General Biotechnology: Lecture Course, Part II - Blinov, V. A. 2004

Large-Scale Microbiological Production of Amino Acids and Organic Acids

♦ Amino Acid Production.

♦ Production of organic acids: lactic, citric, and acetic.

♦ Other organic acids.

Amino acid production. Special attention is paid to this issue worldwide, as amino acid deficiency disrupts METABOLISM/35.html">Protein Biosynthesis, impairs GROWTH AND DEVELOPMENT, and can cause various diseases. At the same time, incorporating just a fraction of a percent of a deficient amino acid into an animal's diet more than doubles the Nutritional Value of the protein. Currently, global amino acid production exceeds 500,050 tons per year, including 200,000 tons of glutamic acid, 160,000 tons of Methionine, 50,000 tons of Lysine, and so on. Amino Acids are produced both microbiologically (~60%) and chemically. For instance, chemical synthesis yields D,L-methionine from acrolein, DL-Tryptophan from indole and nitroacetic ester, sodium L-glutamate from acrylonitrile, L-lysine from cyclohexanone, etc. However, chemical synthesis always produces racemates—mixtures of D- and L-amino acids—which require complex and costly purification to separate. D-Amino acids act as ballast since they are not utilized by the human or animal body, increase raw material consumption coefficients per ton of product, and some are toxic. The exceptions are Glycine, which has no optically active isomers, and methionine, whose DL-forms are utilized equally by the Organism.

Since the 1950s, the ability of auxotrophic mutants of Brevibacterium, Micrococcus, Corynebacterium, and other genera to hyperproduce extracellular amino acids has been known. This served as the basis for establishing large-scale industrial production of L-amino acids. For example, genetically engineered producer strains based on E. coli allow the accumulation of up to 30 g/L of L-Threonine, up to 27 g/L of L-Proline, and up to 22.4 g/L of L-phenylalanine in the medium within 40 hours of Fermentation.

Two Methods are known for obtaining amino acids: single-stage and two-stage processes. In the first method, a mutant polyauxotrophic amino-acid-producing strain is cultivated on an optimal medium for biosynthesis. The target product accumulates in the culture liquid and is subsequently isolated from it. In the two-stage method, During the first stage, the microbial amino acid producer is cultivated in a liquid nutrient medium where the BIOSYNTHESIS OF AMINO acid precursors (intermediates) and the Enzymes catalyzing The formation of the target product takes place. In the second stage, the target product (amino acid) is synthesized with the aid of these enzymes.

Glutamic acid is produced microbiologically in the largest quantities using the Bacteria mentioned above. The hyperproduction of glutamic acid from glucose by these bacteria is based on two biochemical principles: a deficiency of α-ketoglutarate dehydrogenase and the blocking of biotin biosynthesis. Under conditions of biotin limitation in the medium (1–5 µg/L), as well as upon The addition of Antibiotics and detergents, the normal synthesis of membrane Phospholipids is disrupted, increasing its permeability to glutamate. If auxotrophic mutants with low α-ketoglutarate dehydrogenase activity are used (an enzyme that channels the glutamic acid precursor into The Tricarboxylic Acid Cycle), ketoglutarate accumulates and fails to undergo further transformations in the TCA cycle. Bacterial glutamate synthesis yields up to 50% of the product from sugar and can accumulate up to 200 g/L of glutamate in the fermentation medium. METHODS FOR PRODUCING glutamate on ethanol media (up to 60 g/L) or acetate media (up to 98 g/L) are also known.

Let us consider the Technology for Obtaining L-glutamic acid. This is a non-essential amino acid, but many BIOLOGICALLY ACTIVE SUBSTANCES necessary for normal human life activity are synthesized based on it. Monosodium glutamate is widely used as a food additive to improve taste characteristics and to preserve these properties in canned and frozen foods during long-term storage. In medicine, glutamic acid is used to treat conditions associated with Liver and Kidney intoxication.

