KR0169214B1 - Process for removing microorganisms from acrylamide reaction mixture - Google Patents

Abstract

The present invention relates to a method for effectively removing microorganisms by aggregating microbial cells by hydration of acrylamide from acrylonitrile to acrylamide using a microorganism, and then by adding a cationic polyrefining solution to the obtained reaction solution. In the present invention, the amount of cationic polyelectrolyte added to the reaction terminator is in the range of 30-60 mg / L.

Description

How to remove microbial cells from acrylamide reaction solution

The present invention relates to a method for removing the microbial cells from a reaction solution in which acrylonitrile is hydrated with acrylamide using microorganisms.

Acrylamide is a monomer of polyacrylamide, and polyacrylamide is used as a flocculant in wastewater treatment, petrochemical and paper industry, and as a sizing agent in the textile industry.

Acrylamide can be obtained by hydrating acrylonitrile, and the chemical method can be carried out using copper as a catalyst, but there is a problem that is complicated and pollution occurs. To address this problem with chemical methods, biological processes using nitrile hydratase enzymes have been developed (US Pat. No. 4,421,855 and US Pat. No. 5,334,519; Asano et al., Agiic. Biol., 46, 1183). , 1982; Kobayashi et a L., Tibtech., 10, 402, 1992).

Microorganisms that produce the enzyme include aeromonas, agrobacterium, astobacter, brevibacterium, citrobacter, corynebacterium, enterobacter, aeronia, clevsiella, nocardia, pseudomonas, Rizovium, Rhodococcus, Streptomyces, Santobacter and the like have been reported (Digeionimo Antoine, Appl. Environ. Microbiol., 3L, 900, 1976; Harper. Biochem. J., 165 309. 1977; Morimoto et al., EP 530522A; Yamada et al., EP 579907A; Nagasawa et at., Appl. Environ.Microbiol., 8, 87, 1988; Ramakrishna et al., Biotech. Lett., 14, 827, 1992; Li et al., Appl. Biochem, Biotechnol, 36, 171, 1992; Yamada et al, J Ferm, Technol., 57, 171, 1979).

As a result of the continuous microbial screening, it was possible to increase the concentration of acrylamide in the reaction solution by 40%, thereby allowing direct commercialization without concentration (US Pat. No. 5334519; Kobayashi et al., Tibtech. , 10, 402, 1992).

Acrylamide using Brevibacterium microorganism CH2 (KCTC 8462p, Korean Patent Publication No. 91-7850) and Rhodococcus Rhodocross (Korean Patent Publication No. 95-18447, Rhodococcus Rhodocross M33 (IBFM VKM-15150) The present inventors who have studied the hydration reaction in the furnace, when the process of removing the microbial cells is delayed after the hydration reaction is terminated, the microorganism cells are gradually self-decomposed to disperse the contents of the cells, and thus the acryl is It has been found that it is difficult to commercialize the amide-comprising reaction solution as it is.

Therefore, it can be seen that it is required to remove the microorganisms from the reaction solution as soon as possible, and by reducing the time that the microorganisms remain in the reaction solution, a relatively high purity acrylamide product can be obtained.

That is, an object of the present invention is to remove a method for quickly and simply removing microorganisms from a reaction solution of a reaction in which acrylonitrile is hydrated and converted to acrylamide using microorganisms.

The object of the present invention described above is the following steps:

(1) adding a cationic polyelectrolyte to the reaction terminating solution containing acrylamide and microorganisms converted from acrylonitrile to aggregate the microorganisms; And

(2) is achieved by a method for removing microorganisms from an acrylamide-containing reaction solution, comprising the step of separating the microorganisms by centrifugation or filtration of the reaction terminator obtained in step (1).

Other objects, other features and applications of the present invention will become apparent to those skilled in the art by the following detailed description.

Hereinafter, the present invention will be described in more detail.

According to the method of the present invention, by applying microorganisms having nitrile hydratase activity to acrylonitrile and adding the cationic polyelectrolytes to the reaction terminator converted to acrylamide, the microorganisms are effectively aggregated and then filtered or centrifuged. By removing the microorganisms by separation, an acrylamide liquid which can be commercialized without further treatment can be obtained.

Centrifugation of the reaction termination liquid at a normal 2000 g removes only about 40% of the microbial cells, but after aggregation with cationic polyelectrolyte according to the method of the present invention, more than 98% of the microorganisms are removed.

