KR20030071077A - Microbacterium sp. PA5(KCTC 10172BP) capable of degrading lignin - Google Patents

Abstract

PURPOSE: A microorganism Microbacterium sp. PA5(KCTC 10172BP) capable of degrading lignin is provided, thereby effectively purifying organic waste water. CONSTITUTION: A microorganism Microbacterium sp. PA5(KCTC 10172BP) capable of degrading lignin or derivatives thereof is provided, wherein the derivative of lignin is selected from vanillin, phenol, protocatechuic acid, syringic acid, vanillic acid, syringaldehyde, salicylic acid and 2,4-dinitrotoluene; and Microbacterium sp. PA5(KCTC 10172BP) is isolated by the steps of: collecting samples from the soil; culturing the soil samples on plate counting agar at 30 deg. C for 48 hours to isolate 20 strains; inoculating 20 strains in a nutrient medium containing vanillin cophenyl alcohol and culturing them at 30 deg. C for 48 hours to select 8 strains showing good growth activity; and culturing 8 strains in organic waste water containing vanillin cophenyl alcohol to select one strain showing the excellent growth activity and vanillin cophenyl alcohol decomposing activity.

Description

PB5 (BPCT 10172 JP) in microbacterium having lignin degrading activity {Microbacterium sp. PA5 (KCTC 10172BP) capable of degrading lignin}

The present invention relates to a microbacterium genus PA5 (KCTC 10172BP) having lignin-degrading activity, and more particularly, to a new strain microbacterium genus PA5 capable of selectively degrading lignin or a derivative thereof, which is a hardly degradable substance. (KCTC 10172BP) and a method for using this strain to purify wastewater by reducing the chemical oxygen demand (COD) of organic wastewater containing a large amount of hardly degradable material.

The rapid development of the industry and the exponential growth of the population are causing environmental pollution all over the world, and to the extent that it threatens human survival. In addition, as the seriousness of natural destruction such as the increase in the use of fossil fuels and the rapid increase in pollutant emissions is on the rise, research on countermeasures and treatment techniques for directly or indirectly affecting human life is being actively conducted. Industrial wastewater, sewage, and sewage, which are one of the main causes of such environmental pollution, are deteriorating the water quality of rivers and groundwater (Korean Patent Application No. 2000-0022425).

Generally, industrial wastewater treatment technology is divided into three types (chemical, physical, and biological methods), of which biological control method is artificially controlled and managed under certain conditions by using self-cleaning, which is the principle of natural ecosystem material circulation. Compared with physical and chemical treatment methods, secondary pollution is less likely and more economical and efficient. Therefore, it is suitable as a treatment method for organic wastewater (sewage, paper, food wastewater, etc.). Microorganisms involved in such biological treatment include bacteria, yeast, fungi, algae, protozoa, and the like, which are mainly used for wastewater treatment, bacteria and protozoa. The core of biological treatment can be seen as the decomposition of organic materials, hardly decomposable substances, etc. by microorganisms such as bacteria. Even now, researches on the degradation of microorganisms have been conducted in various ways, and in particular, attention has been focused on selecting and using strains having the ability to degrade hardly decomposable substances (Korean Patent Application No. 1998-46886). Protozoa are the final predators of bacteria that degrade BOD (Biochemical Oxygen Demand) or COD-causing agents, are indicators of wastewater treatment, but are more sensitive to changes in the surrounding environment than bacteria, Hard to find

As such, biological treatment of wastewater has many advantages, and many studies are being conducted to develop technology for separating, selecting, dominant and increasing concentration of microorganisms having excellent degradation activity, and applying them to industrial wastewater treatment.

In general, the Fenton treatment method is the most widely used wastewater treatment method for industrial wastewater containing a high concentration of COD or containing a large amount of organic matter, which was published by Fenton in 1894. Oxidation treatment method used. In addition, the wastewater treatment method widely used in paper wastewater, which is currently used for industrial wastewater, can be summarized by the following three chemical reaction equations, which generate iron hydroxide to decompose the hardly decomposable organic substances in the wastewater [Korea Patent Application No. 2001-53003].