Single-stage process. Inoculum, from test tubes up to the seeding bioreactor, is grown under strictly aseptic conditions for 24 hours. For industrial strains of Corynebacterium glutamicum, the nutrient medium composition for seed preparation is (in %): molasses – 8; corn steep liquor – 0.3; ammonium chloride – 0.5; dibasic potassium phosphate – 0.05; magnesium sulfate – 0.03; Water – up to 100%; medium pH – 7.0–7.2. At the biosynthesis stage, instead of corn steep liquor and ammonium sulfate, up to 2% urea is introduced into the nutrient medium, the molasses content is increased to 20%, and up to 1% chalk and 0.1% synthetic antifoam agent are additionally added.

Biomass accumulation up to 6–8 g dry biomass/L is carried out under aerobic conditions, first in 2 m³ inoculators and then in 5 m³ seed bioreactors. Biosynthesis is performed under strictly aseptic conditions in 50 m³ fermenters with a working volume coefficient of 0.7 for 48–52 hours under intensive aeration of 80–85 mg O₂/(L·min). This corresponds to an airflow rate of 1 volume of air per 1 volume of medium per minute. The cultivation Temperature must be maintained constant at 28–30 °C across all stages. By the end of the process, the culture liquid contains up to 45 g/L of glutamic acid. Its yield relative to the consumed sugars is 45–50%.

To obtain glutamic acid for use as food additives or in pharmaceutical formulations, the technological flowchart is supplemented with the following steps:

♦ pretreatment of the culture liquid. To achieve this, quicklime or milk of lime is added, followed by the precipitation of Calcium Ions with phosphoric acid. The precipitate facilitates better Separation of producer Cells and other ballast impurities;

♦ separation of the precipitate. Centrifugation or pressure filtration is employed;

♦ clarification of the filtrate—Treatment with activated charcoal or ion-exchange sorption on an anion exchanger;

♦ concentration of the clarified solution. Vacuum evaporation is performed at 40–60 °C, resulting in the removal of 50–80% of water from the initial solution;

♦ crystallization of glutamic acid at its isoelectric point: the concentrate is acidified with Hydrochloric acid to pH 3.2 (the isoelectric point of glutamic acid) and the solution is cooled to 4–15 °C. A single treatment ensures the crystallization of 77% of the glutamic acid, while repeated treatment increases the yield to 87%. Subsequent recrystallization can raise crystal purity to 99.6%, meeting pharmacopeial requirements;

♦ separation of glutamic acid crystals from the mother liquor. This is achieved using centrifugation, decantation, and returning the mother liquor to the vacuum evaporation stage. The crystals are washed with demineralized water and sent for drying;

♦ drying is conducted in a vacuum or in a stream of heated air at 60–70 °C;

♦ production of monosodium glutamate. To do this, wet crystals of non-recrystallized glutamic acid are treated with sodium hydroxide, followed by subsequent Processing steps in accordance with technical and regulatory documentation.

In accordance with MRTU 18/210-68 requirements, food-grade monosodium glutamate must have the following composition (in %): main substance – not less than 94, sodium chloride – not more than 5, moisture – not more than 1, total nitrogen – not less than 7.02.

Two-stage process. Two variants are used for this purpose:

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In this process, producers of α-ketoglutaric acid include Pseudomonas, Escherichia, Kluyvera citrophila, and Candida. Enzyme producers can be various microorganisms, such as E. coli. Aspartic acid or Alanine serves as the amino group donor.

The technology for producing L-lysine and feed supplements based on it is fundamentally similar. In the Human and Animal body, lysine determines the Biological value of digestible protein, promotes the secretion of digestive enzymes and The transport of calcium into cells, and improves nitrogen balance. Virtually all industrially produced lysine worldwide via microbiological synthesis is used to enrich the diets of farm animals and poultry. It is utilized in the form of liquid lysine concentrate (LLC), lysine feed concentrate (LFC), and highly concentrated lysine feed preparations. Crystalline, highly purified lysine preparations consist of L-lysine monohydrochloride with a main substance content of 97–98%, a moisture content of 0.5%, an ash content of 0.3%, and a melting point of 210 °C.

