KR100463116B1 - Biological method and microorganism for treating waste water containing glycoprotein - Google Patents

Abstract

The present invention is a Pseudomonas sp. CJ-P01 (Pseudomonas sp. CJ-P01), excellent growth and protease activity under aerobic conditions, excellent treatment capacity of saccharide protein wastewater, the isolation method of this strain and the sugar using the strain It relates to a biological treatment of protein wastewater.

Description

Biological method and microorganism for treating waste water containing glycoprotein

The present invention relates to biological wastewater treatment. More specifically, the present invention relates to a method for treating sugar starch wastewater using a novel microorganism Pseudomonas CJ-P01, which has good growth under aerobic conditions of biological wastewater treatment and has excellent protease activity.

Currently, the world is facing serious environmental pollution due to the increased use of fossil fuels due to industrialization, the rapid increase in pollutant emissions, and the natural destruction caused by unreasonable development plans. Such environmental pollution affects human life directly or indirectly through the air or water as a medium, and research on countermeasures and treatment technologies is actively conducted. In other words, there are physical, chemical and biological methods to treat industrial wastewater, but as a method that can decompose and remove various pollutants, there is less possibility of secondary pollution than physical and chemical treatment methods. Research into biotechnological treatment using microorganisms that are being evaluated is actively conducted.

Research on microorganisms is progressing a variety of bacteria, yeasts, molds, protozoa, epigenetics, etc. In particular, there are many studies to select and use strains having the ability to decompose hard-decomposed compounds. That is, a method of decomposing benzene compounds by Xanthobacter (Helmut et al, Appl. Environ, Microbiol. 61, 3884: 1995), a method of decomposing aromatic compounds by immobilizing microorganisms (Rehm et al. , Appl. Microbiol. Biotechnol. 39, 110: 1993). In addition, the use of protozoa and welfare animals (Nathscka et al. 55, 705: 1981), and research on the application to the actual wastewater treatment using microorganisms.

That is, there are mainly three steps as a treatment method using such microorganisms, firstly, activation of existing useful microorganisms which artificially multiply microorganisms existing in the environment of a polluted site, and secondly, decomposing microorganisms separated from nature. Environmental introduction of natural and foreign useful microorganisms to be sprayed on the polluted site by mass cultivation Lastly, it introduces the environment of genetically engineered useful microorganisms obtained artificially degrading ability through chemical manipulation. Therefore, biological treatment has many advantages such as improving treatment water quality, saving facility and energy, and reducing sludge generation. Therefore, it has recently established a technology for separating, selecting, dominant, and concentrating highly active microorganisms with high degradability. Many researches are being conducted to apply wastewater treatment to industrial sites.

In biological treatment using microorganisms, there are many challenges such as stabilizing and improving the treated water quality, minimizing the facilities and conserving energy, reducing the amount of sludge generated and recovering the available resources. What is important in solving this problem is whether it is possible to maintain useful microorganisms in the reactor at high density, and is it possible to establish a system that can actively distribute the developed technology. To establish this technique, the following considerations should be taken into account when separating and identifying microorganisms. In spontaneous generation, it selects and isolates microorganisms that decompose time-consuming organic substances and other chemical substances, repeats the enrichment process at high concentration, and measures and selects and isolates specific enzymes involved in the decomposition of organic substances. It should be able to confirm the treatability and be able to dominate these useful microorganisms at high concentrations and maintain them through continuous monitoring.

Biological wastewater treatment is to remove mixed substrates by mixed cultured microorganisms, and there are aerobic treatment and anaerobic treatment. Aerobic treatment is to obtain energy by oxidatively decomposing organic matter in wastewater by using aerobic microorganisms in the presence of oxygen, as shown by the following reaction route, and synthesize a new cell using a part of this energy.

Organics + O ₂ → CO ₂ + H ₂ O + Energy

Organics + NH ₃ + O ₂ → Cells + CO ₂ + H ₂ O ± Energy

Cell + O ₂ → CO ₂ + H ₂ O + NH ₃ + Energy

BOD sources in wastewater are organic substances and their main components are classified into sugars, fats, and proteins. These compounds are decomposed by redox, hydrolysis, and deamino action of microorganisms. The digestion pathway of sugars, lipids and proteins is summarized as follows.