^{_{Fe 3 + + H 2 O 2}} → Fe 3 + + OH - + OH and

RH + ㆍ OH → R · + H ₂ 0

_{^{Fe 3 + + 3OH - → Fe}} (OH) 3

However, a disadvantage of this process is that the amount of hydrogen peroxide reacts sensitively to the Fenton reaction, thereby reducing the treatment efficiency. In other words, when the amount of hydrogen peroxide is small, oxidative aggregation is weak, and when the excessive amount is added, the aggregated organic material is oxidized again to increase the COD.

In contrast, biological reactions are relatively easy with chemical catalysts, which are difficult to occur under normal in vitro conditions.

One of the difficult problems in the biological treatment of papermaking wastewater was the inability to biodegrade phenolic lignin model compounds, which are lignin degradation products. Phenolic lignin model compounds include catechol, ferulic acid, guaiacol, phenol, protocatechuic acid, syringic acid, and vanillic acid. vanillic acid) [P. Guiraud, R. Steiman, L. Ait-Laydi, and F. Seigle-Murandi. 1999. Degradation of phenolic and chloroaromatic compounds by Coprinus spp. Chemosphere . 38. 2775-2789]. The phenolic compound is a toxic hardly decomposable compound, and in the case of paper wastewater, which is an industrial wastewater in which organic compounds are present, there are many difficulties in reducing COD during treatment [P. Guiraud, F. Seigle-Murandi, R. Steiman, and JL Benoit-Guyod. 1995. Comparison of the toxicity of various lignin-related phenolic compounds towards selected Fungi Perfecti and Fungi Imperfecti . Ecotox. Environ. Saf. 32. 29-33.

Therefore, a lot of researches have recently been conducted to improve the water quality, reduce facilities and energy, and reduce sludge production through biological treatment, and to apply it to the establishment of technology using microorganisms with excellent degradability and the actual industrial wastewater treatment. .

Paper wastewater is a wastewater discharged after a series of processes such as pulp regeneration, processing, and bleaching. It contains a large amount of hardly decomposable polymer compounds, and is used to treat chemical, biological, and physical wastewater treatment. It is true.

Conventional domestic paper wastewater treatment technology has focused on lignin and efforts to remove it [Korean Patent Application No. 2000-7775; AT Martinez, S. Camarero, A. Guitierrez, P. Bocchini, and GC Galletti. 2001. Studies on wheat lignin degradation by Pleurotus species using analyticalpyrolysis. J. Anal. Appl. Pyrolysis. 58/59. 401-411; TK Kirk. 1984. Degradation of lignin. In Microbial Degradation of Organic Compounds. New York, Dekker. 399-437; MSA Leisola, DC Ulmer, R. Waldner, and A. Fiechter. 1984.Role of veratryl alcohol in lignin degradation by Phanerochaete chrysosporium . J. Biotechnol. 1. 331-339.

However, even when these polymer compounds are treated, by-products generated by secondary decomposition remain in the wastewater. In particular, lignin contains a lot of halogen-containing cyclic compounds, and these cyclic compounds are difficult to withstand decomposition chemical reactions by oxidation.

In order to deal with this, additional costs are required such as recirculation and longer residence times in the aeration tank.

Therefore, as the final product is confirmed that vanillin cophenyl alcohol is present in the wastewater through the action of alkaline hydrolysis in the process of papermaking wastewater, a method for reducing it has emerged as a new task.

Therefore, the present inventors have studied to solve the above problems, as a result, it is isolated PA5 (KCTC 10172BP) in the new strain microbacterium that can decompose the lignin or derivatives of the hardly decomposable substance, and the organic wastewater with this strain In particular, the present invention has been completed by purifying the wastewater by removing the hardly decomposable substance contained in a large amount of paper wastewater.

Therefore, an object of the present invention is to purify wastewater by reducing COD of organic wastewater using PA5 (KCTC 10172BP) in the new strain microbacterium that can selectively decompose hardly decomposable substances in organic wastewater.

Figure 1 shows the results of 16s rRNA identification of PA5 (KCTC 10172BP) genus microbacterium.

The present invention is characterized by a microbacterium sp. PA5 (KCTC 10172BP) of the genus Microbacterium genus PA5 having lignin or derivatives thereof.