The amino acid production process is concluded by packaging (in polyethylene bags), labeling, and warehousing of the finished product.

The production of amino acids using immobilized enzymes and cells is economically viable. For instance, the raw Materials for manufacturing L-aspartic acid are fumaric acid (a product of microbial synthesis) and ammonia. Immobilized bacterial cells with aspartase activity catalyze the addition of ammonia across the double bond of fumaric acid. When producer cells are immobilized in a gel, their half-inactivation time is 4 months. The biotechnology of fumaric acid biotransformation can be outlined as follows:

♦ Cell cultivation via submerged fermentation and harvesting by centrifugation;

♦ immobilization of the biocatalyst cells in a gel forming granules 2-3 mm in size;

♦ biotransformation of ammonium fumarate in a packed-bed Column Reactor under continuous flow to yield L-aspartic acid;

♦ crystallization, centrifugation, and crystal washing.

Phenylalanine is produced microbiologically either using a Tyrosine- and methionine-deficient mutant of Brevibacterium lactofermentum or an auxotrophic mutant of E. coli, which can be cultivated in a glucose-phosphate medium that is less complex and costly than the first option. Phenylalanine serves as the precursor for the dietary sweetener aspartame, which is 200 times sweeter than sucrose.

The production of organic acids includes lactic, citric, and acetic acids. Many of these industrial-grade acids are advantageously produced chemically. Organic acids find wide application in the food, chemical, pharmaceutical, and light industries, as well as in household products. For example, the annual production volume of citric acid reaches 400,000 t, utilizing molasses and n-alkanes as raw materials with a yield of 85-140 %, driven by Aspergillus niger and Candida lipolytica as producers. For lactic acid, these parameters are: 30,000 t, 90 % glucose, Lactobacillus delbrueckii; for acetic acid (10 %): 10 million m³, 90-98 % ethanol, Acetobacter aceti; and for propionic acid—unknown, 60 % glucose, Propionibacterium shermanii.

Microbiological processes for organic acid production can be divided into two groups: anaerobic (lactic, propionic) and aerobic (acetic, citric, itaconic, gluconic). All organic acids are intermediate or End products of Carbohydrate Catabolism. Aerobic production of organic acids is carried out using both submerged and surface fermentation methods, whereas anaerobic production utilizes submerged fermentation.

Production of lactic acid. Lactic acid bacteria belong to 4 genera: Lactobacillus, Leuconostoc, Streptococcus, and Pediococcus. The genus Lactobacillus includes three subgenera: Thermobacterium, Streptobacterium, and Betabacterium. The first group does not grow at 15 °C, but tolerates temperatures above 50 °C. Streptobacteria are not thermophilic. Betabacteria produce D,L-lactic acid from glucose. Thermostreptobacteria, streptococci, and pediococci are homofermentative, meaning they produce predominantly lactic acid from hexoses; betabacteria and leuconostocs are heterofermentative. Alongside lactic acid, the latter produce acetic acid, carbon dioxide, and ethanol. Lactic acid bacteria can utilize maltose, lactose, glucose, saccharified starch, and other substrates. Their medium must also contain B-group Vitamins, amino acids, Purines, Pyrimidines, organic acids, etc. Industrial production most frequently employs thermophilic homofermentative species, such as L. delbrueckii strain L-3, which yields 95-98 % lactic acid based on consumed sucrose.

