KR101432425B1 - A novel microorganism Rhodococcus pyridinovorans and Bacillus spp., identified from lugworm and microbial cleaning agent - Google Patents

Abstract

In the present invention, coastal tidal flats and lagoons ( Perinereis aibuhitensis ) was isolated and identified to analyze the decomposition ability of organic matter. The identified microorganisms were able to degrade DNA, agar, starch and tween,
It has high protein, carbohydrate, lipid - degrading ability and resistance to salinity, so it is highly applicable to environmental purification of tidal flat contaminated with organic matter.

Description

A novel microorganism Rhodococcus pyridinovorans and Bacillus spp., Identified from lugworm and microbial cleaning agent, comprising a plurality of strains of microorganisms identified from coastal tidal flats and lagoons,

Coastal tidal and the lug which has the resolution (Perinereis: The present invention, one of endoseolpan and synthetic waste water (2 peptone and glucose ratio 8) of it to make biological environmental cleanup strategy, the main tidal contaminants in the contaminated mud flat from an organic material The present invention relates to a method for purifying an organic substance using a novel strain having salinity tolerance derived from aibuhitensis .

The intertidal zone is continuously exposed to environmental pollution due to its proximity from human settlements and is the most sensitive area in the coastal ecosystem. Therefore, understanding of the degradation of degradable materials by organisms in the intertidal zone is very important from an ecological point of view.

Because of its ability to adapt to stressful environmental conditions, researchers are encouraged to use these worms as an environment watcher, as well as a potentially important ecological species for monitoring programs .

Many benthic organisms that are fed with filtration are commonly used to remove and purify organics from water in homes and ecosystems, and several species of lupins , including Perinereis sp., Are known to be used uncontrolled in shrimp farm environments. In the study of the effects of large invertebrates on bacterial communities in the intertidal zone, oysters, clams, and mollusks are known to be the main repository of Vibrio spp.

On the other hand, endorphanol is an organochlorine insecticide developed in Germany in 1956 and is known by many trade names such as Thiodan, Cyclodan, Malix, and Thiolix. Endosulfan is a digestive-additive insecticide used in crops such as tobacco, mulberry, and cabbage. Domestic production of endosulfan in 2006 was 229 tons, and 2006 shipments were 506 tons. In Korea, in December 2004, the use of edible crops in endosulfan was prohibited in the Pesticide Management Act of 2004, and it was used only for tobacco, mulberry, and soil insect control. However, a large amount is still used for edible crops. In addition, the endotoxin is designated and managed as a substance prohibited to manufacture, import or use by the Toxic Chemical Substances Control Act of the Ministry of Environment.

Endosulfan is cheap and effective, and has been used in production farms for purposes of pest control, despite its high toxicity. Endosulfan not only exhibits significant toxicity to freshwater fish species at very low concentrations but also can interfere with human or animal hormonal systems as an endocrine disruptor, leading to high risks. Nonetheless, endosulfan is an organic chlorine - based insecticide that has been widely used for pest control until recently in Korea and the United States, and is one of the major environmental pollutants.

Endosulfan is an acute toxic, highly toxic pesticide, a highly toxic first class pesticide. Anno sulphate is a fairly stable compound that is not degraded by sunlight in nature, and its half life in soil is very persistent, at least from 6 months to 9 years. Therefore, the remaining endosulfan after it is sprayed on agricultural lands may enter the groundwater, rivers and coasts, and may remain in the soil for a long time and affect the ecosystem.

Although endosulfan has been banned for use in edible crops in Korea, endosulfan has been detected in beef cattle imported from Australia and New Zealand because it is still used in the United States and other countries. Endorphins are also reported to travel long distances in the environment and are reported to be found in the Saharan Desert, even the North Pole. The research on biodegradation of endosulfan is only a few, and its contents are also very limited, so it is urgent to apply it to the field. Endosulfan pesticides were one of the most widely used pesticides in Korea in 2008-2009. Recently, as coastal pollutants have flowed into the sea, coastal tidal flats have been contaminated with organic materials such as endosulfan and synthetic wastewater.

