SU1479515A1 - Method of producing immobilized bacterial cells - Google Patents

Abstract

The invention relates to biotechnology, and specifically to methods for the production of microbial cells-destructors of anionic surfactants. The aim of the invention is to simplify the process and increase the activity of immobilized cells during the destruction of anionic surfactants. The method consists in immobilizing bacterial cells by mixing an aqueous suspension of cells — destructors of the anionic surfactants PSEUDOMONAS AERUGINOSA 1 C or P.ALCALIGENES TR with anion-exchange polyvinyl alcohol or polycaproamide fiber containing flocculated flocculation pattern; , temperature 28-33 ° C for 20-30 minutes. At the same time, polyvinyl alcohol or polycaproamide fiber contains 1-1.5 mol of polyethyleneimine and / or polyepoxidamide per g fiber, and its processing is carried out with an aqueous suspension of microbial cells with a concentration of 0.6-1.0 units. optical density. The method simplifies the process of immobilization of bacterial cells and allows increasing their activity during the destruction of anionic surfactants by 4-6 times. 1 hp f-ly, 1 tab.

Description

(21) 4278459 / 28-13

(22) 06/02/87

(46) 05/15/89. Bksh. Number 18

(71) Leningrad Institute of Textile and Light Industry

them. CM. Kirov

(72) A. B. Lobov, I. I. Shamolin, S. S. Stavsk, L. A. Taranov and O. S. Radchenko

(53) 577.15 (088.8)

(56) Sinitsy A.A. et al. Immobilization of Saccharomyces cerevisiae on aluminoborosilicate glass fibers. Biotechnologists, 1986, K 3,

with. 66-69.

U.S. Patent No. 4,177,107, cl. C 07 G 7/02, 1979.

(54) METHOD FOR OBTAINING IMMOBILIZED BACTERIAL CELLS

(57) The invention relates to biotechnology, specifically to methods for producing microbial cells-destructors of anionic surfactants. The aim of the invention is to simplify the process and increase the activity of immobilized cells during the destruction of anionic surfactants. The method consists in immobilizing bacterial cells by mixing an aqueous suspension of the cells-destructors of the anionic surfactants Pseudomonas aeruginosa 1C or P. alcaligenes TR with anion-exchange polyvinyl alcohol or polycaproamide fiber containing chemically fixed flocculant and conducting linking and conducting linking. 30, temperature 28-33 ° C for 20-30 minutes. At the same time, golivinil alcohol or polycaproamide fiber contains 1-1.5 microns of polyethyleneimine and / or polyepoxide of amine per 1 g of fiber, and its processing is carried out with an aqueous suspension of microbial cells with a concentration of 0.6-1.0 units. optical density. The method simplifies the process of immobilization of bacterial cells and allows to increase their activity during the destruction of anionic surfactants 4-6 times. 1 hp f-ly, 1 taol.

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The invention relates to biotechnology, in particular, to the production of immobilized microbial cells, destinations, anionic surfactants (APAS), which can be used as catalysts with specific and controlled activity in the purification of harmful industrial emissions from a number of chemical plants.

The purpose of the invention is to simplify the process and increase the activity of immobilized cells during the destruction of anionic surfactants.

The method of obtaining immobilized cells of the APAS destructors consists in mixing an aqueous suspension of Pseudomonas alcaligenes TR cells (VKPM B-2981) or Pseudomonas aeruginosa 1C with a porous adsorbent - anion

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exchange polypentine alcohol or pyramide amide fiber containing chemically fixed floc nt polyethylenimine and / or polyispoxide amine in the amount of 1.0-1.5 mol per 1 g of adsorbent, and immobilization is carried out at a bath module of 20-30, temperature 28-Z3 C for 20-30 minutes It is preferable to use an aqueous suspension of cells with a concentration of 0.6-1.0 units of optical density.

