RU2125607C1 - Continuous method of producing citric acid and its salts - Google Patents

Abstract

FIELD: biotechnology, microbiological and food industries. SUBSTANCE: citric acid is produced by fermentation of fungus Aspergillus niger VKPM F-718 on molasses nutrient medium for two stages. On the first stage molasses is used at pH 4.0-5.0 and fermentation is carried out at 32-34 C. Then in 30-48 h process involves continuous feeding molasses solution and nutrient salts at dilution rate 0.025-0.03 h^-1. On the second stage method involves utilization of microorganism suspension overflowing from the first stage and a process is carried out at 30-32 C and at pH 2.0-3.0. Microorganism suspension is removed at the same rate. Mother liquors forming at citric acid crystallization are used for additional producing citric acid salts. For preparing the sowing material fungus mycelium A. niger F-718 is grown additionally for 16-20 h at 32-35 C and at pH 4-5 on molasses nutrient medium. EFFECT: intensified process, improved utilization of mother liquors at crystallization stage. 3 cl, 3 tbl

Description

 The invention relates to the microbiological industry, in particular to a continuous method for producing citric acid and its salts, and can be used in the food industry, medicine, pharmacology.

 A known method for the production of citric acid by cultivating Aspergillus niger fungus on molasses culture medium with the continuous introduction of a nutrient solution and removal of the final product (A.S. USSR N 510509, C 12 P 7/48, 1976). In this method, a small increase in the average daily productivity of citric acid is provided in comparison with the periodic management of the process.

 A known method of producing citric acid and its salts from fermentation solutions obtained by culturing yeast on hydrocarbon substrates (A. S. USSR N 998504, C 12 P 7/48, 1983). The disadvantage of this method is the conduct of the fermentation process in a batch mode, as well as the use of basic fermentation solutions for the production of citric acid salts (sodium or potassium citrate).

 Closest to what is proposed in its technical essence is a continuous method for producing citric acid, which includes stepwise fermentation of Aspergillus niger fungus on molasses nutrient medium containing carbon sources, nitrogen, mineral salts, by supplying the required amount of nutrient medium in a continuous stream, followed by isolation of citric acid from the culture liquids (A.S. USSR N 432186, C 12 P 7/48, 1974). Since the known method provides for the use of a battery of series-connected fermenters, it is characterized by low economic efficiency.

 The objective of the invention is to intensify and increase the economic efficiency of the method for producing citric acid.

The problem is solved in that in the known continuous method for producing citric acid, which includes stepwise fermentation of Aspergillus niger fungus on molasses nutrient medium containing carbon sources, nitrogen, mineral salts, followed by isolation of the product from the culture fluid, the fermentation process is carried out in two stages, and in the first step with molasses pH value of 4.0 - 5.0, the fermentation is conducted at a temperature of 32 - 34 ^o C, then after 30 - 48 hours, after reducing the intensity of the mycelium biomass formation start indirect discontinuous supply of molasses to the fermentation medium and the nutrient salt solution at a rate of dilution medium 0.025 - 0.03 h ^-1, in the second stage is used as overflow from the first stage slurry fermentation microorganisms, the process is conducted at a temperature of 30 - 32 ^o C and the medium pH 2.0 - 3.0, the suspension of microorganisms from the second stage of fermentation is carried out with a medium dilution rate of 0.025-0.03 h ^-1 , and in the process of product isolation, mother liquors formed during crystallization are used to additionally produce citric acid salts.

 As a seed for fermentation, a highly productive strain of fungus A.niger F-718 is used.

To obtain seed, the mycelium of the fungus A.niger F-718 is grown for 16-24 hours at a temperature of 32 - 35 ^o C on molasses nutrient medium with a pH value of 4-5, containing reducing substances (PB) - 2%, sucrose - 0 , 5%, ethyl alcohol - 0.1%, KH ₂ PO ₄ - 0.02%, MgSO ₄ - 0.02%, ZnSO ₄ • 7H ₂ O - 10 mg / l, CoSO ₄ • 7H ₂ O - 1 , 0 mg / l, Na ₂ MoO ₄ - 0.5 mg / l.

 Growing mycelium on a nutrient medium containing molasses, sucrose, ethyl alcohol and trace elements (Zn, Co, Mo), improves the quality of the seed.

 The mother liquors obtained during the crystallization of citric acid are used to prepare citric acid salts. Moreover, pure salts of food or medical value are obtained from the first or second mother liquors, and more contaminated mother liquors (second or third) are used to obtain technical salts of citric acid, for example sodium citrate, which is widely used in the manufacture of detergents as a substitute for polyphosphates.

 The technical result of the invention, which consists in intensifying and increasing the economic efficiency of production, is achieved through the use of a highly productive strain of the fungus A.niger F-718, conducting the process in a continuous mode in two stages, subject to the stated fermentation conditions and the use of uterine formed during crystallization of citric acid , to obtain citric acid salts.

