KR102255930B1 - Phlebiopsis crassa NNIBRFG4544 strain isolated from freshwater having lignin degradation activity and uses thereof - Google Patents

Abstract

Disclosed are microorganisms that is not known as microorganisms exhibiting lignin decomposition activity to be utilized as a novel use and new strains, a composition for decomposing lignin using the same, and a method for decomposing lignin using the same. The present invention provides Phlebiopsis crassa and a new strain Phlebiopsis crassa NNIBRFG4544 strain having lignin degradation activity, a composition for decomposing lignin using the same, and a method for decomposing lignin using the same.

Description

Phlebiopsis crassa NNIBRFG4544 strain isolated from freshwater having lignin degradation activity and uses thereof}

The present invention relates to a microorganism having a lignin decomposing activity, and more particularly to a microorganism having a decomposing activity for lignin contained in industrial wastewater and discharged in the paper industry.

The paper industry is a representative industry that generates a large amount of industrial wastewater, and the number of industrial wastewater discharging businesses is 1,960, accounting for only 3.8% of the total, but the daily wastewater generation amount is 487,000㎥/day, which is 9.6% and the amount of wastewater discharge is 340,000㎥/day. It reaches 9.0%.

Wastewater generated by the paper industry contains a large amount of non-degradable organic substances, and a representative non-degradable organic substance is lignin. Lignin is a material that acts like an adhesive that connects the fibers that make up wood and is a high molecular organic compound essential for maintaining the structure of wood. Due to these characteristics, a process of removing lignin from wood is indispensable when producing pulp in the paper industry, and most of the lignin removed in this way is recovered through a recovery process and used in the production of other materials. However, lignin that has not been recovered in this process is included in the wastewater of the paper mill and is discharged.The lignin introduced into the river through the wastewater deteriorates the light transmittance of the river due to its unique color, thereby interfering with the photosynthesis of underwater plants. As a result, the dissolved oxygen is depleted. In addition, it can be said that it is very important to prevent lignin from entering the river because it also causes deterioration in water quality due to toxicity of lignin itself or high COD.

Since the Nakdong River Water System Act was enacted in 2002, the Ministry of Environment has invested more than 2 trillion won to improve the water quality of the Nakdong River until 2017.Through this, the concentration of biodegradable pollutants such as BOD, nitrogen, and phosphorus has improved considerably, but is not measured as BOD It was investigated that the number of non-degradable pollutants measured only by COD was rather increased. This is because most of the water quality improvement projects have been conducted with the aim of reducing BOD, and it can be seen that the proportion of non-degradable pollutants contained in industrial wastewater has increased due to changes in the industrial structure.

Microorganisms used in most biological wastewater treatment processes are bacteria, which have a long history of process development and utilization research, but are relatively easy to handle and have high decomposition efficiency of biodegradable pollutants. However, in the case of bacteria, not only the decomposition efficiency of non-degradable contaminants is very low, but depending on the type of contaminant, it is also hit to the point of causing a problem in the stability of the process.

In the case of fungi to which fungi or mushrooms belong, the rate of decomposition of general organic substances is slower than that of bacteria, but due to the nature of feeding organic substances having a complex structure, there is a high possibility as a resource that can treat non-degradable contaminants. In fact, fungi that decompose non-degradable organic substances such as PVA (polyvinyl alcohol) and PAH (polyaromatic hydrocarbon) have been steadily reported since the late 1990s, and studies on enzymes involved in decomposition are also actively progressing.

Studies on the degradation of lignin using fungi have been mainly focused on white rot fungi. White rot is a fungus commonly found in decaying trees, and is a representative fungus that has lignin-degrading enzymes such as laccase and manganese oxidase (MnP). Phanerochaete chrysosporium (Phanerochaete chrysosporium) species, which has been the most studied lignin decomposition, has many studies on lignin decomposition activity and pollutant decomposition using the same.Patent No. 0797540 is white Disclosed is a technology for providing a lacase gene, a lignin-degrading enzyme, using winter umbrella mushrooms, which are swelling bacteria.

However, there is a need to discover a variety of fungi that can be applied as microorganisms having lignin-decomposing activity, but little research has been done on this, and further, fungi having lignin-decomposing activity other than white rot is not suggested.

Accordingly, the present invention is to present a microorganism and a new strain, a composition for decomposing lignin using the same, and a method for decomposing lignin using the same, a microorganism and a new strain that are not known as microorganisms exhibiting lignin decomposing activity until now.

In order to solve the above problems, the present invention provides a Phlebiopsis crassa having a lignin decomposition activity.

In order to solve the another problem, the present invention provides a novel microorganism Phlebiopsis crassa NNIBRFG4544 (accession number: KCTC13958BP) strain.

In addition, the strain provides a Pleviopsis crassa NNIBRFG4544 (accession number: KCTC13958BP) strain, characterized in that it has a lignin degrading activity.