In the present invention, microorganisms using endosulfan as a carbon and energy source are separated from endosulfan and organic matter-degrading microorganisms through concentration cultivation in coastal tidal flats, and their characteristics are analyzed in order to prepare a biological environment purification strategy for coastal tidal flats , And the possibility of its application as a purification agent for coastal tidal organics using isolated strains.

Korean Patent Publication No. 10-03289130 relates to Rhodococcus koreensis DNP505, a new species in Rhodococcus capable of degrading 2,4-dinitrophenol, Dinitrophenol, which is a harmful substance to humans and other organisms, can be degraded. When applied to wastewater, sewage or soil contaminated with 2,4-dinitrophenol, the biodegradation process using microorganisms A composition for Rhodococcus coriensis DNP505 is disclosed, which is a new species of Rhodococcus that can be used to efficiently purify 2,4-dinitrophenol in pollutants. Korean Patent Publication No. 10-03252520 discloses a novel microorganism capable of decomposing an aromatic compound, Rhodococcus pyridinovorans PDB9, a method for decomposing an aromatic compound using the same, and a method for decomposing aromatic compounds such as wastewater, sewage, Or soil treatment agent, which is a novel microorganism capable of decomposing aromatic compounds having a toxic effect on the human body and other organisms, and a method for decomposing pyridine having high toxicity among aromatics Which is used to purify wastewater, sewage or soil containing pyridine. Korean Patent Registration No. 10-04352310 discloses a Rhodococcus sp. YHLT-2 (KCTC 10203BP) strain which decomposes oil such as crude oil, diesel and gasoline in a very wide temperature range of 1 to 37 ° C, And a method for biological purification of soil and ground water contaminated with oil used. In addition, Korean Patent Registration No. 10-08507430 discloses a Bacillus subtilis strain having antimicrobial activity against Staphylococcus aureus and a composition for a probiotic using the same. However, the strains disclosed in the above are strains derived from terrestrial plants or animals And is not identified on the basis of marine animals resistant to salt as in the invention.

The present invention is to provide a biological environment purification strategy of contaminated mud flat from an organic material, the strain and the lug on the coast tidal origin (Perinereis aibuhitensis ), which is known to be closely related to the digestion and bio-immunity of nutrients, and to provide an organic cleaning agent which can be useful for purifying tidal environment contaminated with organic matter.

In order to isolate microorganisms using endosulfan as a carbon and energy source in coastal tidal flats, an inorganic nutrient medium of 25 ml BM medium containing endosulfur at a concentration of 10 ppm was prepared, and then 5% of a sea water sample or a coastal soil sample was inoculated at room temperature (10 to 20 ^° C). Using a single strain isolated from the enriched culture, the resolving power was analyzed by HPLC on a BM medium supplemented with 10 ppm of endosulfan as a single carbon source and an energy source, and a strain with excellent resolution was selected.

In addition, the lagoons ( Perinereis aibuhitensis ) were analyzed based on the culture method. Bacillus sp. Bacillus sp. Bacillus sp .

Six strains isolated from endosulfan concentrate culture were identified on the basis of 16S rRNA base sequences. As a result, five strains of six genera were identified as Echinicola vietnamensis ), Rhodococcus ( Rhodococcus pyridinivorans), to Lu Liao Mobilis (Ruegeria mobilis), Tala consumption gel mouse's Latino borane (Thalassobius gelatinovorus), Tala Sophie La Te PD pillars (Thalassospira tepidiphila), Tena Bar Coolum ahip Interceptors (Tenacibaculum It was investigated by aiptasiae).

As a result of identification of 31 colonies selected from the nematodes , the 11 strains contained in the genus Bacillus were isolated from the 16S rRNA sequence, and Bacills As a result of analysis of the diversity of 11 strains contained in genus, Bacillus alcicola algicola), Bacillus anthraquinone system (Bacills anthrasis), Bacillus Aquitania Maris (Bacills aquimaris), Bacillus Barbary kusu (Bacills barbaricus), Bacillus Hwajinpo N-Sys (Bacills hwajinpoensis), Bacillus nanhi N-Sys (Bacills nanhiensis), Bacillus Viet Nam N-Sys (Bacills of Bacillus spp vietnamensis) including all seven kinds (species). The lug (Perinereis aibuhitensis ).