Polyvinyl alcohol or polycaproamide fiber, 1 g of which contains 1-1.5 moles of chemically fixed flocculant, has an increased affinity for microbial destructive cells of anionic surfactants due to the inclusion of polyethylenimine and / or polyepoxide of amine in the support structure. This allows the cells to be immobilized for 20-30 minutes under mild conditions without using the complex equipment necessary for irradiation. The microbial cell is fixed at some distance from the fibrous matrix through the molecules of chemically bound polyethylenimine and / or polyepoxidamine contained in the fiber. provides this cell with greater mobility than if the latter were in the polymer mass, improves the access of the substrate to it, contributes to the efficient operation of the enzyme system of the immobilized micro hydrochloric cells and complete preservation of its activity.

Using the proposed method allows to simplify the process by (eliminating the stage of radiation polymerization, as well as increasing the activity of immobilized cells during the destruction of anionic surfactants from 1.5-3.6 (with complete preservation of cell activity) to 9.2-15 , 3 mg of degraded APAS per 1 mg of cells, i.e., 4-6 times.

The method is illustrated by the following examples.

Example 1. Anion-exchange polyvinyl alcohol fiber (1 g) containing chemically fixed polyethyleneimine in an amount of 1 mol / g is treated for 20 minutes with an aqueous suspension of microbial culture of P. caligenes TR (VKPM B-2981) (20 ml) with concentration cells 0.6 units optical density at 28 C. Fiber

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air is dried, and washed with distilled water. The content of cells immobilized on the fiber is 12 mg / g. The activity of cells immobilized on the fiber, 9.2 mg of degraded anionic surfactants per mg of cells (table).

PRI mme R 2. Anion-exchange polyvinyl alcohol fiber (1 g) containing 1.3 mol / g of chemically fixed polyotyleneimine is treated for 25 minutes with an aqueous suspension of microbial culture of P. alchigenes TR (25 ml) with a cell concentration of 0.8 units. optical density at a temperature of 30 ° C ... The fiber is dried in air, and then washed with distilled water. The content of cells immobilized on the fiber is 15 mg / g. The activity of cells immobilized on the fiber is 9.6 mg of the destructed anionic surfactants per 1 mg of cells.

PRI me R 3. Anion-exchange polyvinyl alcohol fiber (1 g) containing chemically fixed polyethylenimine in an amount of 1.5 mol / g is treated for 30 minutes with an aqueous suspension of microbial culture of P. alcienes TR (30 ml) with a cell concentration of 1.0 units. optical density at 33 ° C. The fiber is dried in air and then washed with distilled water. The content of cells immobilized on the fiber is 21 mg / g. The activity of cells immobilized on the fiber is 11.8 mg of the destructed anionic surfactants per 1 mg of cells.

PRI me R 4. Anion-exchange Polyvinyl alcohol fiber (1 g) containing chemically fixed polyethylenimine in an amount of 0.6 mol / g is treated for 20 minutes with an aqueous suspension of microbial culture of P. alcaigenes TR (20 ml) with a cell concentration 0.6 units optical density at 28 ° C. The fiber is dried in air and then washed with distilled water. The content of cells immobilized on the fiber is 6 mg / g. The activity of cells immobilized on the fiber is halved - to 5.0 mg of degraded anionic surfactants per mg of cells.

Anion-exchange Polyvinyl alcohol (polycaproamide) fiber containing flocculate in an amount of more than 1.5 mol per 1 gram, has low strength and increased fragility,

no-irony is u h iT) it; w microbial microbial cells seem impractical.

Example 5 Anion-abonance poly-phytonic (1 g) fiber containing 1.3 mol / g chemically fixed polyethylenimine is treated for 30 minutes with an aqueous suspension of microbial culture of P. aeruginosa 1C (30 ml) with a cell concentration 0.9 units optical density at 30 ° C. The fiber is dried in air and then washed with distilled water. The content of cells immobilized on the fiber is 23 mg / g. The activity of cells immobilized on the fiber is equal to 15.5 mg of degraded anionic surfactants per 1 mg of cells.

