Microbiological Synthesis

in microorganisms, the synthesis of structural elements or metabolic products by the cells’ enzyme systems.

In microbiological synthesis, as in any organic synthesis, complex substances are formed from simpler compounds. Microbiological synthesis should be distinguished from fermentation, which also results in various metabolic products (alcohols, organic acids) but which is predominantly an effect of the decomposition of organic matter. Many of the products formed by microbiological synthesis are physiologically active and of economic value.

Microbiological synthesis embraces a broad range of processes, including the accumulation of a microbial mass for use as a protein-vitamin supplement to fodders; as a source of proteins, lipids, enzymes, toxins, vitamins, and antibiotics; as an agent for animal and plant parasite control; and as a carrier of enzymic activity in the transformation of organic compounds. Also among the processes classified under microbiological synthesis is the production of metabolites that accumulate outside the cells (for example, enzymes, toxins, antibiotics, amino acids, vitamins, and nucleotides).

Microbiological synthesis occurs within the cell upon the activation of low-molecular-weight components (for example, coenzyme A) and the participation of nucleotide phosphates (most often, adenyl derivatives). Many metabolites are then eliminated from the cell. A characteristic feature of microorganisms is their capacity for supersynthesis—that is, the formation of certain metabolic products (many amino acids, nucleotides, and vitamins) in excess of the cells’ requirements. For example, in Micrococcus glutamicus cultures, glutamic acid may accumulate in amounts greater than 10 mg per milliliter (ml) of medium, and in cultures of the fungi Eremothecium ashbyii and Ashbya gossipii, vitamin B2 may accumulate to 1–2 mg per ml, instead of the usual hundredths or even thousandths of a mg.

The capacity for the supersynthesis of particular compounds is species characteristic; indeed, such microorganisms are, as a rule, used as producers in the microbiological synthesis of their characteristic metabolites. The cultures used either have been selected from natural sources or are specially bred mutant strains for which the supersynthesis is a result of mutagen-influenced metabolic disturbances. The use of mutants makes it possible to increase substantially the yield of a number of products. For example, cultures have been developed for a high level of supersynthesis of lysine, inosinic acid, and certain vitamins. Mutants have made it possible to increase penicillin biosynthesis 100–150 times. Mutant strains are also used in the manufacture of other antibiotics.

A number of products are obtained in the process of microbiological synthesis from the most diverse compounds of carbon and nitrogen, thanks to the great variety of microbial enzyme systems. Thus, in synthesizing proteins, nucleic acids, and other metabolites, cells may use different inorganic sources of nitrogen, depending on the culture. For carbon compounds, they may use various carbohydrates, organic acids (including acetic), or liquid, solid, or gaseous hydrocarbons. Certain species that are capable of chemosynthesis or photosynthesis can assimilate carbon dioxide as a carbon source. The selection of appropriate cultures makes it possible to obtain the desired substances from inexpensive and readily available raw materials by means of microbiological synthesis, which for some cases, such as antibiotics, is in fact the only economically efficient means of production.

Some of the products of microbiological synthesis, such as baker’s yeast, have long been used by man, but the method has attained broad industrial application only since the 1940’s and 1950’s. Progress in the field has been due primarily to the discovery of penicillin, which spurred detailed research on the physiologically active metabolic products of microorganisms. The organization of penicillin production on an industrial scale led to the solution of many microbiological, technological, and engineering problems. Together with the broadening of yeast production as a protein-vitamin fodder supplement, this served as the basis for the development of industrial microbiological synthesis. The development of a special apparatus, the fermenting chamber, which is equipped with devices for mixing the medium and supplying sterile air, has made it possible to conduct industrial biosynthesis without contamination by foreign microorganisms.

Technologically, modern microbiological synthesis consists of a series of operations. The most important of these stages are preparing the necessary cultures of the producer-microorganisms, preparing the nutrient medium, growing the inoculation material, culturing the producer under the prescribed conditions (the synthesis stage, often called fermentation), filtering and separating out the biomass, isolating and purifying the desired product (when necessary), and drying.

The method used in the processes of isolation and purification, which often occupy an important place in other technological operations, is determined by the chemical nature of the substance obtained and may involve extractive and chromatographic methods, crystallization, or sedimentation. The most advanced method is continuous operation, in which there is an uninterrupted supply of nutrient medium and uninterrupted removal of the products (for example, a microbial biomass, or nutritive yeast). However, the continuous method is far from developed for all processes of microbiological synthesis, and most metabolites (amino acids, antibiotics, vitamins) are obtained by the periodic method—that is, the products are removed at the end of the process.

In some cases (for example, in the production of a number of enzymes), the producers are grown not in fermenting chambers (the deep method) but on the surface of the nutrient medium (the surface method).

The microbiological industry, which can already supply a large assortment of compounds of many classes, was developed in the USSR for the manufacture of various products. Work in microbiological synthesis is being conducted in almost all of the industrially developed countries. In many of them the products of microbiological synthesis are an important component of the economy (in Japan, enzymes and amino acids, and in Hungary, medicinal preparations).

Antibiotics, historically among the first products of microbiological synthesis, are widely produced for medicine and agriculture. Most antibiotics accumulate outside the cells of the producer microorganisms (principally actinomycetes and certain, mostly mutant, fungi and bacteria). The antibiotic preparations that are used predominantly for medicine are distinguished by their high degree of purity. Animal fodder usually includes a concentrate of the medium, in which the producer microorganism has been grown. In addition, fodder sometimes includes the biomass, which contains considerable amounts of additional producer metabolites, including vitamins, amino acids, and nucleotides. Some antibiotics (phytobacteriomycin, trichothecin, polymyxin) are used to protect plants from phytopathogens.

