KR100457071B1 - Development of decolorization and biodegradation techniques for dyeing waste waters treatment by bacterial consortium - Google Patents

Abstract

The present invention relates to a method for culturing chromatographic microorganisms based on soil in a dye wastewater discharge area and a method for treating dye wastewater using the same, and to isolate and incubate microorganisms native to soil in a dye wastewater discharge area, thereby causing major contamination. It was created for the purpose of detoxification and mineralization of dye wastewater by inducing color removal of phosphorus wastewater and complete mineralization of intermediate decomposition products.

The dyeing wastewater chromatolysis microorganisms take the soil in the wastewater discharge area and then suspended it with distilled water at a ratio of 1:10, and then, after standing for 30 minutes, extract 2% of the suspension,

The extracted suspension is,

Inoculated into a sterile complex culture medium having a volume of 99 times the extracted suspension,

In the inoculated medium,

Layered mineral oil having 1/99 volume relative to the volume of the medium, and incubated at 30 ° C. Characterized by separating the bacillus to produce.

Description

Development of decolorization and biodegradation techniques for dyeing waste waters treatment by bacterial consortium

The present invention relates to a method for culturing chromatographic microorganisms based on soil in a dye wastewater discharge area, and more specifically, to cultivate chromatolytic microorganisms in soil in a dye wastewater discharge area, thereby decolorizing the dye wastewater and To achieve waste water purification.

In general, dyeing wastewater is only about 4.5% of total wastewater discharge in terms of wastewater generation and wastewater characteristics.

Pollution loads from wastewater (pre-treatment BOD concentrations) account for about 24% of the total wastewater, which can have a significant impact on public waters.

Such dyeing wastewater has a different dyeing method depending on whether the forge to be treated is natural or synthetic fiber,

In addition, even if the same fibers are different from the dyes and mordants used in the dyeing method and the color tone of the dyeing, the investigation of the components is generally complicated, and the daily variation of the water quality is very large according to the operating conditions of the work process.

As described above, various treatment methods and combinations thereof have been examined because of the complex composition of the dyeing wastewater and the change in water quality. However, the establishment of accurate wastewater treatment system has not been established.

As mentioned above, dyeing wastewater treatment facilities have problems that need improvement due to subsidies of proper treatment facility operation and management, design of inappropriate treatment facilities, and fluctuation of quantity and quality of wastewater.

In particular, in addition to satisfying the water quality standards as the emission standard of dyeing wastewater is strengthened, chromaticity removal, which is the biggest problem of dyeing wastewater, has recently emerged as a major concern.

The chromaticity of the dyeing wastewater remains in the chromaticity of various colors even after the wastewater purification, it is impossible to recycle the purified water,

In addition, the discharge of purified water caused the coloration phenomenon according to the residual chromaticity in rivers, sea areas, resort districts and urban landscapes.

Thus, various methods and facilities have been devised and used to remove the chromaticity contained in the dyeing wastewater.

Among them, chlorine-based oxidation method, chlorine-based oxidation-ultraviolet method, fenton reagent method, ozone (O ₃ ) method of decolorization, and chemical methods such as filtration, natural sedimentation method, pressure flotation method, etc. are largely classified.

However, the above chemical and physical methods have the following problems,

① It is impossible to decolorize various colors because it is applied only to specific coloring materials.

② It is uneconomical due to excessive initial cost or operation cost depending on the coloring material.

③ There is no clear guarantee of discoloration efficiency.

The effectiveness is extremely low due to the back.

Therefore, the present invention was created in order to solve the above problems, and isolate and incubate microorganisms native to the soil of the dye wastewater discharge area, thereby inducing color removal of wastewater, which is the main cause of pollution, and complete mineralization of intermediate decomposition products. The purpose is to detoxify and mineralize the dyeing wastewater.

1 is a graph showing the chromatic decomposition state of the red color according to the acidity during the treatment of dye wastewater through the present inventors chromatographic microorganisms,

Figure 2 is a graph showing the chromatic decomposition state for the yellow color according to the acidity when the dye wastewater treatment through the chromatographic microorganism of the present invention,

Figure 3 is a graph showing the chromaticity decomposition state for the blue color according to the acidity in the treatment of dye wastewater through the present inventors chromatographic microorganisms,

Figure 4 is a diagram showing the type of dye used for bacilli separation of the present inventors chromatographic microorganisms.

Hereinafter, the microbial culture process and chromaticity decomposition process of the present invention will be described with reference to the accompanying drawings.