It also includes a method for purifying wastewater containing lignin or derivatives thereof using the strain.

The present invention will be described in more detail as follows.

The present invention relates to chemical oxygen of organic wastewater containing PA5 (KCTC 10172BP) in a new strain microbacterium capable of selectively degrading lignin or a derivative thereof, and a large amount of hardly decomposable substances using the strain. A method of purifying wastewater by reducing the required amount of COD.

First, water and soil around the industrial wastewater treatment plant and streams from which the wastewater flows out are collected and used as a sample for strain separation.

Finally, 8 strains were selected, and microorganisms having the highest resolution of lignin derivatives were identified. As a result, they were identified as mycorrhizal bacteria having similar properties to the genus Microbacterium sp. Microbacterium sp. PA5). It was deposited on February 16, 2002 to the Korea Biotechnology Research Institute Gene Bank. The accession number is KCTC 10172BP.

The strain has excellent growth under aerobic conditions, and has an excellent ability to degrade lignin or its derivatives. Lignin derivatives include vanilline, cophenyl, phenol, protocatechuic acid, syringic acid, vanillic acid, syringaldehyde, Salicylic acid, 2,4-dinitrotoluene, and the like are preferable.

The strain is used to measure the removal efficiency of lignin or derivatives thereof in the organic wastewater, and to determine the treatment efficiency by measuring the COD value in the wastewater.

The strain can be usefully used for wastewater treatment by reducing the COD value of the wastewater containing a large amount of hardly decomposable substances.

Hereinafter, the present invention will be described in detail based on the following examples, but the present invention is not limited thereto.

Example 1 Microbial Separation

About 10 strains of soil and sludge near the paper wastewater treatment plant were purified by using plate counting agar medium. 0.1 g of the sample was suspended in 9.9 ml of physiological saline (NaCl 0.85%), diluted 10 ⁵ times, and plated in agar medium (tryptone 0.5%, yeast extract 0.25%, glucose 0.1%, agar 1.8%), followed by 48 at 30 ° C. Time incubation. The microbial colonies formed after the cultivation were purely isolated from the strains by a screening method in agar medium.

About 20 strains isolated from pure isolates were inoculated in nutrient medium containing vanillin cophenyl alcohol, shaken and cultured at 30 ° C. for 48 hours, and finally, eight strains having excellent growth degree were finally selected by measuring absorbance at 660 nm.

Among the 8 selected strains, strains with excellent cell growth and vanillin cophenyl alcohol degradability in actual organic wastewater containing vanillin cophenyl alcohol were selected and named as PA5.

Example 2: Identification of Microorganisms

Types of strains obtained from microbial microorganisms with excellent enzymatic activity and proliferation degree for sewage treatment can be referred to the Manual of Determinative Bacteriology and Manual for the Identification of Medical Bacteria. Strain identification was performed after examining the physiological, physiological and biochemical properties.

As a result, the isolated strain PA5 forms a circular colony in a solid medium, and the colony color was pale yellow. This isolate is a Gram-positive bacterium with a growth pH range of 6 to 8, a growth temperature range of 25 to 35 ° C, a cultivated anaerobic bacterium capable of growing under aerobic and anaerobic conditions. . In addition, the physiological characteristics are as shown in Table 1 below, and comparing the DNA homology [Fig. 1], 99% (472/474), microbacterium ( Microbacterium schleiferi ) (ATCC Y17237) and micro It was confirmed that it has 98% (497/504) homology with PCOB-2 in the bacterium (ATCC Y07842).

From the above results, the isolated strain was identified as mycorrhizal fungus with similar properties to the genus Microbacterium, which was genus Microbacterium. PA5 (Microbacteriumsp. It was named PA5) and was deposited with the Korea Biotechnology Research Institute Gene Bank (KCTC) on February 16, 2002 and was given accession number KCTC 10172BP.