Of the vitamins, provitamins, and coenzymes, microbiological synthesis is basically used in the manufacture of vitamin B₁₂. To some degree, it is also involved in the production of 82 and its coenzyme flavin adenine dinucleotide (FAD); of carotenoids; and of ergosterol. The manufacture of various other compounds of this type (nicotinamide coenzymes) is also being developed.

Vitamin B₁₂ is obtained almost exclusively by microbiological synthesis. Its principal producers are propionic-acid bacteria, actinomycetes, and a complex of methane-forming bacteria that make use of by-products of the fermentation industry (alcohol and acetone-butyl residues) and that are used principally for fodder concentrate (dried medium with the producer biomass). Many microorganisms are capable of the supersynthesis of vitamin B2, actively discharging the product into the medium; however, the most active cultures—mainly the fungi Eremothecium ashbyii and Ashbya gossipii—are used as industrial producers. E. ashbyii produces both the free vitamin and FAD. Although beta-carotene, a provitamin of vitamin A, can be obtained by several methods (extraction from carrots, chemical synthesis), it is formed, along with other carotenoids, in the cells of many microorganisms and imparts a characteristic color (from yellow to red) to the biomass.

Of the greatest practical interest is a culture of Blakeslea trispora, the most active synthesizer, which is used principally as a producer in industrial biosynthesis. Ergosterol, a provitamin of vitamin D₂, is contained in the cells of many yeasts. Baker’s yeast is the principal source for its industrial manufacture. However, yeast cultures with a substantially higher level of ergosterol accumulation have already been developed. In addition, during yeast development a complex of vitamins and coenzymes is synthesized and is accumulated in the yeast biomass, which is attracting increasing attention as a source of these compounds.

Enzymes that can be synthesized by microorganisms, as well as enzyme preparations based on them, have become very important in the national economy, particularly in the food industry. Many mycelial fungi and certain actinomycetes and bacteria serve as producers of enzymes (proteases, amylases, phosphatases, cellulases, pectinases, glucose oxidase, lipases, catalase). Either the microbial mass or a microbe-free filtrate is treated, depending on the localization of the enzyme. The production of pure enzyme preparations is associated with considerable technological difficulties. Such preparations are usually very expensive; for this reason, compound preparations (containing, for example, protease and lipase or protease and amylase) are used in industry.

The amino acid deficiency observed in the human and animal diet in many countries has led to the industrial production of these substances by several methods, including microbiological synthesis. In this case, the main advantage of microbiological synthesis over chemical synthesis is that the products can be obtained directly in the form of their natural isomers (L-forms). The most important amino acids obtained by microbiological synthesis are lysine and glutamic acid. Bacterial cultures of the genera Brevibacterium and Micrococcus usually serve as the producers. Most auxotrophic mutants that are supersynthesizers of a particular amino acid, which is released into the medium, are used industrially.

The microbiological synthesis of nucleotides (in particular, of inosinic, guanylic, and other acids) has been widely developed in Japan, where the substances are used chiefly as supplements to specific products in oriental cuisine. In the future, nucleotides will probably gain importance as regulators of many of the enzymic and hormonal processes in the animal body. Nucleotide accumulation occurs mainly in the culture fluid, that is, outside the cells of the producers. Biochemical mutants that show pronounced supersynthesis of the given compound are used in the microbiological synthesis of nucleotides, as well as in the synthesis of amino acids.

The microbial biomass has special significance as a source of protein. The production of such a biomass on an inexpensive raw material is regarded as a means of eliminating the growing protein deficit in human and animal nutrition. Industrial methods of microbiological synthesis of nutritive yeasts, used in the form of a dry biomass as a protein and vitamin source in animal husbandry, have undergone the most intensive development. Nutritive yeasts contain a substantial amount of protein (up to 50–55 percent), which, in turn, contains essential amino acids, such as lysine, tryptophan, and methionine. In addition, the yeasts are rich in vitamins and a number of microelements. Inexpensive raw carbohydrates (the hydrolysates of by-products of the wood products industry; inedible plant materials, such as sunflower-seed husks and corn cobs; caustic sulfites; and various types of residues) have been used for growing these yeasts. The large-scale industrial production of yeasts on hydrocarbons (n- alkanes, gas oil, and various petroleum fractions) is being organized. Large supplies of the raw material make it possible to set up huge-capacity microbial biomass production. The possibility of using bacteria to obtain a protein-vitamin biomass is under study. Many bacteria grow well on hydrocarbons (especially gases, such as methane) and other carbon sources, such as methanol and acetic acid. Hydrocarbons and their derivatives are also of interest as raw materials for the microbiological synthesis of certain physiologically active compounds (amino acids, vitamins, and nucleotides, for example).

Also among the products of microbiological synthesis are certain plant protection agents, such as bacterial entomopathogenic preparations (the Soviet products Entobakterin, Insektin, and Dendrobatsillin), which destroy harmful insects and prevent their mass proliferation. The same effect is produced by “protein crystals”—carriers of toxicity located in microbial cells. Many bacterial fertilizers are also obtained by microbiological synthesis.

A special case of microbiological synthesis is the microbiological transformation of organic compounds. Because of the high activity of specific enzyme systems, microorganisms are capable of effecting a number of reactions on the molecules of organic compounds without changing their basic structure. Reactions on molecules of steroid compounds have received the most study. Under strictly defined conditions, dehydrogenation, deacetylation, and hydroxylation can occur, altering the physiological activity of the initial steroid compound. Owing to the selection of appropriate microorganisms—carriers of specific enzyme systems—the use of the microbiological transformation method is becoming increasingly widespread.