In addition, the strain provides a Phlebiopsis crassa NNIBRFG4544 (accession number: KCTC13958BP) strain, characterized in that it is isolated from fresh water.

In order to solve the above another problem, the present invention provides a composition for decomposing lignin containing the strain or a culture material thereof as an active ingredient.

In order to solve the another problem, the present invention provides a method of decomposing lignin by treating the composition in a solution containing lignin.

According to the present invention, it is possible to provide a microorganism Phlebiopsis crassa and a new strain Phlebiopsis crassa NNIBRFG4544 strain that can be used for new purposes having lignin-degrading activity in addition to the existing white decay bacteria. .

These microorganisms and new strains according to the present invention have excellent lignin decomposition performance and are very suitable for a biological treatment process of papermaking or dyeing wastewater, and have a very high value as a biological resource that can be used in related industries.

1 is a photograph showing the result of confirming that the NNIBRFG4544 strain according to the present invention is a pure culture strain by subculturing it on a PDA plate medium;
2 is a view showing the LSU nucleotide sequence of the NNIBRFG4544 strain according to the present invention,
Figure 3 is a view showing the results of analyzing the phylogenetic diagram for molecular biological identification of the NNIBRFG4544 strain according to the present invention based on the LSU nucleotide sequence,
5 is a graph showing the measurement result of lignin degradation activity of the NNIBRFG4544 strain according to the present invention,
Figure 6 is a photograph showing a culture solution filtered after culturing the NNIBRFG4544 strain according to the present invention and the control and other comparative strains in a liquid medium containing lignin for 7 days.

Hereinafter, the present invention will be described in detail through examples. Prior to this, terms or words used in the specification and claims should not be construed as being limited to their usual or dictionary meanings, and the inventors appropriately explain the concept of terms in order to explain their own invention in the best way. Based on the principle that it can be defined, it should be interpreted as a meaning and concept consistent with the technical idea of the present invention. Therefore, the configuration of the embodiments described in the present specification is only the most preferred embodiment of the present invention, and does not represent all the technical spirit of the present invention, and various equivalents and modifications that can replace them at the time of the present application It should be understood that there may be.

The present inventors obtained samples from various freshwaters nationwide to find freshwater fungi having lignin-degrading activity, and among them, NNIBRFG4544 strain isolated from freshwater in Danjang-myeon, Miryang-si, Gyeongnam, was obtained, and to conduct molecular biological identification of purely isolated freshwater fungi, LSU As a result of nucleotide sequence analysis, it was identified as Phlebiopsis crassa. The strain was cultured in a lignin-containing medium and the lignin degradation rate was measured to confirm that the lignin degradation activity was very excellent, and the present invention was reached.

Accordingly, the present invention discloses a lignin (lignin) decomposing activity of Pleviopsis crassa (Phlebiopsis crassa), and a novel microorganism Phlebiopsis crassa (Plebiopsis crassa) NNIBRFG4544 (accession number: KCTC13958BP) strain.

The present invention is to present a new use of a specific microorganism, Phlebiopsis crassa having lignin-degrading activity, and therefore, as a strain of Phlebiopsis crassa , it is used for decomposing lignin. It should be seen that the strain, the composition for decomposing lignin using the same, or the method for decomposing lignin using the same, all belong to the technical concept of the present invention.

It was confirmed that the strain according to the present invention has excellent lignin degradation activity.

According to an application example of the present invention, there is provided a composition for decomposing lignin containing the strain or a metabolite thereof as an active ingredient.

In the present invention, the strain or its metabolite may be used in the state of the cells of the strain, the culture medium of the strain, the culture filtrate of the strain, and the like. Here, the microbial cells of the strain mean all of the microbes themselves, and the culture medium means a culture solution containing the microbial cells, and the culture filtrate means a culture solution containing no cells.

According to another application example of the present invention, the method of decomposing lignin may be performed by treating an effective amount of the strain or a metabolite thereof in a solution containing lignin.

Hereinafter, the present invention will be described in detail by way of examples.

Example 1: Isolation of freshwater fungus NNIBRFG4544 strain having lignin degradation activity

In the present invention, freshwater was collected in Danyang-myeon, Miryang-si, Gyeongsangnam-do, in 2017, as shown in the strain collection information in Table 1, in order to select freshwater fungi having lignin-degrading activity. To purely separate freshwater fungi from the collected fresh water, filter the filter paper with a filter paper with 0.2 µm pores and place the filter paper on 1.5% water agar medium supplemented with antibiotics (streptomycin, ampicilin) at 25℃, in dark conditions. Cultured in. After 1 day of cultivation, the filter paper was removed, and the grown mycelium was separated from the water agar medium, inoculated into a 24-well plate V8 medium, and cultured, and the fungi grown in the V8 medium were again subcultured in PDA medium several times. Pure separation.