In the present invention, the strains isolated from the worms of the present invention have excellent protein, carbohydrate, and lipid-resolving ability and are resistant to salt, and the isolates isolated from the coastal tidal flats are salt tolerant and are used as a microorganism using endosulfan as a carbon and energy source. Therefore, it is effective to purify tidal environment contaminated with organic matter such as endosulfan.

1 shows a process of separating endosulfan degrading microorganisms by concentrated culture.
FIG. 2 is a schematic diagram showing the identification process of a Bacillus strain derived from a nematode.
Fig. 3 shows the 16S rDNA sequence of Rhodococcus pyridinovorans EDB2 strain isolated from the tidal flats.
Figure 4 shows the results of the systematic analysis of EDB2 by the 16S rRNA sequence.
FIG. 5 shows the nucleotide sequence of the 16S rDNA gene of Bacillus strain CBW4 isolated and isolated from the nematode.
6 shows the nucleotide sequence of the 16S rDNA gene of Bacillus strain EBW4 isolated and isolated from the nematode.
Fig. 7 is a schematic diagram showing the systematic relationship of the Bacillus strain derived from the identified wormwood.
Figure 8 shows the results of biochemical characterization of EDB2 by API 20E.
Fig. 9 shows the results of biochemical characterization of EDB2 by API 20 NE.
Fig. 10 shows the results of biochemical characterization of EDB2 by API CHB / E.
Fig. 11 shows the results of biochemical characterization of EDB2 by API ZYM.
Figure 12 shows the results of pure culture of strain EDB-2, which can use Ann and Sul plate in solid and liquid media.
Fig. 13 shows the results of the endosulfan degradation test of the strain EDB2 by HPLC.
FIG. 14 shows a process for producing a microbial preparation using a composite strain derived from a tidal flat.
14 shows a microbial preparation prepared using a mite-derived complex.
Figure 15 shows an example of a microbial preparation prepared using a Bacillus sp.

The present invention relates to a salt tolerance selected from a strain of the deposit No. KACC91762P isolated and identified by inoculating a coastal tidal flats and seawater into an endosulfuric acid thickening culture and cultivating the concentrated culture and growing at a temperature of 25 to 42 DEG C and a pH of 5 to 10, , Carbohydrates, lipids, and surfactants, salinity tolerant Rhodococcus ( Rhodococcus spp. pvridinivorans ) and Bacillus strains cultured from Bacillus sp. strains of KACC91722P and KACC91749P, which have been identified from the EDB2 strain and the nematode, and which have salinity tolerance and ability to decompose organic matters such as proteins, carbohydrates, lipids and surfactants The present invention provides a microorganism purifying agent for a tidal flat used for purification.

The present invention also relates to a microorganism preparation for the purification of coastal tidal flats containing the culture of the strain as an active ingredient and a method for degrading at least one organic substance selected from endosulfan, protein, carbohydrate, lipid and surfactant deposited on coastal tidal- ≪ / RTI >

The Rhodococcus pvridinivorans EDB2 strain derived from coastal tidal flats identified in the present invention is aerobic, gram-positive, rod-shaped and pinkish. The growth temperature range was 20 ℃ ~ 45 ℃ and the optimal temperature was about 20 ℃ ~ 42 ℃. It showed no decomposition ability of casein and cellulose but showed decomposition ability of DNA, agar, starch and tween. Therefore, it showed high lipid degradation ability and is highly applicable to lipid - contaminated environmental purification.

Accordingly, the present inventors deposited the above-identified strain of the present invention to the National Institute of Agricultural Science and Technology (KACC) on Nov. 22, 2012 and received the deposit number KACC91762P.

In addition, CBW2 and CBW4 of the genus Bacillus were isolated and identified for the first time by the present inventors, and they were identified as Perinereis aibuhitensis ) was isolated and identified for the first time based on the culture method.