Therefore, screening microorganisms with excellent enzymatic activity that breaks down carbohydrates (starch, fiber), fat, and proteins, which are the main components of these organic substances, and removing these organic substances using these microorganisms together improves the treatment efficiency of organic wastewater. It can be called a method.

The inventors of the present invention were in charge of Pseudomonas CJ-P01 during the testing of isolates of microorganisms in various industrial wastewater treatment plants and livestock and slaughterhouses to measure the activity of each enzyme involved in the decomposition of organic substances. The microorganisms identified as having excellent protease enzymatic activity and the ability to treat glycoprotein wastewater completed the present invention.

The present invention provides a novel microorganism Pseudomonas CJ-P01, which has good growth under aerobic conditions, has excellent protease activity, and is excellent in treating glycoprotein wastewater. The strain of the present invention was deposited with the Korean spawn association on accession number KFCC-11052 on October 24, 1998.

The present inventors separated the microorganisms by using water and soil around the stream from which industrial wastewater treatment plants, livestock and slaughterhouses, and livestock slaughterhouses run out in Seoul, Gyeonggi-do, Gangwon-do, and Chungcheong-do. From the isolated microorganisms, the activity of protease, an enzyme involved in organic degradation, was measured, and the microorganisms that showed excellent activity were firstly screened. Then, the microorganisms having high removal rate of biochemical oxygen demand (BOD) for the glycoprotein wastewater were selected. Final selection identified Pseudomonas CJ-P01.

Microorganism Pseudomonas CJ-P01 according to the present invention forms round and convex colonies on the composite medium and its properties are described in Table 1, Table 2 and Table 3 below.

Morphological and Cultural Characteristics of Pseudomonas CJ-P01 Item Characteristics Gram dyeing - shape Bacillus motility +

Physiological and Biochemical Properties of Pseudomonas CJ-P01 Item Characteristics Hemolytic + Oxidase + Catalase + Oxygen composition Aerobic Indole formation - Reduction of nitric acid to nitrite + Urease formation + Methyl red test + Starch hydrolysis - Gelatin hydrolysis + Sight rate test +

Utilization of Organic Compounds of Pseudomonas CJ-P01 Item Characteristics Glucose + Fructose + Xylose + Glycerol + Sorbitol + Mannitol + Raffinose + Ramrose + m-inositol +

In another aspect, the present invention provides a method for treating glycoprotein wastewater using microorganism Pseudomonas CJ-P01, which has good growth under aerobic conditions and has excellent protease activity.

In another aspect, the present invention provides a method for separating Pseudomonas CJ-P01 strain, characterized in that the detection with a mercury chloride solution in a medium consisting of nutrient broth, sodium chloride and gelatin.

The following examples illustrate the invention. However, these examples are intended to explain the invention in more detail and should not be understood as limiting the scope of the invention as these.

(Example 1)

Pseudomonas genus CJ-P01 microorganism with excellent protease activity and excellent treatment of glycoprotein synthetic wastewater

Isolation of Strains and Measurement of Enzyme Activity

In order to isolate microorganisms with excellent protease enzyme activity and excellent treatment of glycoprotein synthesis wastewater, samples from wastewater treatment plants in meat processing factories in Seoul, Gyeonggi-do, Gangwon-do and Chungcheong-do are acclimated to the glycoprotein synthesis wastewater for a certain period of time. The microorganisms were suspended in sterile saline, and the LB medium was plated in a plate medium made by dissolving LB medium in 1 liter of meat wastewater (filtered with a sterile filter at 0.2 μm). The petri dishes were incubated for 1 to 3 days in an incubator at 25 to 30 ° C. to isolate 30 types of microorganisms showing excellent growth on plate medium. In order to measure the protease enzyme activity against isolated microorganisms, one platinum tooth was inoculated into a plate medium made by dissolving the protease enzyme activity measuring medium (nutrition juice juice 8g, sodium chloride 5g, gelatin 10g, agar 16g) in 1 liter. When done, add one or two drops of mercuric chloride solution to the cells. A few seconds later, when the surrounding cells did not respond to the mercury chloride solution and formed a transparent area, the strain was determined and selected as a protease activity. Afterwards, five strains with relatively broad regional bands were selected. On the other hand, inoculated with one platinum in a culture test tube containing 5 ml of LB medium of the above composition except for agar, and incubated for 24 hours at 25 ~ 30 ℃ in a shaking rotation incubator and the culture medium in a centrifuge Cells were recovered by centrifugation for 5 minutes. Some of the recovered microorganisms were used in subsequent experiments and some were lyophilized and stored.