Collection area Year of collection water system Separation Danyang-myeon, Miryang-si, Gyeongsangnam-do 2017 Nakdong River (Danjangcheon) fresh water

Example 2: Identification of freshwater fungus NNIBRFG4544 strain having lignin degradation activity

1 is a photograph showing the result of confirming that the NNIBRFG4544 strain according to the present invention is a pure culture strain by subculturing it on a PDA plate medium. To carry out molecular biological identification of freshwater fungi isolated as shown in FIG. 1, the LSU (Large subunit of ribosomal DNA) nucleotide sequence was analyzed, and the results are shown in FIG. 2 (SEQ ID NO: 1). The primers used are shown in Table 2 below.

primer
name Forward (5'-3')
Primer sequence primer
name Reverse (5'-3')
Primer sequence LR0R ACCCGCTGAACTTAAGC LR7 TACTACCACCAAGATCT

As a result of BLAST of NCBI for the LSU sequence, the NNIBRFG4544 strain was identified as Phlebiopsis crassa. In addition, as shown in Fig. 3, in the phylogenetic analysis using the LSU nucleotide sequence of a related species, it showed 99.91% similarity with Phlebiopsis crassa strain KKN-86, and morphological observation according to mycelial growth, spore shape, size, etc. Finally, the strain was named as Phlebiopsis crassa NNIBRFG4544 strain, and it was entrusted to the Korean Collection for Type Cultures (KCTC) on September 23, 2019, and the accession number KCTC13958BP was established. Was granted.

Example 3: Confirmation of lignin degradation activity of Phlebiopsis crassa NNIBRFG4544 strain

In order to confirm the lignin degrading activity of the Phlebiopsis crassa NNIBRFG4544 strain isolated and identified in Examples 1 and 2, culture in PDA medium containing lignin (dark culture at 25°C for 7 days) It was confirmed whether or not a bleached area in which brown peculiar to lignin disappears occurred, and the results are shown in FIG. 4.

Referring to Figure 4, in the range in which the NNIBRFG4544 strain grows, a bleaching region in which the color peculiar to lignin disappears is clearly identified, from which it is proved that the NNIBRFG4544 strain according to the present invention has excellent lignin decomposition activity.

Example 4: Pleviopsis crassa ( Phlebiopsis crassa ) Measurement of lignin degradation activity of NNIBRFG4544 strain

In order to measure the lignin degradation activity of the Phlebiopsis crassa NNIBRFG4544 strain isolated and identified in Examples 1 and 2, agar of the NNIBRFG4544 strain was added to 150 ml of PDB medium containing lignin at a concentration of 0.5 g/L. Five plugs (agar plug) were inoculated and cultured for 7 days at 25°C and 150 rpm. During the incubation period, 2 ml of the culture solution was taken once a day, filtered using a 0.45 μm syringe filter, and the absorbance at a wavelength of 245 nm was measured. As a control, a lignin-containing PDB medium not inoculated with the strain was treated under the same conditions, and the absorbance measurement was performed in the same manner, and the results are shown in FIG. . For comparison, the results of measuring the activity of the Phlebiopsis crassa NNIBRFG4544 strain and other strains (see Table 3 below) isolated together with the NNIBRFG4544 strain according to the present invention are shown together.

Strain name Microorganism name Strain name Microorganism name NNIBRFG4291 Ganoderma carnosum NNIBRFG6074 Peniophora incarnata NNIBRFG4524 Bjerkandera sp. NNIBRFG6481 Paraphaeosphaeria sporulosa NNIBRFG4544 Phlebiopsis crassa NNIBRFG6483 Penicillium sp. NNIBRFG4547 Bjerkandera sp. NNIBRFG6496 Pholiota adiposa NNIBRFG4639 Fomitopsis sp. NNIBRFG6506 Paraphaeosphaeria sporulosa NNIBRFG4928 Lenzites betulina NNIBRFG6507 Penicillium sp. NNIBRFG6047 Penicillium brevicompactum - -

5 and 6, in the case of the Phlebiopsis crassa NNIBRFG4544 strain according to the present invention, the lignin concentration in the medium decreases to 0.167 g/L, resulting in a lignin decomposition rate of 0.048 g/L·day. It can be seen that the lignin degradation activity is significantly superior compared to the control and other strains.

The preferred embodiments of the present invention described above are disclosed to solve the technical problem, and those of ordinary skill in the art to which the present invention pertains will be able to make various modifications, changes, additions, etc. within the spirit and scope of the present invention. , Such modifications and changes should be seen as falling within the scope of the following claims.

Korea Research Institute of Bioscience and Biotechnology KCTC13958BP 20190923

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Claims (6)

delete A novel microorganism having lignin decomposing activity Phlebiopsis crassa NNIBRFG4544 (Accession No.: KCTC13958BP) strain. delete The method of claim 2,
The strain is Phlebiopsis crassa NNIBRFG4544 (accession number: KCTC13958BP) strain, characterized in that isolated from fresh water. A composition for decomposing lignin containing the strain of claim 2 or 4 or a culture material thereof as an active ingredient. A method of decomposing lignin by treating the composition of claim 5 in a solution containing lignin.

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