The 16S rDNA sequences of the isolated strains were analyzed by molecular genetic analysis. As a result, the systematic analysis of the 16S rRNA sequences of eleven Bacillus strains revealed that they were divided into five groups, Among the middle strains CBW2 and CBW4, Bacillus vietnamensis 15-1 ^T and Bacillus anthracis ATCC 14578 ^T and And 90.7% and 94.2%, respectively. EBW4 showed homology to Bacillus hwajinpoensis SW-72 ^T and 96.8% homology, indicating that it was a new species.

Therefore, the present inventors named the newly isolated strain of the present invention, which has the 96.8% homology with Bacillus hwajinpoensis SW-72T, as "Bacillus sp. EBW4", and as of October 26, 2012 And deposited with the National Center for Agronomy and Genetic Resources (KACC) of the National Academy of Agricultural Science and received the deposit number KACC91749P.

The present inventors have named the new strain of the present invention as "Bacillus sp. CBW4" and deposited it on the National Institute of Agricultural Science and Technology (KACC) as of June 8, 2012 and assigned the deposit number KACC91722P received.

Hereinafter, the identification method and biochemical characteristics of the lagoon and coastal tidal flats, which are excellent in organic matter-resolving ability such as endosulfan, will be described with reference to the following examples.

1. Isolation and Identification of Strain from Coastal Tidal Flats and Rugwort

1.1. Endorphin  And organic degradation strain isolation and culture conditions

In order to separate microorganisms using endosulfan as carbon and energy source, 25 ml of BM medium (10 ml of 1 M phosphate buffer, 0.25 g of MgSO ₄ .7H ₂ O, 0.02 g of CaCl ₂ .2H ₂ O, 0.02 g of FeSO ₄ · 7H ₂ O, 2.38 g of (NH ₄ ) ₂ SO ₄ , 30 g of sea salt, and 990 ml of distilled water) was prepared and then 5% of the seawater samples or the coastal soil samples were inoculated. The incubation was carried out at room temperature (10 to 20 ^° C), and 1 ml of the culture solution was inoculated to the new medium three times at intervals of one week to continue the subculture (FIG. 1). In order to isolate the various endosulfan degrading microorganisms, a separate concentrated culture was carried out by the same method as described above five times using a new sample.

In the case of endosulfan, the culture was incubated for 7 days, and then 1 mL of the culture was inoculated into each new medium and the second and third culturing were repeated under the same conditions. The synthetic wastewater degrading ability was investigated for endosulfan degrading microorganisms, so no concentrated culture was carried out separately. The resolution of synthetic wastewater was tested by inoculating BM medium containing 10,000 ppm of synthetic wastewater to microorganisms degrading endosulfan.

Using a single strain isolated from the enriched culture, the resolving power was analyzed by HPLC on a BM medium supplemented with 10 ppm of endosulfan as a single carbon source and an energy source, and a strain with excellent resolution was selected.

1.2. Isolation and culture conditions of strains from the worms

In the present invention, the lupine ( Perinereis aibuhitensis ) were harvested from the paddy field in Suncheon Bay. FIG. 2 is a schematic diagram showing the identification process of a Bacillus strain derived from a nematode.

In order to isolate the microorganisms present inside the barnacle, living rats were placed in BM buffer (Mikesell et al ., 1993) , then washed three times in vortex and then briefly in 70% ethanol again. The washed ridge was cut with a sterilized razor blade and then cut into several very short segments. The ridge was vortexed in a conical tube containing 50 mL of BM buffer, and the endogenous microorganism of the rug was allowed to escape into the buffer.

The pellet was collected by centrifugation at 12,000 rpm, and 2 mL of BM buffer was added thereto. The microbial cells were inoculated into 100 μl of the solid Bacto marine agar 2216 (MA) medium. MA medium was used to increase the microbial diversity that can be analyzed by the culture method using both the 1:10 and 1: 2 diluted medium and the undiluted medium at the same time. LB or LB agar medium was used as the primary culture medium, and detailed culture conditions were detailed in the growth test according to the environmental condition, because all strains isolated were able to grow in LB .