The technological process for obtaining L(+)-lactic acid is as follows. A molasses medium containing 5-20 % sugar, malt sprout extract, Yeast extract, vitamins, and ammonium phosphate is inoculated with L. delbrueckii. Fermentation proceeds at 49-50 °C with an initial pH of 6.3-6.5. As lactic acid accumulates, the medium is periodically neutralized with chalk. The entire fermentation cycle takes 5-10 days; the resulting culture liquid contains 11-14 % calcium lactate and 0.1-1.5 % sucrose. Bacterial cells and chalk are removed by filtration (waste), and the filtrate is evaporated to a concentration of ~ 30 %, cooled to 25 °C, and sent for crystallization, which lasts 1.5-2 days. Calcium lactate crystals are treated with sulfuric acid at 60-70 °C, precipitating gypsum. Potassium ferricyanide is added to the supernatant at 65 °C to remove iron ions, followed by sodium sulfate to eliminate heavy metals. Coloring matter is removed using activated charcoal. Subsequently, the lactic acid solution is vacuum-evaporated to 50 % or 80 %. This partially purified lactic acid is used for technical purposes. Higher-purity lactic acid can be obtained by distilling its methyl esters or through countercurrent extraction with isopropyl ether in packed columns.

Lactate is widely used as an acidulant in the manufacture of jams, jellies, confectionery, liqueurs, extracts, and in vegetable canning. Lactic acid is applied to adjust the pH of beer wort, in the leather, textile, and pharmaceutical industries, for manufacturing Solvents and plasticizers, varnishes, drying oils, detergents, and for treating natural fabrics. However, it should be noted that lactic acid is a strong corrosive agent.

Homo- and Heterofermentative lactic acid bacteria have long been used in baking. Their associations with Yeasts, which are beneficial for developing aroma, flavor, porosity, texture, and freshness, are known as starters (sourdoughs). Lactic acid fermentation underpins fodder ensiling and the pickling of vegetables (cabbage, cucumbers), fruits, and berries (olives, apples). Lactobacilli are the basis for preparing Fermented milk products, cottage cheese, and cheeses. Finally, lactic acid bacteria are components of prophylactic and therapeutic medications (bifidumbacterin, bifidocol, colibacterin, lactobacterin). Lactobacilli are antagonistic to putrefactive, acetic acid, and butyric acid bacteria, as well as enterobacteria, but not to yeasts.

Modern methods include producing lactic acid using Streptococcus thermophilus in a fluidized-bed bioreactor. In this bed, activated carbon beads coated with a biofilm move continuously. In the lower section, the carbon adsorbs the substrate, while in the upper section, it adsorbs lactic acid. The medium contains glucose, yeast extract, sodium acetate, dibasic ammonium citrate, dibasic potassium phosphate, and magnesium and manganese sulfates. The productivity of the system is 12 g/(L·h) of lactic acid.

Production of citric acid. Previously, citric acid was extracted from citrus plants. Currently, surface and submerged cultivation methods using microbial producers have been developed. Modern production employs selected strains of A. niger, specifically strain P-3, which yields 98-99 % citric acid relative to consumed sucrose. This strain exhibits high osmotolerance (up to 12 % sugar in the nutrient medium). The primary raw material for citric acid production is molasses, which consistently has a high iron content. Iron is precipitated using potassium hexacyanoferrate(II) - K₄[Fe(CN)₆]. Fungal spores (conidia) are produced in a separate department in three stages. In The First stage, A. niger is grown on Agar slant medium in test tubes; in the second and third stages, it is propagated on solid or liquid media in Erlenmeyer flasks or aluminum trays measuring 8.5-12 dm² with side heights of 7 to 20 cm. Each stage lasts 2-4 days at a temperature of 32 °C. During the formation and maturation of conidia, the colorless mycelium turns black. Conidia are harvested by vacuum pump aspiration, dried in a thermal cabinet at 28-30 °C, mixed with sterile activated charcoal (1:2), packaged in sterile vials, and stored for 1.5-2 years. Up to 4-5 g of dry conidia can be obtained from 10 dm² of nutrient medium in trays.

Surface liquid-phase fermentation. Citric acid production is carried out in fermentation chambers. Trays are placed on racks, 8-10 units high. Each tray is equipped with a drainage fitting at the bottom. These chambers are outfitted with supply and exhaust ventilation that ensures a uniform flow of sterile air at a specified temperature and humidity (3-4 m³/m² of mycelium·h). The temperature in the chambers is 34-36 °C, and the depth of the liquid molasses nutrient layer is 6-12 cm.