1 is a graph showing the chromaticity decomposition state for red color according to acidity when dyeing wastewater treatment using the chromatolytic microorganism of the present invention, Figure 2 is chromaticity decomposition for yellow color acidity when treating the dyeing wastewater through the chromatographic microorganism of the present invention Figure 3 is a graph showing the state of chromatic decomposition for blue according to the acidity when dyeing wastewater treatment through the chromatolytic microorganism of the present invention, the color of the present invention, Figure 4 The types are illustrated together in a diagram.

In order to achieve the above object, the method of culturing chromatin-decomposed microorganisms based on the soil of a dye wastewater discharge area according to the present invention is to perform microorganisms treating dye wastewater through growth and sanction of bacteria rather than conventional filamentous fungi,

To this end, the soil of the wastewater discharge area is collected, suspended in distilled water at a ratio of 1:10 (soil-1g, distilled water-10ml), and then allowed to stand for 30 minutes, and 2ml of the suspended suspension is added with dye. Inoculated into 198 ml of sterile complex culture medium,

The dye added to the complex culture medium,

As shown in FIG. 4, the dye was generally used in dyeing plants based on 63 types of eight colors of blue, yellow, red, black, orange, green, brown, and purple.

The mixed culture medium is a 2% LB medium (DIFCO, Lennox) and dyes are mixed in equal proportions and used at a concentration of 100 mg of dye in 1 L of sterile distilled water, mineral oil after inoculation of supernatant to set the culture conditions to microaerobic conditions. Layered to 2 ml and incubated at 30 ° C.

The culture medium as described above was sequentially diluted in a 98well microtiter plate (BECTON DIKINSON) and plated on an agar plate medium added with 1.5% agar to isolate a bacillus producing a transparent ring.

In order to determine the biochemical and physiological characteristics of the isolated bacillus prior to its industrial application,

In order to examine the cell wall pattern and morphology of the bacillus, staining is performed using a Gram staining kit,

To determine the spore formation ability of the isolated bacilli, spore staining was performed by incubation for 24 hours in LB plate medium for 7-10 minutes by heating with spore staining reagent (Malachite green, sapranin) and

Substrate utilization and physiological biochemical tests were performed using API (Analytical Profile Index) 20A to identify isolates.

The bacterium that has undergone various experiments as described above leads to the following experimental results,

Positive identification of URE (urease), GEL (gelatin), SPOR (spore), and GRAM (gram staining) showed identification of IDID (20020003) with 99.6% homology with CLOSTRIDIUM spp. .

The bacillus isolated from the culture described above was identified as CLOSTRIDIUM spp., Which is an absolute anaerobic bacillus having excellent decolorization and resolution.

As described above, the decolorization and resolution using the CLOSTRIDIUM spp., Which is an absolute anaerobic bacterium with excellent decolorization and resolution of the dyeing wastewater, will be described in detail with reference to Examples.

First, to see the decolorization and decomposition reactions through the isolated bacilli,

The same culture medium as that used for separating the bacilli is required, and pH culture additives are added to the culture medium to create the same environment as that of the actual dye wastewater.

The culture medium applied to the decolorization and decomposition reaction of such bacilli,

198 ml is prepared by adding a wastewater sample having a mixing ratio of dye and distilled water having a concentration of 100 mg / l and a pH formulation for setting acidity to a culture medium having a concentration of 2% LB.

These culture media are each equipped with acidity pH5 to pH12, and the dye added to the wastewater sample is performed based on three types of reactive dyes, Reactive Red-4, Reactive Blue-2, and Reactive Yellow-2.

Example 1

First, 2 ml of isolated bacilli are inoculated into 198 ml of the culture medium to which the Reactive Red-4 dye is added among the culture media prepared for each color and acidity, and then cultured for 12 hours, 24 hours, and 36 hours.

After centrifugation of the culture broth at each hour for 3 minutes at 14,000rpm as described above, the supernatant was taken and measured at 3nm intervals to measure absorbance and chromaticity reduction rate through the maximum absorption wavelength of the dye.

Absorbance and chromaticity reduction rate for each culture time period measured through this example were derived as shown in the table below.

Table 1

Absorbance Reduction and Discoloration Rate of pH of Reactive Red-4 by CLOSTRIDIUM spp.

12 hours later 24 hours later 36 hours later pH Control absorbance Experimental group Decolorization% Control absorbance Experimental group Decolorization% Control absorbance Experimental group Decolorization% pH5 1.296 1.291 0.39 1.288 1.277 0.85 1.294 1.294 0 pH6 1.301 1.298 0.23 1.292 0.195 84.91 1.299 0.042 96.77 pH7 1.294 1.087 16 1.283 0.103 91.97 1.282 0.053 95.87 pH8 1.286 0.483 62.44 1.276 0.042 96.71 1.273 0.044 96.54 pH9 1.291 0.165 87.22 1.275 0.034 97.33 1.28 0.042 96.72 pH10 1.288 0.122 90.53 1.278 0.037 97.1 1.274 0.042 96.7 pH11 1.274 0.037 97.1 1.254 0.036 97.13 1.257 0.047 96.26 pH12 1.208 0.704 41.72 1.2 0.066 94.5 1.201 0.051 95.75

Based on the above experimental results, as the absorbance decreases and the decolorization reaction proceeds, the intermediate metabolite is not observed on the graph, and the absorbance gradually decreases.