1.3. Total genomic DNA  Separation and purification

Each strain was inoculated into 50 mL of LB medium, cultured at 30 ° C. at 180 rpm for 24 hours, and harvested by centrifugation (6,300 × g, 10 min, 4 ° C.). The recovered cultured cells were resuspended in 5 mL of TES buffer (0.1 M Tris-HCl (pH 7.0), 0.01 M EDTA, 1 M NaCl), 200 μl of lysozyme (10 mg / mL) Lt; / RTI > 100 μl of 10% SDS was added and mixed slowly to prevent physical DNA cleavage. Then, 50 μl proteinase K (20 mg / ml) and 5 μl RNase were added and reacted at 37 ° C for 30 minutes.

Add 5 ml of TES buffer, add 10 ml of phenol, and mix well. The supernatant was removed from the supernatant, transferred to a new tube, treated twice with phenol / chloroform / isoamyl alcohol and once with chlorofrom. The supernatant was transferred to a new tube, and 1/10 volume of 3M sodium acetate (PH.54) and 2-3 volumes of 100% ethanol were added and left at room temperature for 30 minutes.

After the centrifugation, the supernatant was discarded, the salts were washed with 70% ethanol, and centrifuged again. The supernatant was discarded, ethanol was removed from the air, and the precipitate was dissolved in sterilized water to be used in the next experiment. After electrophoresis on 0.8% agarose gel, the DNA band was identified and quantified using a UV-spectrophotometer (F 800, Beckman Coulter, Fullerton, USA). For purification of total DNA, crude DNA was dissolved in 10 ml of cesium chloride solution, ethidium bromide was added, dialyzed against 102,200um membrane filter for 30 minutes, and the DNA solution was recovered and concentrated. Then, a sample for amplification or cloning of a specific gene Were used.

1.4. Microbial separation result

1.4.1. In the enrichment culture,  by Endorphin  Decomposition microorganism separation result

Six strains isolated from the endothelium-enriched culture were five genera and six species, Echinicola var. vietnamensis), Rhodococcus flute Dino's borane (Rhodococcus pyridinivorans), to Lu Liao Mobilis (Ruegeria mobilis), Tala consumption gel mouse's Latino borane (Thalassobius gelatinovorus), Tala Sophie La Te PD pillars (Thalassospira tepidiphila), Tena Bar Coolum ahip Interceptors (Tenacibaculum aiptasiae ) .

The strain EDA5 isolated in the present invention has a high possibility of being a new genus which has not yet been reported. The most highly related strain of the 16S RNA sequence is Echinicola vietnamensis KMM 6221 ^T showed only 92.2% homology (Table 1). Also, the strains EDA2 and EDSW1 are likely to be new species. In the present invention, the resolving power of the respective microorganisms constituting the concentrated culture solution was determined to be the highest in Rhodococcus sp. EDB2 was selected and its characteristics were analyzed.

Microorganisms isolated by endosulfan enrichment culture based on 16S rRNA base sequence Strain Closese Relatives Similarity (%) Diff / Total nt EDA2 Thalassobius gelatinovorus IAM 12617 ^T 96.9 42/1360 EDA3 Ruegeria mobilis NBRC 101030 ^T 98.8 16/1361 EDA5 Echinicola vietnamensis KMM 6221 ^T 92.2 110/1414 EDB2 Rhodococcus pyridinivorans PDB9 ^T 99.0 14/1428 EDSW1 Tenacibaculum aiptasiae a4 ^T 97.6 33/1394 EDSW2 Thalassospira tepidiphila 1-1B ^T 98.4 22/1380

1.4.2. Results of Culture-Dependent Lumbricus Endogenous Microorganism Segregation

Bacterial isolates were obtained by inoculating 0.1 mL of BM buffer containing the extract of Rubus coreus into MA medium prepared by adjusting the concentration of three kinds of nutrients by dilution method. Thirty colonies were screened and cultured according to the color and shape of the colonies grown on the MA medium. 11 colonies were identified on the basis of the 16S rRNA nucleotide sequence. As a result, 11 strains were identified as Bacillus 1 genus, 1 strain of Micrococcus , 1 strain of Pantoea , 7 strains of Shewanella genus, and 10 strains of genus Vibrio (Table 2).