Maximum heat generation is reached by the 5th day (500-550 kJ/m²·h), with an initial sugar concentration averaging 12 %. During the first 24 hours, the pH drops from 6.8-7.0 to 4.5, and by days 8-9 down to 3.0. Under these conditions, peak acid production occurs on days 5-6 and then stabilizes at 50-60 g/m²·h. Typically, 6-7 days after fermentation begins—when the sugar concentration drops to 3-4 %—a sterile molasses solution without nutrient salts is added, equal to 30-35 % of the initial volume. This technique extends fermentation to 12 days, increasing The amount of processed medium for the target product by 30-35 %.

The harvested culture liquid contains a mixture of citric, gluconic, and oxalic acids along with residual sugar in a ratio of 40-50:3:1:7, meaning The Citric Acid content ranges from 80 to 90 %. To isolate it, milk of lime [Ca(OH)₂] or chalk (CaCO₃) is added to the culture liquid heated to 100 °C, adjusting the pH to 6.8-7.0. Tricalcium citrate, which is less soluble in hot water than in cold water, precipitates out along with calcium oxalate. The precipitate is filtered off, washed with hot water, and hydrolyzed with sulfuric acid, leaving free citric acid in solution. This solution is purified, vacuum-evaporated, and crystallized. The acid crystals are dried and packaged.

Solid-state fermentation. This method is relatively simple. Fermentation of the A. niger strain is carried out on moistened rice or wheat bran placed in trays. The conditions are identical to those used for agar or liquid nutrient media. After cultivation is complete, the bran is extracted with water, into which the acids pass. Calcium citrate and pure citric acid are then isolated following the scheme described above.

The submerged method of citric acid production is economically viable when the plant capacity exceeds 2,500 t of acid per year. It utilizes specialized cultures of A. niger—specifically strain No. 288/9 in Russia. First, conidia are grown. Then, the inoculum is propagated in an inoculator and seed tank on a medium containing 3-4 % sugar. After 1-1.5 days, the inoculum is transferred from the seed tank to the main fermenter, and the process is run for 5-7-10 days, with three successive additions of a molasses solution (25-28 % by sugar content) to bring the final sugar concentration to 12-15 %. Upon completion of fermentation—indicated by a drop in acid production—the fungal mycelium is filtered off, and the culture liquid is processed According to the standard scheme.

Currently, citric acid production has been established via biosynthesis using yeasts cultivated on paraffins and lower alcohols (ethanol). The product yield ranges from 80-140 %.

Citric acid enhances pancreatic activity, stimulates appetite, and promotes nutrient assimilation. It is used in cooking as well as in the production of soft drinks, marmalade, wafers, and pastilles. It is included in the recipes of certain types of sausages and cheese. It is applied in winemaking, vegetable oil refining, and condensed milk production. Furthermore, it preserves the natural taste and aroma of meat and fish during long-term frozen storage.

It is used in the formulation of shampoos and detergents. The latter holds significant environmental importance because citric acid readily undergoes microbial degradation during wastewater treatment.

The mycelium of the citric acid producer is used to isolate the enzyme pectinase and flavins, or it is dried and supplied as feed for livestock and poultry.

Production of acetic acid. Vinegar has been known since 7,000 BC. Acetic acid fermentation is caused by Acetobacter oxidans, A. aceti, A. xylinum, etc. However, for acetic acid fermentation to take place, the substrate sugar must first be converted into ethyl alcohol; in other words, Alcoholic Fermentation precedes acetic acid fermentation. Alcoholic fermentation is best carried out by selected strains of wine yeast (Saccharomyces ellipsoideus). In addition to ethanol, they produce metabolic byproducts that enhance the flavor and aroma of vinegar. Vinegar is graded according to The Nature of the fermented substrate, such as apple, grape, pear, and other varieties. The aroma and flavor of vinegar are determined by esters (such as ethyl acetate), higher alcohols, and organic acids.