PRI me R 6. Anion exchange popivinyl alcohol fiber (1 g) containing 1.2 mol / g of chemically fixed polyepoxidamine is treated with an aqueous suspension of microbial culture of P.alcaligenes TR (25 ml) for 25 minutes cells 0.8 units optical density at 30 ° C. The fiber is dried in air and then washed with distilled water. The content of cells immobilized on the fiber is 18 mg / g. The activity of cells immobilized on the fiber is 11.2 mg of the destructed anionic surfactants per 1 mg of cells.

Example 7. Anion-exchange polycaproamide fiber (1 g) containing 1, 1 mol / g chemically fixed polyepoxidamine in an amount of 1a / 1 is treated with an aqueous suspension of microbial culture of P.aeruginosa 1C (20 ml) with a cell concentration of 0.7 units . optical density at 28 ° C. The fiber is dried in air and then washed with distilled water. The content of cells immobilized on the fiber is 13 mg / g. The activity of cells immobilized on the fiber is 9.1 mg of degraded anionic surfactants per mg 1 cells.

PRI me R 8. Anion-exchange polycaproamide fiber (1 g) containing chemically fixed polyepoxidamine in an amount of 1.0 mol / g is treated for 20 minutes with an aqueous suspension of microbial culture of P.alcaligenes TR (25 ml) with a cell concentration 0.6 units optical density

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at 28 ° C. The fiber is dried in air and then washed with distilled water. The content of cells immobilized on the fiber is 12 mg / g. The activity of cells immobilized on a fiber is 9.3 mg of degraded anionic surfactants per 1 mg of cells.

PRI me R 9. Anion-exchange polycaproamide fiber (1 g) containing 1.2 mol / g of chemically fixed polyethyleneimine is treated for 25 minutes with an aqueous suspension of microbial culture of P.alcaligenes TR (30 ml) with a concentration of cells 0.8 units optical density at 30 ° C. The fiber is dried in air and then washed with distilled water. The content of cells immobilized on the fiber is 15 mg / g. The activity of the cells immobilized on the fiber is 9.9 mg of degraded anionic surfactants per mg of cells.

Example 10. Anion-exchange polycaproamide fiber (1 g) containing 1, 4 mol / g of chemically fixed polyethylenimine is treated for 30 minutes with an aqueous suspension of microbial culture of P.aeruginosa 1C (30 ml) with a cell concentration of 1.0 units . optical density at 33 ° C. The fiber is dried in air and then washed with distilled water. The content of cells immobilized on the fiber is 22 mg / g. The activity of cells immobilized on the fiber, 14.2 mg of degraded ALAV per 1 mg of cells.

Example 11. Anion-exchange polyvinyl alcohol fiber (1 g) containing chemically fixed polyethylenimine and polyepoxidoamine in an amount of 1.3 mol / g, is treated for 25 minutes with an aqueous suspension of microbial culture of P.alcaligenes TR (28 ml) with a cell concentration of 0.9 units. optical density at 30 ° C. The fiber is dried in air and then washed with distilled water. The content of cells immobilized on the fiber is 21 mg / g. The activity of cells immobilized on the fiber is 12.6 mg of the destructed anionic surfactants per 1 mg of cells.

Example 12. Anion-exchange polycaproamide fiber (1 g), containing chemically fixed polyethyleneimine and polyepoxidamine in the amount of 1.2 mol / g, is treated

in ic iPHiif1} () million water suspensions of microbial fluid P.aeruginosa 1C (26 ml) with a concentration of 1.0 counts of optical density at 33 ° C. Air dry and then rinse with distilled water. The content of cells immobilized on fiber fibers is 20 mg / g. The activity of cells immobilized on the fiber is equal to 13.4 mg of degraded LPLV per mg of cells.

Example s 13-24. Immobilization is carried out as described in examples 1-12, varying the content of PEI and / or IDA in the composition of the hycaproamide (13-18 examples) of polyvinyl alcohol (19-24) fibers.