Selection of Strains with Glucoprotein Synthetic Wastewater Treatment Capacity

After suspending the first five selected microorganisms in sterile distilled water, each microorganism was inoculated by 1% (v / v) to the glycoprotein synthetic wastewater adjusted to 200ppm, 1000ppm, 3000ppm (BOD) and shaken. The incubator was incubated at 25-30 ° C. The growth was then observed, and a certain amount of culture was aseptically collected and processed in a centrifuge, and then BOD was measured using the supernatant to compare the removal ability of organic matter.

As shown in FIG. 1, the growth of the saccharide protein synthetic wastewater adjusted to 200 ppm, 1000 ppm, and 3000 ppm (BOD) was observed for 24 hours. As a result, microorganisms identified as Pseudomonas CJ-P01 were superior to other microorganisms. In addition, the removal ability of the organic matter was compared with respect to the saccharide protein synthetic wastewater adjusted to the 200 kinds, 1000 ppm, 3000 ppm (BOD) of the five kinds of microorganisms. The composition of the glycoprotein synthetic wastewater used by the present inventors was 20 g / l of glucose, 10 mg / l of yeast extract, 0.1 g / l of sodium chloride, 10 g / l of ammonium sulfate, 0.5 g / l of potassium phosphate, 0.2 g / l of magnesium sulfate, and 5 mg of iron chloride. / g, calcium chloride 50mg / l based on the composition of peptone 3g / l, tryptone 3g / l, beef extract 3g / l was used and diluted to the appropriate concentration as needed.

After the cells collected in the centrifuge were suspended in sterile distilled water, 1% (v / v) of each microorganism was inoculated into the saccharide protein synthetic wastewater adjusted to 200 ppm, 1000 ppm, and 3000 ppm (BOD). After inoculation, aseptically collect a certain amount of the culture solution at 25 to 30 ℃ in a shaker incubator, dilute the appropriate amount, measure the BOD using the supernatant treated in a centrifuge, and treat the organic matter of each microorganism in each wastewater. Was determined. As a result, as shown in Fig. 2, the microorganisms identified as Pseudomonas CJ-P01 exhibited excellent BOD removal rate for the saccharide protein synthetic wastewater adjusted to 200ppm, 1000ppm, and 3000ppm (BOD). Final screening.

Pseudomonas CJ-P01's glycoprotein wastewater treatment ability, separated and screened at industrial wastewater treatment plants, can be used for the treatment of on-site wastewater, and can be applied to various bioremediation technologies including environmental treatment agents using microorganisms. It is expected to be.

1 is a graph showing the growth rate of the saccharide protein synthetic wastewater of the novel microorganism Pseudomonas sp. CJ-P01 of the present invention.

Figure 2 is a graph showing the removal rate of biochemical oxygen demand (BOD) when treating the glycoprotein synthesis wastewater using the novel microorganism Pseudomonas CJ-P01 of the present invention.

Figure 3 is an electron micrograph (x8000) of the novel microorganism Pseudomonas CJ-P01 of the present invention.

Claims (3)

Microbial Pseudomonas genus CJ-P01 with good growth under aerobic conditions, good protease activity and excellent treatment of saccharide starch wastewater (Deposited date: October 24, 1998; Accession No .: KFCC-11052). Method for treating glycoprotein wastewater using the microorganism of claim 1. A method for separating the microorganism of claim 1, characterized by detecting with a mercury chloride solution in a medium consisting of nutrient broth, sodium chloride and gelatin.