Typically, acetic acid bacteria are immobilized (adsorbed) on wood shavings, activated charcoal, coke, etc. By passing an ethanol solution through such generators, a 10-15 % acetic acid solution is obtained. In practice, 90 L of acetic acid are produced from 100 L of ethanol.

More than 100,000 tons of acetic acid are produced worldwide every year. Half of this amount is synthesized chemically as technical-grade acetic acid, which is used to manufacture acetone, acetylene, synthetic Dyes, Pharmaceuticals (such as aspirin, antipyrine, and phenacetin), flavoring agents (coumarin and vanillin), and as a substrate for microbiological biotransformation. Acetic acid is particularly widely used in the food industry. Acetic acid producers of the genus Acetobacter develop On the surface of the medium to form a mucous pellicle consisting of Cellulose (90%) and bacterial cells. This pellicle is harvested, dried, and processed to produce medical-grade biofilms. When used to cover burn wounds, such biofilms promote healing within 7-8 days.

Other organic acids. Glucose serves as the carbon source for propionic acid bacteria. Propionic acid bacteria are Gram-positive, spore-free, non-motile rods, all belonging to the family Propionibacteriaceae. P. freudenreichii and P. acidipropionici are utilized for propionic acid production, whereas P. freudenreichii and P. acnes are used to obtain vitamin B₁₂.

Acid biosynthesis is carried out in simple media containing, for example (in %): carbohydrate — 1-2, ammonium sulfate — 0.3, potassium hydrophosphate — 0.2, cobalt chloride — 0.0001, biotin — 0.00001, pantothenate — 0.1, and thiamine — 0.01. The biosynthesis of propionic acid using cells immobilized in a gel matrix, such as PAAG, is becoming increasingly widespread.

Often, propionate and acetate are not separated as the end products of fermentation, since both acids possess preservative properties. Separated cells are used to obtain superoxide dismutase (SOD), catalase, peroxidase, and vitamin B₁₂. The dried powdered extract is used in the food industry as an antioxidant and vitamin Supplement.

Gluconic acid is produced by strains of Aspergillus niger cultivated in fermenters under intensive aeration and agitation, maintaining a constant temperature (30 °C) and a medium pH of 6.0-7.0. The hardware setup for the industrial production of gluconic acid is similar to that of submerged citric acid production.

The fungal mycelium is used to extract glucose oxidase, which finds application in the food and pharmaceutical industries. The selected A. niger strain produces almost exclusively gluconic acid; therefore, to recover it, the culture liquid is filtered from the mycelium, evaporated, and dried. Gluconic acid and its salts are widely used in practice. For instance, calcium gluconate accelerates Blood clotting, sodium gluconate is used in the manufacture of detergents, and gluconic acid is applied in photography, lithography, dye production, and metal cleaning, among other uses.

Itaconic acid is produced by Aspergillus itaconicus and A. terreus. The technological process for producing itaconic acid is similar to that of citric acid — the producer undergoes fermentation via either surface or submerged methods. Itaconic acid is used in the Chemical synthesis of high-quality resins, Pitron-type fibers, detergents, pharmaceuticals, dyes, and other Organic compounds.

Malic acid is used in organic synthesis, such as in the synthesis of uracil. It is produced either chemically from maleic acid or via microbiological synthesis. Chemical synthesis yields a racemic mixture of D,L-malic acid, whereas the L-isomer is typically required. The latter is obtained from fumaric acid using immobilized fumarase, which catalyzes the Hydration reaction to yield L-malic acid. This process does not require the isolated enzyme, but simply cells containing fumarase. Japanese companies utilize carrageenan gel, a seaweed polysaccharide, as a cell carrier. Granules of the immobilized cells are packed into a column through which a fumaric acid solution is passed. An L-malic acid solution is collected at the column outlet. The inactivation period for such a column is 160 days.