RU2062788C1 - Method of continuous exopolysaccharide preparing - Google Patents

Abstract

FIELD: microbiological industry. SUBSTANCE: method involves growing producing strain under aeration on nutrient medium using fermenter cascade. In the first fermenter strain is grown for period including time for maximal cell growth rate attainment in this fermenter and for 2-4 hr after this index attainment; then obtained biomass is fed to the second fermenter which is filled preliminary with nutrient medium where biomass is occurred for period including time for maximal cell growth rate attainment and for 2-4 hr after this index attainment. Content of the second fermenter is fed equally to the third and fourth fermenters which are filled preliminary with nutrient medium; in the third fermenter strain is grown for period including time for maximal cell growth rate attainment and for 2-4 hr after this index attainment. Then biomass from the third fermenter is fed to the second and fifth fermenters at amount that is necessary for ready product preparing in working volume of sown fermenters; from biomass remained in the third fermenter and fed to the fourth fermenter the end product is obtained after that process is repeated from the first fermenter. EFFECT: improved method of exopolysaccharide preparing. 1 dwg, 2 tbl

Description

 The invention relates to the microbiological industry and for the production of biopolymers, for example, exopolysaccharides.

 Known methods for producing exopolysaccharides using various strains: Acinetobacter sp. VKPM B-3243 (auth. St. N 1579059), Bacillus polymyxa VKPM B-3015 (auth. St. N 1698293), Candida tropicalis yeast consortium CMPM U-507 and bacterial strain Acinetobacter species CMPM B-3243 (auth. N 1311256).

Strains growing on a rich organic medium under aeration conditions at 28-30 ^o C for 72-96 hours produce a polysaccharide, turning the culture fluid into a gel.

The cultivation of a strain of Acinetobacter sp. on a liquid mineral medium of the following composition: wt. KH ₂ PO ₄ 0.55; NaHPO ₄ 1.0; NH ₄ Cl 0.06; MgSO ₄ 0.019; CaCl ₂ 0,00075; FeSO ₄ 0.0006, water the rest.

 As a source of carbon nutrition use sucrose at a concentration of 4% or glucose at a concentration of 2% or ethanol in an amount of 1 vol.

The seed culture of B. polymyxa (10 vol.) Contribute to a nutrient medium containing, about. hydrolyzed corn starch 3; BVK 0.5; KH ₂ PO ₄ 0.05; K ₂ HPO ₄ • 3 H ₂ O 0.05; MgO ₄ • 7 H ₂ O 0.04; CaCl ₂ 0.02.

 Periodic cultivation of exopolysaccharide strains is carried out in devices of the ANKum type with a volume of 10 l, a working volume of 4 l.

 A common disadvantage of the known methods for producing exopolysaccharides is the low yield of the target product, the frequency of the process, the duration of the process.

 Closest to the proposed invention is a continuous method of culturing microorganisms, for example, strains for producing exopolysaccharides, including growing its producing strain under conditions of aeration in a nutrient medium using a cascade of reactors (ed. St. N 206491).

 The use of reactors cascade of strains in the process of cultivation allows to intensify the process of growing strains.

 The disadvantage of the prototype is to obtain exopolysaccharides with variable properties, the inability to obtain the optimal combination of useful properties, the duration of the process.

 To obtain exopolysaccharides with stable properties, it is proposed to grow the strain in the first fermenter for a time consisting of the time to reach the maximum cell growth rate in this fermenter and 2-4 hours after reaching this indicator, after which the resulting biomass enters the second fermenter, previously filled with nutrient medium , which is for a time consisting of the time to reach the maximum cell growth rate in the second fermenter and 2-4 hours after reaching this about indicator. Then the contents of the second fermenter are distributed equally to the third and fourth fermenters, pre-filled with nutrient medium, while in the third fermenter the strain is grown for a time consisting of the time to reach the maximum cell growth rate in this fermenter and 2-4 hours after reaching this indicator, after which, from the third fermentor, the biomass enters the second and fifth fermenters in the amount necessary to obtain the finished product in the working volume of the seeded fermenters, and from the rest the resulting biomass in the third fermenter and fed into the fourth fermenter receive the final product, after which the process is repeated from the first fermenter.

 The method is as follows.

 1. Preparation of seed.

Exopolysaccharide was obtained using two types of strains. The liquid nutrient medium for growing culture in flasks on a rocking chair has the following composition:
For Acinetobacter sp strain
KH ₂ PO ₄ 0.55
NaHPO ₄ 1.0
NH ₄ Cl 0.06
MgSO ₄ 0.019
CaCl ₂ 0,00075
FeSO ₄ 0.0006
water the rest.

For Bacillus polymyxa strain
hydrolyzed corn starch 3
BVK 0.5
KH ₂ PO ₄ 0.05
K ₂ HPO ₄ • 3 H ₂ O 0.05
MgO ₄ • 7 H ₂ O 0.04
CaCl ₂ 0.02
water the rest.