Examples 25-32. Immobilization was carried out, as described in examples 1-12, by modifying the wave modulus (suspension volume of 1 mg, added per 1 g of fiber), temperature, duration of immobilization, and cell concentration in suspension.

The dialysis data of example 25 shows that reducing the treatment module (respectively, the suspension volume in 1 ml of 1 g of fiber required for processing) to 15 leads to a decrease in the number of cells attached to the fiber due to insufficient treatment suspension and uneven processing of the carrier. An increase in the modulus to 35 (example 26) is impractical because with an increase in the volume of the suspension processing fiber (other things being equal immobilization) it absorbs — due to its anion-exchange capacity and structural features — the same number of cells as in module 30 (example 3) .

Reducing the concentration of cells in suspension to 0.4 units. wholesale The density of the processing module 20 (Example 27) does not provide a high content of cells on the fiber due to their insufficient number in the initial suspension, while using immobilization of suspensions with an increased concentration of cells (Example 28), firstly, the process of immobilization due to aggregation The bacteria in concentrated solutions, secondly, in any case, can not be fixed on the fiber Kue.Tim g, more than the number of pulp and structures

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The duration of treatment is determined by the period after which an equilibrium occurs between cells immobilized on the fiber and in a free state in suspension. Therefore, reducing the contact time of the fibers with the cell suspension (Example 29) does not allow such an equilibrium to be achieved, as a result of which the necessary number of cells is fixed on the fiber. After equilibrium, the continuation of immobilization (an increase in the time of cell contact with the fiber — Example 30) is impractical because the carrier has already associated such a number of cells that is maximum possible under other equal conditions (Example 3).

Similar patterns are observed with a decrease (below 28 ° C) or with an increase (above 33 ° C) immobilization temperature (example 31 and 32).

The described examples (for convenience of comparison) are given for PVA fiber containing PEI in the amount of 1.0 and 1.5 mol / g, and microbial culture of P.alcaligenes TR. However, such patterns apply to all other types of fibers, which are given in examples 1-24, as well as both types of microbial cultures.

The activities of the bacteria-destructors of anti-surfactants are determined by the change in the concentration of anti-surfactants (sodium dodecyl sulfate for P.aeruginosa 1C culture and alkylbenzenesulfonate for P.alcaligenes TR culture) in an aqueous solution colorimetrically by reaction with methylene blue and expressed as 1 mg reduction. the concentration of the corresponding anionic surfactants per 1 mg of free or immobilized cells. The content of cells in an aqueous solution of anionic surfactants is chosen equal to the number of cells immobilized per g of fiber, i

Thus, the use of the proposed method allows to simplify the process by eliminating the stage of radiation polymerization and increase the activity of immobilized cells during the destruction of anionic surfactants 4-6 times - from 1.5-3.6 to 9.2-15.3 mg of degraded AAL on 1 mg of cells, since the activity of the cells in the well-known procedure at PMmpmbdin remains, and in the proposed increases by 4-6 times.

Claims (2)

1. A method of producing immobilized bacterial cells, which consists in mixing an aqueous solution of bacteria cells with a porous ion-exchange synthetic adsorbent, incubating the mixture to fix the cells on the adsorbent and washing the adsorbent with an aqueous solution, characterized in that, in order to simplify the process and increase the activity of immobilized cells during destruction anionic surfactants 0 Substances immobilized are cells of Pseudomonas aeruginosa 1C or P.alcaligencs VKPM B-2981, anion exchange polyvigash alcohol or polycaproamide fiber containing 1.0-1.5 mol polyethylenimine and / or polyepoxide of amine per 1 g of fiber is used as an adsorbent The incubation is carried out for 20-30 minutes at 28-33 ° C, the ratio of the cell suspension to the adsorbent is set to 20-30 ml per 1 g of the adsorbent. 2. The method according to claim 1, wherein the concentration of cells in the aqueous suspension is set to 0.6-1.0 units of optical density.