The addition of insoluble pyridinium or polyelectrolyte is based on the principle of coagulation and sedimentation of microorganisms by mechanisms such as neutralization, cross-linking by polymers, and mutual hydration reactions. (Hushes et al., Biotech. Tech, 4, 55, 1990: Mukhopadhyay et al., Ibid, 4, 121, 1990).

In the present invention, the aggregation-precipitation performance of microorganisms having nitrile hydratase activity was examined for electrolytes classified as neutral, anionic and cationic. As a result, polyelectrolytes having a cation in the production of acrylamide were examined. In particular, it was experimentally confirmed that the most effective when using cationic polymer materials such as polyethyleneimine or polyacrylamide.

Microorganisms that can be removed by applying the method of the present invention include all conventional microorganisms having nitrile hydratase activity, as described above, and in particular, a brine having the ability to convert acrylonitrile to a high concentration of acrylamide. Non-bacterium CH2 (KCTC 8462p), Rhodococcus Rhodocross M33 (KBFM VKM-15150), Rhodococcus Rhodocross M8 (KBFM VKM-15150) and Rhodococcus Rhodocross ATCC 33278 are advantageously used.

Conversion of acrylonitrile to acrylamide using these microorganisms can be carried out by conventional methods well known in the art, for example, see Lee et al., Enzyme Miciob. Technol., 15, 980, 1993 Can be used.

The amount of cationic polyelectrolyte added to the acrylamide-containing reaction terminator thus obtained may be appropriately selected within the range of about 17.5-110 mg / l or less, preferably about 30-60 mg / l.

Microorganisms flocculated-precipitated by the addition of polyelectrolytes can be removed, for example, by centrifugation at about 2000 x g for about 2 minutes or by filtration using filter paper of the kind such as Whatman 5A or 40A.

In this way, the final reaction solution from which microorganisms were removed contains acrylamide in the range of 20-40% (w / v), and can be commercialized according to various purposes and uses without any additional treatment such as a concentration step.

The present invention will be described in more detail with reference to the following Examples, but the present invention is not limited to these Examples.

Reference Example 1. Culture of Microorganisms

Microorganisms used in the present invention are Brevibacterium microorganism CH2 (KCTC 8462p), Rhodococcus Rhodocross M33 (IBFM VKM-15150; Korean Patent Application No. 94-12239), Rhodococcus Rhodocross M8, Rhodococcus Rhodocross ATCC 33278 It was.

Brevibacterium microorganism CH2 contains 15g of glucose, 3g of yeast extract, 3g of malt extract, 5g of bactopeptone, 1g of potassium dihydrogen phosphate, 1g of potassium dihydrogen phosphate, 1g of salt, 0.2g of magnesium sulfate, and 0.02g of iron sulfate. Incubated for 25 hours at 30 ℃ in medium dissolved in (Lee et al., Enzyme Microb. Tcchnol., 15, 980, 1993).

Rhodococcus microorganism M33 (glucose 5g / l, N3NO ₃ 1g / l, KH ₂ PO ₄ 0.6g / l, K ₂ HPO, 0.5g / l, MgSO ₄ 0.5g / l, FeSO ₄ 0.005g / l, COC1 ₂ 0.004 g / L) was incubated for 48 hours at 30 ℃, pH 7 (Korean Patent Publication No. 94-12239).

Each culture was centrifuged at 10000 x g to separate microorganisms.

Reference Example 2. Preparation of Acrylamide

80 liters of deionized water was charged to a 120 liter reaction tank controlled at a temperature of 10-25 ° C., and microbial cells were added at a concentration of 0 4-0.6 g / l, and the acrylonitrile concentration of the reactor was 0.05% (w / v) or less Hydration was carried out by adding 15-35 kg of (99.95% purity; East-West Petrochemical Co., Ltd.) to be adjusted to give an acrylamide furnace.

After 24 hours, Brevibacterium microorganism CH2 produced a reaction solution containing 20% acrylamide, and Rhodococcus Rhodocrose M33, Rhodococcus Rhodocrose M8, and Rhodococcus Rhodocrose ATCC 33278 were 40 after 8 h each. A reaction solution containing%, 40%, and 30% acrylamide was produced. Therefore, the reaction liquid containing acrylamide obtained by the method of the present invention has a concentration of 20 to 40% of acrylamide.

