is proteinaceous in nature and shaped like an 8-sided crystal which, upon entering the insect gut, breaks down into toxic components and induces paralysis.

Bacillus thuringiensis acts as an antagonist to 150 species of insects.

AGAT-25K is a biofungicide, immunostimulant, and plant growth regulator created on the basis of soil bacteria Pseudomonas aureofaciens and their metabolic products.

Rhizoplan is an ecologically clean biological preparation, harmless to humans and animals, whose active element consists of living cells of rhizosphere bacteria capable of effectively suppressing The Development of phytopathogenic bacteria and fungi.

Khetomik is a microbial preparation designed to protect winter wheat, barley, sunflower, potatoes, lupine, soybeans, tomatoes, and sugar beets from root rot pathogens.

Entomopathogenic fungi have not found as widespread use as bacterial preparations. This is because they exhibit activity only under conditions of high and stable humidity. The best-known preparations include boverin, entomophthorin, vertalec, and mykotal.

Boverin is a preparation for controlling the Colorado potato beetle and other insects, consisting of conidia of the fungus Beauveria bassiana. Upon entering the insect's body, the conidia germinate, causing its death.

Vertalec is a preparation based on Aschersonia alearoides intended for controlling aphids and whiteflies.

To control rodents, microbial preparations containing Salmonella enteritidis are used—for example, bactorondecid, which can be produced in two forms: grain-based (on wheat, barley, or oat grains) and bone-meal-based (on bone shavings).

The natural Immunity of rodents to salmonellae varies among species. It is most pronounced in larger species (rats, susliks, etc.), whereas smaller species possess lower immunity. Acquired immunity is unstable and short-lived. These bacteria exhibit strict Specificity, meaning selective pathogenicity. They are safe for humans, domestic animals, and wildlife.

The preparation BPS-44, based on Bacillus subtilis, is used to increase the productivity of farm animals and poultry.

Silage making is a complex microbiological process that takes place with the participation of lactic acid bacteria inhabiting plants. Plant juices serve primarily as the nutrient medium for bacterial development.

Plant mass is placed into a silo Structure and packed tightly. This leads to the almost complete die-off of aerobic bacteria. Microorganisms capable of developing under conditions of insufficient oxygen actively proliferate: enterobacteria, Clostridium, Bacillus, Streptococcus, Leuconostoc, Pediococcus, and Lactobacillus.

During the first days following the placement of the plant mass (up to 8 days), cocci—especially streptococci—dominate among the lactic acid bacteria. They compete with Gram-negative bacteria during the initial stage of ensiling. The total count of microorganisms can reach 10⁹/g.

Over the period from 8 to 15 days, the population of pediococci, leuconostocs, and homofermentative and heterofermentative lactobacilli increases. The leading role at this time belongs to Lactobacillus brevis and Lactobacillus plantarum. Lactic acid predominantly accumulates, and the pH drops to 4.0, which suppresses the development of putrefactive and other undesirable microorganisms.

During the 15–60 day period, the microbial count reaches 10^б/g. The ensiling process is accelerated by adding lactic acid bacterial starters and nutrients (molasses, malt). Lyophilized cultures of Lactobacillus plantarum, Lactobacillus casei, and Lactococcus lactis are used, which are diluted with water prior to application to the plant mass.

PRODUCTION OF SINGLE-cell protein. Microbial biomass is an excellent Supplement for the diet of livestock, fish, and poultry. It is rich in Essential Amino Acids, vitamins, Trace Elements, and Unsaturated Fatty acids.

The following are used for the industrial PRODUCTION OF MICROBIAL protein:

- Yeasts such as Pichia guilliermondii and Candida tropicalis, cultivated on various raw Materials containing CARBOHYDRATES and Hydrocarbons (n-alkanes);

- bacteria such as Methylophilus methylotrophus, grown on methanol;

- Algae.

Raw materials for Yeast biomass production can include:

- agricultural wastes: rice husks, corn, grain, potatoes;

- food industry wastes: molasses, molasses-alcohol vinasse, whey;

- hydrolyzates of plant materials: wood hydrolyzates, Hydrolysis-alcohol vinasse, sulfite liquors;

- n-alkanes.

Following the cultivation of yeast on carbohydrate-containing media, they are separated from the culture liquid. The cells are inactivated by treatment with dry steam for 45 minutes. The biomass is then irradiated with ultraviolet light, which converts ergosterol into vitamin D₂. Dry biomass is obtained using spray drying.

The accumulation of yeast biomass on n-alkanes has several distinctive features. The yeast is cultured on highly purified, straight-chain saturated hydrocarbons of varying lengths. The hydrocarbon concentration should be 2%; higher concentrations lead to incomplete assimilation and accumulation within the cells. Centrifugate from the culture liquid is used instead of water. Mineral components are added to the medium: phosphates, KCl, MgSO₄, (NH₄)₂SO₄, NH₃, and trace elements. Cultivation is carried out at a temperature of 50-60 ⁰С with enhanced aeration. The commercial name for feed yeast produced on hydrocarbons is paprin.