The fermentation of the fungus in the first stage at a pH of 4.0 - 5.0 and a temperature of 32 - 34 ^o C helps to optimize the growth of the fungus and accelerate the spontaneous decrease in pH of the medium to the values optimal for the biosynthesis of citric acid (2.0 - 3.0).

The greatest accumulation of citric acid in the medium is achieved when the process is conducted in a continuous mode with a dilution rate of the medium 0.025 - 0.03 h ^-1 . Maintaining the pH of the medium in the second stage of fermentation in the range of 2.0 - 3.0 and a temperature of 30 - 31 ^o C helps to increase the acid-forming ability of the fungus A.niger F-718.

 The invention is illustrated by the following examples.

 Example 1

Sterile molasses medium with a pH of 5.0, containing PB - 2%, sucrose - 0.5%, ethyl alcohol - 0.1%, KH ₂ PO ₄ - 0.02%, MgSO ₄ - 0.02%, ZnSO ₄ • 7H ₂ O - 10 mg / l, CoSO ₄ • 7H ₂ O - 0.1 mg / l, Na ₂ MoO ₄ - 0.5 mg / l, seeded with fungus mycelium A. niger F-718. Growing is carried out in flasks on a rocking chair at a temperature of 35 ^o C for 24 hours. The grown mycelium is used as a seed for fermentation.

Fermentation of the fungus A.niger F-718 is carried out continuously in a molasses culture medium in two stages. Fermentation in the first stage is carried out at an initial pH of 4.0 and a temperature of 32 ^o C. After 30 hours from the start of fermentation after reducing the intensity of mycelium formation, the process is switched to continuous mode by continuously feeding molasses solution (with a PB content of 8%) into the fermentation medium and nutrient salts with a dilution rate of the medium (D) = 0.025 h ^-1 . The flowing suspension of microorganisms at the same rate enters the second stage (in the second fermenter), where the fermentation is carried out at a temperature of 30 ^o C. The pH in the second stage is reduced to 2.0.

 After the second stage of fermentation, the fermentation solution enters the collector, then the culture fluid is separated from the biomass of the fungus in any way, for example by centrifugation or filtration, citric acid is also isolated in the usual way, for example by precipitation in the form of calcium citrate.

 The concentration of citric acid in the medium is 73 g / l. The yield of citric acid from PB is 91.25%.

 The mother liquors formed during crystallization of citric acid are neutralized to pH 7.0 with sodium hydroxide, evaporated and crystallized. The crystals are separated, washed and dried.

The output of sodium trisodium citrate (in terms of 100% Na ₃ C ₆ H ₅ O ₇ • 5.5 H ₂ O) is 95% of the salt content in the solution.

 Example 2

Fermentation seed is obtained by inoculating A. niger F-718 fungus mycelium in a sterile fermentation medium containing 2% PB, 0.5% sugar, 0.1% ethyl alcohol and salts according to Example 1. Growing is carried out in flasks on a rocking chair at a temperature of 32 ^o C and a pH of 4.0 for 16 hours. The grown mycelium is used as a seed for fermentation.

Fermentation of the fungus A.niger F-718 is carried out continuously in two stages. The fermentation in the first stage is carried out at an initial pH of 5.0 and a temperature of 34 ^o C. After 48 hours from the start of fermentation, the process is transferred to a continuous mode according to example 1. The supply of nutrients and removal of the suspension of microorganisms is carried out with a dilution rate of medium (D) - 0 , 03 h ^-1 . The flowing suspension of microorganisms at the same rate enters the second stage, where the fermentation is carried out at a temperature of 31 ^o C. The pH in the second stage is reduced to 3.0.

 The concentration of citric acid in the medium is 75 g / l. The yield of citric acid from PB is 93.7%.

 The trisubstituted sodium citrate is obtained from the mother liquors of Example 1. The yield of citric acid salt is 93%.

 Example 3

The seed for fermentation is prepared according to Example 2. As a producer of citric acid, the A.niger 228 fungus strain is used. Fermentation is carried out continuously in molasses culture medium in two stages. After 36 hours from the start of fermentation, a continuous supply of molasses solution (with a PB content of 8%) and nutrient salts at a dilution rate of medium (D) = 0.03 h ^{-1 is started} . The flowing suspension of microorganisms at the same rate enters the second stage, where fermentation is carried out at a pH of 2.0.

 After separation of the biomass of the fungus, the culture fluid is used to produce citric acid.

 The concentration of citric acid in the culture fluid is 60 g / l. The yield of citric acid from PB is 75%.

 The mother liquors formed after separation of the citric acid crystals are neutralized with potassium hydroxide, evaporated and crystallized, the crystals are washed and dried.

 The output of potassium citric acid trisubstituted is 92% of the salt content in the solution.

 Example 4

 Seed for fermentation is obtained according to example 1. As a producer of citric acid, a strain of fungus A.niger 945 is used. Fermentation of the fungus and transfer of the process to continuous operation is carried out as in example 1. The pH in the second stage of fermentation is maintained at 2.5. The separation of the biomass of the fungus from the culture fluid and the selection of citric acid from the solution are carried out by conventional methods.