This is the case of the red series, as shown in Figure 1 pH condition shows the best decolorization rate from pH9 to pH11, it can be seen that almost no decoloration under pH5 conditions.

Example 2-

2 ml of isolated bacterium was inoculated into 198 ml of the culture medium to which the Reactive Yellow-2 dye was added, and the cultures were incubated for 12 hours, 24 hours, and 36 hours.

After centrifugation of the culture broth at each hour for 3 minutes at 14,000rpm as described above, the supernatant was taken and measured at 3nm intervals to measure absorbance and chromaticity reduction rate through the maximum absorption wavelength of the dye.

Absorbance and chromaticity reduction rate for each culture time period measured through this example were derived as shown in the table below.

TABLE 2

Absorbance and Decolorization Rate of Reactive Yellow-2 by pH by CLOSTRIDIUM spp.

12 hours later 24 hours later 36 hours later pH Control absorbance Experimental group Decolorization% Control absorbance Experimental group Decolorization% Control absorbance Experimental group Decolorization% pH5 10078 1.066 1.11 1.078 1.08 0 1.095 1.103 0 pH6 1.105 1.097 0.72 1.109 0.924 16.68 1.127 0.525 53.42 pH7 1.149 1.12 2.52 1.154 0.845 26.78 1.175 0.311 73.53 pH8 1.166 1.032 11.49 1.169 0.583 50.13 1.188 0.156 86.87 pH9 1.144 0.85 25.7 1.152 0.246 78.65 1.169 0.1 91.45 pH10 1.104 0.76 31.16 1.114 0.176 84.2 1.132 0.109 90.37 pH11 1.086 0.895 17.59 1.102 0.323 70.69 1.127 0.125 88.91 pH12 1.066 0.996 6.57 1.086 0.626 42.36 1.103 0.114 89.66

Reactive Yellow-2, a yellowish color of dyeing wastewater, was almost impossible to decolorize and decompose under pH5 conditions. It was found that pH9-pH10 was most suitable.

Example 3-

2 ml of the isolated bacterium was inoculated into 198 ml of the culture medium to which the Reactive Blue-2 dye was added, and incubated for 12 hours, 24 hours, and 36 hours, respectively.

After centrifugation of the culture broth at each hour for 3 minutes at 14,000rpm as described above, the supernatant was taken and measured at 3nm intervals to measure absorbance and chromaticity reduction rate through the maximum absorption wavelength of the dye.

Absorbance and chromaticity reduction rate for each culture time period measured through this example were derived as shown in the table below.

TABLE 3

Absorbance and Decolorization Rate of Reactive Blue-2 by CLOSTRIDIUM spp.

12 hours later 24 hours later 36 hours later pH Control absorbance Experimental group Decolorization% Control absorption Experimental group Decolorization% Control absorption Experimental group Decolorization% pH5 0.915 0.915 0 0.924 0.914 1.08 0.909 0.927 0 pH6 0.92 0.92 0 0.917 0.896 2.29 0.932 0.787 21.3 pH7 0.924 0.95 0 0.922 0.855 7.27 0.93 0.719 22.69 pH8 0.921 0.922 0 0.928 0.797 14.12 0.932 0.498 46.57 pH9 0.927 0.83 10.46 0.93 0.71 23.66 0.936 0.361 61.43 pH10 0.936 0.787 15.92 0.936 0.477 49.04 0.952 0.113 88.13 pH11 0.936 0.803 14.21 0.936 0.407 56.52 0.953 0.089 90.66 pH12 0.907 0.899 0.88 0.918 0.462 49.67 0.939 0.145 84.56

Reactive Blue-2, which is a blue series, shows the best effect between pH10-pH12 on the pH condition for decolorization and decomposition reactions, while it shows little decolorization at pH5.

In the experiment, the degree of decolorization reaction was quantified by converting the degree of optical density reduction of each dye into a percentage.

Light density measurement was measured for 12 hours, 24 hours, and 36 hours of incubation at pH 1 intervals under pH-pH12 conditions for chromatic decomposition, respectively, and then measured for absorbance reduction using Spectronic GENESYS 5 (MILTON ROY). By doing so.