Glycosylation of Isolated Strain PA5 (BIOLOG) Item result Item result water - p -hydroxy phenylacetic acid - α-cyclodextrin - 3-methyl-glucose - dextrin + α-methyl-D-glucoside - glycogen - α-keto glutaric acid - Tween40 + α-keto valeric acid - Tween80 - L-lactic acid v β-cyclodextrin - α-methyl-D-mannoside - N-acetyl-D-glucosamine - propionic acid - inulin - palatinose + L-arabinose - D-ribose - D-arabitol - salicin - cellobiose + succinic acid - mannan - sedoheptulosan - D-fructose + succinamic acid - L-fucose - stachyose - D-galactose - alaninamide - gentiobiose - D-alanine - α-D-glucose + L-alanine - m-inositol - L-alanyl-glucine - α-D-lactose - L-asparagine - lactulose - D-tagatose - maltose + L-glutamic acid - D-mannitol + D-xylose + D-mannose + glycyl-L-glutamic acid - D-melibiose - lactamide - β-methyl-D-glucoside - D-lactic acid methyl ester - D-psicose - D-malic acid - D-raffinose - L-malic acid - L-rhamnose - methyl pyruvate - D-sorbitol - mono-methyl succinate + sucrose + L-pyroglutamic acid - D-trehalose + pyruvic acid - turanose + L-serine - xylitol - 2,3-butanediol - methyl pyruvate - adenosine v mono-methyl succinate + 2-deoxy adenosine v acetic acid - adenosine-5-monophosphate - amygdalin - inosine - arbutin - uridine - maltotriose + thymidine - D-melezitose + thymidine-5-monophosphate - D-galacturonic acid - putrescine - D-gluconic acid v uridine-5-monophosphate - α-methyl-D-galactoside - fructose-6-phosphate - β-methyl-D-galactoside - glycerol - α-hydroxy butyric acid - D, L-α-glycerol phosphate - β-hydroxy butyric acid v glucose-1-phosphate - γ-hydroxy butyric acid - glucose-6-phosphate - Note), negative (less than 20%); v, variable (20-80%); +, positive (over 80%)

Example 3: Paper Wastewater Component Analysis

First, in order to know how much lignin is present in the wastewater and how it remains, it was collected and analyzed on a regular basis to find an industry that produces paper by recycling pulp.

Raw water from paper wastewater from P's wastewater treatment plant located in J city was supplied and used as a sample. Samples were analyzed by GC-Mass (HP 5890 series II Plus, Pyrolyzer CDS PYROPROBE 2000, MASS HP 5989A) [analysis conditions: Pyr. temp. (600 ° C., 5 seconds), Inj. temp. (270 ° C.), Det. temp. (290 ° C.), Oven temp. {40 (3) -8-290 (5)}].

P, which is currently subject to sampling, is a plant that treats wastewater using the activated sludge method, which is a biological wastewater treatment method, and the COD concentration of the discharged water, which is the final treated water, was confirmed to be about 100 mg / l. The wastewater was analyzed to find the reason for no further COD treatment efficiency, and lignin derivatives were detected instead of lignin present in the water system [Table 2]. When wastewater is treated using the activated sludge method, which is a biological wastewater treatment method, large molecules of lignin are decomposed and secondary products are generated and are present in the water system.

Component Analysis of Papermaking Wastewater Using GC Mass Detection substance Retention time (minutes) phenol 11.071 Benzeneamine 11.657 2-methylphenol 12.537 3- (1-methylethyl) phenol 15.833 1,2-dihydro-1,5,8-trimethyl naphthalene 18.587 Bis (1,1-dimethylethyl) phenol 20.153 5-acetyl-6-methyl-benzimidazolone 24.290

As shown in Table 2, the secondary compound, which is not lignin, but its derivatives was detected in the actual papermaking wastewater, and these substances are classified as hardly decomposable substances, have a slight toxicity, and are a source of COD in the wastewater. Will work. Therefore, when the treatment of such secondary by-products means that the removal efficiency can be higher than the existing COD concentration.

Example 4 Removal Efficiency of Lignin Derivatives by New Strains

Using the strain of the present invention and 8 kinds of isolated strains, the artificial wastewater was prepared and treated similarly to the lignin component present in the papermaking wastewater, and then the concentration of the lignin derivative was measured to determine the removal efficiency of the lignin derivative by the isolate. It is shown in Table 3 below.