Identification of lugid-derived bacteria based on 16S rRNA sequence Strain Closest Relatives Similarity  (%) Diff / Total nt CBW1 Shewanella marisflavi SW-117 ^T 98.4 23/1441 CBW2 Bacillus vietnamensis 15-1 ^T 90.7 127/1362 CBW3 Bacillus barbaricus V2-BIII-A2 ^T 96.8 46/1419 CBW4 Bacillus anthracis ATCC 14578 ^T 94.2 76/1305 CBW5 Bacillus nanhaiensis JSM 082006 ^T 99.2 11/1429 CBW6 Shewanella marisflavi SW-117 ^T 98.8 18/1441 CBW7 Bacillus nanhaiensis JSM 082006 ^T 99.6 5/1401 CBW8 Vibrio azureus LC2-005 ^T 99.2 10/1298 CBW9 Bacillus algicola KMM 3737 ^T 98.5 22/1443 CBW10 Vibrio azureus LC2-005 ^T 98.8 15/1298 CBW11 Micrococcus yunnanensis YIM 65004 ^T 99.0 13/1393 CBW12 Vibrio azureus LC2-005 ^T 99.2 11/1298 CBW13 Vibrio azureus LC2-005 ^T 99.2 10/1298 CBW14 Bacillus barbaricus V2-BIII-A2 ^T 99.0 14/1419 CBW15 Bacillus vietnamensis 15-1 ^T 98.0 27/1311 CBW16 Shewanella marisflavi SW-117 ^T 98.6 20/1439 CBW17 Shewanella marisflavi SW-117 ^T 97.5 36/1435 CBW18 Pantoea septica LMG 5345 ^T 99.6 5/1340 CBW19 Shewanella marisflavi SW-117 ^T 99.7 4/1416 CBW20 Bacillus aquimaris TF-12 ^T 99.4 8/1403 CBW21 Bacillus aquimaris TF-12 ^T 99.2 11/1401 EBW1 Vibrio azureus LC2-005 ^T 98.5 19/1297 EBW2 Vibrio azureus LC2-005 ^T 99.2 10/1298 EBW3 Vibrio azureus LC2-005 ^T 99.4 8/1295 EBW4 Bacillus hwajinpoensis SW-72 ^T 96.8 47/1446 EBW5 Vibrio azureus LC2-005 ^T 99.2 11/1298 EBW6 Shewanella aquimarina SW-120 ^T 98.0 30/1438 EBW7 Shewanella aquimarina SW-120 ^T 98.0 29/1440 EBW8 Vibrio azureus LC2-005 ^T 99.2 11/1298 EBW9 Vibrio azureus LC2-005 ^T 99.2 10/1298

1.5. 16S rRNA  Analysis of gene sequence and systematic relationship

Bacterial 16S rDNA sequencing was performed by culturing the isolates in liquid medium at 30 ° C for 2 days to 3 days. The chromosomal DNA extracted from the chromosomal DNA was used as the template for the PCR reaction and the primers were Eub27F: 5'-AGA GTT TGA TCC A set of TGG CTC AG- 3 'and Eub 1522R: 5' -AAG GAG GTG ATC CAR CCG CA- 3 'was used.

The PCR reaction was performed using GeneAmp PCR System 9700 (Applied Biosystems, CT). The reaction conditions were 95 ° C for 5 minutes, 1 minute at 95 ° C, 1 minute at 55 ° C, 1 minute at 72 ° C 30 times and finally at 72 ° C for 1 minute and then stored at 4 ° C. The amplified DNA was recovered by gel extraction kit (SolGent) after electrophoresis on 1.0% agarose gel.

The recovered DNA is then ligation in pGEM-T easy vector (Promega, Madison, Wisconsin) E. coli JM109. The plasmid DNA of the transformed clones was isolated and DNA sequencing was performed bidirectionally using an ABI 377 automated sequencer (Applied Biosystem, Foster, USA) using a promoter primer or SP6 promoter primer.