In addition, the pH agent is added to the culture medium after autoclaving, adjusted using NaOH and HCl solution, and filtered using a filtration kit.

As above

CLOSTRIDIUM spp., An absolute anaerobic bacillus isolated from the soil in the dye wastewater discharge area, shows excellent discoloration and degradability after 36 hours of reaction time in acidic acid with pH9-pH12. .

In order to apply CLOSTRIDIUM spp., A bacterium with excellent chromatic resolution of dyeing wastewater, directly to the dyeing wastewater treatment process,

First, the inoculated CLOSTRIDIUM spp., Isolated and cultured, is inoculated in LB solid or liquid medium, and then cultured for 24 hours at 37 ° C under anaerobic conditions.

In addition, the conditions of the dyeing wastewater are set so that the bacterium cultured together with the culture of the bacterium can exhibit active color resolution in the dyeing wastewater.

To this end, 2 Ｘ LB liquid medium is added to the dyeing wastewater in the same amount and stirred, and 6NHCl (hydrochloric acid) is added to the stirred dyeing wastewater to set the conditions of the dyeing wastewater to have a hydrogen ion concentration of 10.

As described above, the bacterium precultured in the dyeing wastewater with chromatic decomposition conditions was added in an amount corresponding to 2% of the total dyeing wastewater volume and maintained at 37 ° C. in an anaerobic state for 24 hours to incubate variously in the dyeing wastewater. Dissolve the chromaticity to discolor

The method of culturing chromatographic microorganisms based on the soil in the dye wastewater discharge area of the present invention as described above removes various chromaticities of the dye wastewater, which occupy a considerable part of the pollution degree due to the total wastewater discharge,

It is a useful invention that can lead to the conversion to environmentally friendly industrial structure, thereby improving competitiveness and suppressing water pollution throughout the industry.

Claims (5)

In the culture method of the dye wastewater chromatographic microorganism; The dyeing wastewater chromatographic microorganism, After collecting the soil in the wastewater discharge area and suspending with distilled water at the ratio of 1:10, it was allowed to stand for 30 minutes, and 2% of the suspended suspension was taken out. The extracted suspension is inoculated into a culture group complex culture medium having a volume of 99 times the extracted suspension, In the inoculated medium, the mineral oil having a volume of 1/99 volume relative to the volume of the medium was layered and left to culture at 30 ℃, Diluting the culture solution in a 98well microtiter plate (BECTON DIKINSON) sequentially and spreading on agar plate containing 1.5% agar added to the affected agar plate medium to separate the bacterium producing the transparent ring based on the soil of the dye wastewater discharge area. Cultivation method of one chromatographic microorganism. The method of claim 1; The isolated bacilli are, Gram dyeing work, Spore staining by heating for 7-10 minutes using spore staining reagent (Malachite green, sapranin) after incubation in LB plate medium for 24 hours, For the identification of isolated bacterium, it is based on the soil of the dye wastewater discharge area characterized by CLOSTRIDIUM spp., Which is an absolute anaerobic bacterium identified by performing substrate utilization and physiological biochemical tests using API (Analytical Profile Index) 20A. Cultivation method of one chromatographic microorganism. The method of claim 1; The complex culture medium is 2% LB medium (DIFCO, Lennox), A method for culturing chromatolytic microorganisms based on soil in a dye wastewater discharge area, wherein the added dye is mixed with 1 liter of sterile distilled water at a ratio of 100 mg of dye. The method of claim 1 or 2; In the chromaticity decomposition action remaining in the dye wastewater of the isolated bacilli CLOSTRIDIUM spp., CLOSTRIDIUM spp., The bacterium, is characterized in that, after 36 hours of incorporation into the dyeing wastewater, all three colors of the red, yellow, and blue discoloration rates of 90% or more of the basic structure are expressed under strongly acidic conditions between pH 9 and pH 12. Method of culturing chromatographic microorganisms based on soil in wastewater discharge area. The dye wastewater treatment process using the bacillus CLOSTRIDIUM spp. CLOSTRIDIUM spp., The isolated cultured bacilli, was inoculated in LB solid or liquid medium, and then cultured for 24 hours at 37 ° C. under anaerobic conditions. To the dyeing wastewater, add 2 μLLB liquid medium in the same amount and stir, 6NHCl (hydrochloric acid) was added to the stirred dyeing waste water to form a hydrogen ion concentration of 10, Based on the soil in the wastewater discharge area, characterized in that the pre-cultivated bacillus is added to the amount corresponding to 2% of the total wastewater wastewater incubation for 24 hours under anaerobic conditions at 37 ℃ temperature Dyeing wastewater treatment method using one chromatographic microorganism.