The sterilization mode of the nutrient medium for flasks 118 ^o C for 30 minutes To obtain seed in storage flasks, the liquid nutrient medium is inoculated with a culture suspension from one jamb. The suspension is prepared by pouring on each jamb 10 ml of sterile physical. solution. The culture in the storage flasks is carried out on a rocking chair at 28 ^o C for 19-22 hours. The stirrer speed is 220 rpm.

2. The cultivation of seed is carried out in an inoculator with a capacity of 0.1 m ³ . 35 l of water are loaded into the apparatus with the mixer operating, then the remaining components of the nutrient medium. The planted seed in the amount of 1.2-1.5 l under aseptic conditions is introduced into the device through the dispenser.

 3. Maintaining the technological process of growing seed in five fermenters.

The first fermenter (volume 16 l) is loaded with a nutrient medium as described above. Then the finished seed is transferred from the inoculator to the 1st fermenter. The process is carried out at 28 ^o C with a working stirrer, air flow 1-0.8 V air / 1 V environment. To control the sterility and development of the producer, samples are taken for sterility, microscopy and pH at least 1 time in 4 hours. In addition, the growth curve of the culture in various phases is recorded. The cell concentration (g / l) is determined by the optical density of the solution (see graph).

 And the beginning of the exponential, the end of the logarithmic phase. Uniform small cells of rounded shape, located by irregular groups, less often singly or in short chains.

 In the exponential growth phase, in the groups of young cells, a sign of active division, the maximum growth rate (the inflection point of the curve).

 From the beginning of the stationary phase of growth, small, homogeneous vegetative cells.

 D steady stationary phase of growth, the population is heterogeneous.

 VV 'the moment of reseeding of the culture from one fermenter to another.

 The 2nd fermenter (volume 100 l) is loaded with a nutrient medium. Then from the first fermenter comes the seed in the explosive growth phase '. In the apparatus (for the process conditions, see the 1st fermenter), the process of growing seed material occurs. At the same time, the cell growth curve is taken. Having reached inflection point B (maximum cell growth rate), the growth process is continued for another 2-4 hours, after which the contents are fed in equal proportions to the 3rd and 4th fermenters with a volume of 250 l, pre-filled with nutrient medium.

 In the 3rd fermenter, the growing process is carried out similarly to the 1st and 2nd fermenter. The culture growth curve is also taken. Bringing the process to the inflection point (B) for this apparatus (maximum growth rate of the culture), another 2-4 hours continue to conduct the process of growing the culture. Then, from the 3rd fermenter, a portion of the biomass is transferred by the dispenser to the 2nd and 5th fermenters, previously filled with nutrient medium. The volume of the 5th fermenter is 250 l.

 In each of these fermenters (2nd and 5th), biomass was transferred in the amount of 5-20% of the volume of sown apparatus.

 The final product is prepared from the remaining biomass in the 3rd fermenter. In the 4th fermenter, the process is also conducted until the final product is completely obtained.

 After this, the process is repeated from the 1st fermenter. The culture growth curve is removed only in the start-up period to determine the time of the maximum culture growth rate for this apparatus.

 The finished product should have the following indicators (see table 1).

 The results of experiments on obtaining exopolysaccharides by the proposed method are summarized in table. 2.

 The transfer of biomass from the 1st, 2nd and 3rd fermenters in the explosive growth phase (see the curve) allows to obtain an exopolysaccharide with the required rheological characteristics (kinematic viscosity) required in the oil field.

 The carbohydrate content of up to 0.8% can be obtained by the proposed method, while in the prototype this indicator is not higher than 0.2%. In this phase, there are no signs of specific bacteriophagy of the culture. The process time according to the claimed embodiment is 40-44 hours.

 When the installation was in prototype mode, when the cascade of reactors is used only to increase the working volume of the process, it amounted to 72-74 hours. TTT1 TTT2

Claims (1)

 A method for the continuous production of exopolysaccharides, including growing the strain producing it under aeration in a nutrient medium using a cascade of fermenters, characterized in that in the first fermenter the strain is grown for a time consisting of the time to reach the maximum cell growth rate in this fermenter and 2-4 hours after reaching this indicator, after which the resulting biomass is fed into a second fermenter, pre-filled with nutrient medium, in which the biomass is for a belt, including the time to reach the maximum cell growth rate in the second fermenter and 2-4 hours after reaching this indicator, then the contents of the second fermenter are fed equally to the third and fourth fermenters pre-filled with nutrient medium, and in the third fermenter the strain is grown for a time consisting from the time to reach the maximum cell growth rate in this fermenter and 2-4 hours after reaching this indicator, after which the biomass is fed from the third fermenter to the second and fifth farms ntatory in an amount necessary to obtain the final product in the working volume fermenters sown, and from the remaining biomass in the fermenter third and fourth received fermenter a final product and the process is repeated starting from the first fermenter.

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