Removal Efficiency of Lignin Derivatives by Isolated Strains division Removal efficiency (%) Sirinic acid vanillin Sirinaldehyde Vanillic acid Control - - - - PK2608 26.3 22.6 17.5 18.3 PK262 37.7 31.9 31.7 29.2 PAK2603 13.5 19.4 17.2 18.6 PK265 25.1 20.4 22.8 20.1 PA5 95.2 99.1 95.9 99.2 PAK2601 13.1 19.1 11.4 17.8 PAK2607 15.4 15.3 15.1 16.0 PK264 10.6 16.1 12.2 11.4

As shown in Table 3, after treating the papermaking wastewater using about eight kinds of excellent activity, the concentration of the lignin derivative was measured, and the genus microbacterium. Removal efficiency of PA5 (KCTC 10172BP) was found to be the best among the strains investigated at 95 to 99%.

Example 5: Lignin Removal Efficiency by New Strains

Since wood, which is a raw material in pulp wastewater, contains lignin, tar, sugars, and the like, it is meaningful to quantify lignin that is difficult to decompose in water.

Therefore, this experiment measured the lignin and lignin derivatives using the Nitroso method (Pearl-Benson method), a quantitative method that pays attention to lignin [APHA, AWWA, WPCF. 1985. Standard Methods for the Examination of Water and Wastewater. 607.].

In this experiment, the influent from the site was sampled, and the control group used the current aeration tank sludge, and the experimental group was inoculated at a volume ratio of 5% using PA5 (KCTC 10172BP) in the microbacterium. The same was done.

As a result of three repeated experiments using PA5 (KCTC 10172BP) in the microbacterium according to the present invention, it was directly administered to the papermaking wastewater, and as shown in Table 4, the average removal efficiency of the control group was 78%.

Lignin Removal Efficiency in Paper Wastewater Using New Bacteria division Control 1 time Episode 2 3rd time Average Lignin removal efficiency (%) 18.2 83.6 63.6 88.5 78.6

Example 6: COD Treatment Efficiency Using New Bacteria

In order to see the effect of the removal efficiency of the lignin or lignin derivatives on the COD source using the microorganism PA5 (KCTC 10172BP) in the strain of the present invention, was carried out as in Example 5 and investigated the COD treatment efficiency The results are shown in Table 5 below.

COD Treatment Efficiency in Paper Wastewater Using New Bacteria division Control 1 time Episode 2 3rd time Average COD treatment efficiency (%) 6.3 24.9 19.5 25.7 23.4

As shown in Table 5, when the lignin component is removed using the microbacterium PA5 (KCTC 10172BP) according to the present invention, it was seen that the COD concentration was also removed. The microbacterium genus PA5 according to the present invention (KCTC 10172BP) showed an average treatment efficiency of 23%, and showed superior treatment performance compared to the control.

Therefore, the lignin removal efficiency of the isolated strain microorganism PA5 (KCTC 10172BP) obtained by the present invention was somewhat different depending on the type of wastewater, but showed an average removal efficiency of 78% or more for the control of the wastewater. In addition, it was determined that lignin derivatives, which are secondary by-products present in papermaking wastewater, affected COD sources.

As described above, the present invention, PA5 (KCTC 10172BP) in the new strain microbacterium having excellent degradation activity of lignin or its derivatives, which is a hardly decomposable substance, further improves the rate of wastewater treatment, and expands the range of treated wastewater. The present invention relates to a waste water purification method.

Therefore, the new strain according to the present invention is very useful for improving and restoring the contaminated natural environment.

Claims (3)

PA5 of the genus Microbacterium having lignin or its derivative degradation activity (KCTC 10172BP). The method of claim 1, wherein the derivative is vanilline (phenol), phenol (phenol), protocatechuic acid (protocatechuic acid), syringic acid (syringic acid), vanillic acid (syringaldehyde) (syringaldehyde), salli PA5 (KCTC 10172BP) in the microbacterium, characterized in that it is selected from salicylic acid and 2,4-dinitrotoluene. A method of purifying wastewater, characterized by reducing chemical oxygen demand (COD) of wastewater containing lignin or derivatives thereof using PA5 in the microbacterium (KCTC 10172BP).