Acrylamide and acrylonitrile were quantified using gas chromatography (Model 3300 from Varian). Chromosolve W (80-100 mesh) was quantified using a column with 10% Carbowax 20M. The quantitative conditions were: detector, injection port temp = 210 ° C, column temperature temp.) = 160 ° C. and a flow rate of 30 ml / min FID helium gas (Lee et al., Enzyme Miciob. Technol., 15, 980, 1993).

[Example 1-8 and Comparative Example 1-6 flocculant administration effect]

2.5 ml of the reaction solution obtained by applying Rhodococcus Rhodocrose M33 in Reference Example 2 was added to the flocculant shown in Table 1 at a concentration of 30 mg / L to form flocculation, and centrifuged at 2000 x g for 2 minutes. The separated supernatant was measured for absorbance at 540 nm. The results are shown in Table 1 below. The microbial removal efficiency was calculated from the measured absorbance and the results are shown in Table 1 below.

(Initial absorbance of the reaction solution: 2.0 OD (540 nm))

In Table 1, PEI (polyethyleneimine) has a molecular weight of 7.5 × 10 It is made by Sigma Co., Ltd. and PAA (Polyacrylamide) series is made by Yiyang Chemical (Ulsan). , Cationic YCX is an acrylic acid product, molecular weight 6-8 × 10 , C is a methacrylic acid product, each having the following molecular weight: C-100P 7-9 × 10, C-001P 2-4 × 10 , C-021P 1-3 × 10 , C-100PM 2-4 × 10 , C-l01P 2-4 × 10 .

In the case of diatomaceous earth and activated carbon, microorganism aggregation was not observed with the naked eye, but by centrifugation, more than 70% of the microorganisms were removed. As can be seen from the results of Table 1, in the case of the anionic flocculant and the neutral flocculant, the flocculation effect was not so good.

Example 9-12

It carried out similarly to the method of Example 1, changing the kind of microorganism and the kind of flocculant into the kind of Table 2 below, and measuring the absorbance at 540 nm.

The results are shown in Table 2.

From the results in Table 2, it can be seen that the cationic flocculants used according to the present invention sufficiently cause the flocculation regardless of the type of microorganism.

Example 13

In the same manner as in Example 1, PAA C-101P was added to the reaction terminating solution in the amount of 10, 20, 30, 40, 50, 60, or 70 mg / L as a flocculant to precipitate the microorganisms, centrifugation and microorganisms The removal rate was calculated. The results are shown in Table 3 below.

From the results in Table 3, it can be seen that the amount of the flocculant added to the reaction terminating liquid is preferably 70 mg / L or less, particularly 40 mg / L or less.

Example 14

In the same manner as in Example 13, instead of centrifugation was filtered using Whatman 40A filter paper to measure the filtration rate and the absorbance of the filtrate. The results are shown in Table 4.

Example 15

Except for using PEI as the flocculant, the same procedure as in Examples 13 and 14 was carried out, and the absorbance after centrifugation (540 nm) and microbial removal rate (5), and the absorbance after filtration (540 nm) and filtration rate (ml / min ) Was measured and the results are shown in Table 5 below.

Example 16

In Examples 13 and 15 the amount of flocculant was increased and the highest microbial removal efficiency (99.9% or more) was investigated. The results are shown in Table 6.

From the results in Table 6, the amount of flocculant added was maintained in a linear relationship with the concentration of microorganisms in order to show a microbial removal effect of 99.5 or more. The slope was 10 for PEI and 6.5 for the PEI, and the slope for PAA C-101P. 12.8, and 7.8.

Claims (4)

Following steps (1) to (2); (1) adding a cationic polyelectrolyte to a reaction terminator containing acrylamide and microorganisms converted from acrylonitrile to aggregate the microorganisms; And (2) separating the microorganisms by centrifugation or filtration to separate the microorganisms obtained in the step (1). The method of claim 1 wherein the cationic polyelectrolyte is polyethyleneimine or polyacrylamide. The method according to claim 1 or 2, wherein the cationic polyelectrolyte is added in an amount of 17.5-110 mg / l or less with respect to the reaction termination solution. The method according to claim 3, wherein the cationic polyelectrolyte is used in an amount of 30 -60 mg / l based on the reaction termination solution.