 The concentration of citric acid in the culture fluid is 66 g / l. The yield of citric acid from PB is 82.5%.

 Example 5

Obtaining seed is carried out analogously to example 1. As a producer of citric acid using a strain of the fungus A.niger F-718. Fermentation is carried out in a continuous mode on molasses nutrient medium in one stage at an initial pH of 5.0 and a temperature of 34 ^o C. After 30 hours from the start of fermentation, the molasses (with a PB content of 8%) and nutrient salts are continuously fed into the fermenter with a dilution rate of the medium (D) = 0.025 h ^-1 . The temperature of the fermentation is reduced to 30 ^o C, the pH of the medium to 2.0 and maintained at this level throughout the process.

 The suspension of the fungus from the fermenter is filtered, the culture fluid after separation is used to isolate citric acid.

The concentration of citric acid in the culture fluid is 56 g / l. The yield of citric acid from PB is 70%
Thus, studies on the selection of active strains - producers of citric acid (examples 1-4), as well as on the optimization of the modes of its preparation show that the fungus strain A.niger F-718 has the greatest acid-forming ability.

 The maximum yield of citric acid is achieved by fermentation of the fungus in a continuous mode in two stages.

 Examples 6-10.

 Obtaining seed and fermentation of the fungus A.niger F-718 is carried out analogously to example 1 at optimal pH, temperature, dilution rate of the medium, etc., but the transfer to continuous operation is carried out after the specified in table. 1 time from the start of fermentation.

 Studies have shown that the growth of the fungus culture A.niger F-718 slows down after 30-36 hours from the start of fermentation, which indicates the advisability of transferring the process to continuous mode after the specified time. With an increase in the time of cultivation of the culture without supplying food components for more than 40 hours, the growth rate of the culture decreases due to the lack of nutrient components in the medium.

 The results presented in table. 1 show that the greatest yield of citric acid from the concentration of the substrate occurs when the process is transferred to a non-continuous mode after 30 - 48 hours from the start of fermentation.

 Examples 11 to 15.

Repeat the process described in example 1, but the rate of dilution of the medium is changed in the range from 0.02 to 0.04 h ^-1 (table. 2).

The results presented in table. 2 show that with an increase in the rate of dilution of the medium (D), the yield of citric acid decreases. The most effective process in continuous mode at D = 0.025 - 0.03 h ^-1 .

 Examples 16 to 20.

 Repeat the process described in example 2, but the pH of the medium in the first and second stages of fermentation is set equal to the values indicated in the table. 3.

 A.niger fungus develops in a wide pH range - from 2.0 to 9. The initial pH value has a significant effect on the biosynthesis of citric acid. Conidia and mycelium germinate better at pH 6–7, however, they swell strongly, burst a certain amount, resulting in an increased risk of extraneous microflora. In addition, at the indicated pH values, predominant biosynthesis of oxalic and gluconic acids is observed.

 As can be seen from the table. 3, the most appropriate cultivation of the fungus at an initial pH of 4.0 to 5.0. At these pH values, as a result of rapid self-acidification of the medium, the fungus predominantly synthesizes citric acid.

 Maintaining the pH of the medium in the second stage of fermentation in the range of 2.0 - 3.0 is also important, because at these pH values, the fungus does not actually grow, and its acid-forming ability increases sharply.

 The proposed method may contain other advantages arising from the presented description and obvious to specialists in this field.

Claims (3)

1. A continuous method for producing citric acid, including stepwise fermentation of Aspergillus niger fungus on molasses nutrient medium containing carbon sources, nitrogen, mineral salts, followed by isolation of the product from the culture fluid, characterized in that the fermentation process is carried out in two stages, and the first stages use molasses with a pH of 4.0 - 5.0, the fermentation is carried out at a temperature of 32 - 34 ^o C, then after 30 - 48 hours after the intensity of the formation of biomass of mycelium is reduced, continuous feeding into the fermentation begins the ionic medium of a solution of molasses and nutrient salts with a dilution rate of 0.025 - 0.03 h ^-1 ; in the second stage, a suspension of microorganisms flowing from the first stage of fermentation is used; the process is carried out at a temperature of 30 - 32 ^o C and a pH of 2.0 - 3, 0, the suspension of microorganisms from the second stage of fermentation is carried out with a dilution rate of 0.025-0.03 h ^-1 , and in the process of isolating the product, the mother liquors formed during crystallization of citric acid are used to produce citric acid salts.  2. The method according to claim 1, characterized in that use the strain of the fungus Aspergillus niger VKPM F-718. 3. The method according to claims 1 and 2, characterized in that before fermentation the seed is grown for 16 to 20 hours at a temperature of 32 - 35 ^o C on molasses nutrient medium with a pH of 4-5 containing 2% reducing substances, sucrose 0.5%, ethyl alcohol 0.1%, KH ₂ PO ₄ 0.02%, MgSO ₄ 0.02%, ZnSO ₄ • 7H ₂ O 10 mg / l, CoSO ₄ • 7H ₂ O 0.1 mg / l, Na ₂ MoO ₄ 0.5 mg / l.

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