The determined nucleotide sequence was analyzed using a database of Ribosomal Database Project-II (http://rdp.cme.msu.edu) GenBankhttp: //ncbi.nlm.nih.gov). The systematic relationship of the microorganisms determined by the nucleotide sequence was shown to be related to the 16S rNA nucleotide sequence of the type strain using EZtaxon.

1.5.1. Endorphin Decomposition strain EDB2 16S of rRNA  Gene analysis result

Figure 3 shows the 16S rDNA sequence of the isolated Rhodococcus pyridinovorans EDB2 strain. Figure 4 shows the results of the systematic analysis of EDB2 by the 16S rRNA sequence. In a molecular phylogenetic analysis based on the 16S rDNA sequence, the strain designated as strain EDB2 was phylogenetically identified as Rhodococcus . Rhodococcus (99.6%) and R. rhodochrous (98.5%) with biphenyl-resolving R. pyridinivorans . The strain EDB2 was named as R. pyridinivorans EDB2 (Fig. 3). R. pyridinivorans R04, which has the highest homology with strain EDB-2, is known to decompose biphenyl, a highly toxic aromatic compound. R. sp. WTZ-R2 has been shown to be highly capable of removing di (2-ethylhexyl) phthalate (DEHP).

Therefore, the strain R. pyridinivorans isolated in the present invention Since EDB2 is expected to be useful for removing various aromatic and aliphatic compounds that are pollution - resistant to tidal flats, the following characteristics were investigated for the removal of various aromatic and aliphatic compounds which are degradable tidal flats.

1.5.2. Origin of lug Bacillus  Analysis of strain diversity and phylogenetic relationships

31 colonies selected according to the color and shape of the colonies grown on the MA medium were separately selected and cultivated in pure form and then identified on the basis of the 16S rRNA sequence. As a result, Bacillus alcicola algicola), Bacillus anthraquinone-shi (Bacillus anthrasis), Bacillus Aquitania Maris (Bacillus aquimaris), Bacillus Barbary kusu (Bacillus barbaricus), N-Sys Hwajinpo Bacillus (Bacillus hwajinpoensis , Bacillus a strain of 11 contained in the Bacillus such nanhiensis), Bacillus Viet Nam N-Sys (Bacillus vietnamensis) was isolated.

In the analysis of the phylogenetic relationships based on the 16S rRNA nucleotide sequences of 11 Bacillus strains identified in the present invention, they were divided into five groups. Among them, Bacillus strain Bacillus anthracis (hereinafter referred to as "CBW4") and We analyzed the characteristics of Bacillus cannabinoensis (hereinafter referred to as "EBW4"). In particular, the strains CBW2 and CBW4 are Bacillus vietnamensis 15-1 ^T and < RTI ID = 0.0 &^gt; Bacillus anthracis ATCC 14578 ^T and And 90.7% and 94.2%, respectively. EBW4 showed homology to Bacillus hwajinpoensis SW-72 ^T and And 96.8% homology, indicating a new species. FIG. 5 shows the nucleotide sequence of the 16S rDNA gene of Bacillus strain CBW4 isolated and isolated from the worms, and FIG. 6 shows the nucleotide sequence of the 16S rDNA gene of Bacillus strain EBW4 isolated and isolated from the worms. FIG. 7 shows the results of the identification of Bacillus sp. Is a schematic diagram showing the systematic relationship of the strain. The invention of microbes endogenous to the worms has been found for the first time in the present invention, and an in-depth analysis of the functions of these microbes needs to be performed.

In the present invention, Perinereis information on a variety of Bacillus appeared as a dominant species of bacteria separated from aibuhitensis) was investigated in detail the characteristics of the two Bacillus strains CBW4 EBW4 and a superior resolution, the organic substance selected as the noticed that first reported in the present invention.

2. Coastal tidal flats and From the lobster  Growth Characteristics of Identified Strains by Environmental Conditions

2.1. Strain Growth Test by Environmental Condition

In the present invention, the growth of the strain was examined by varying three environmental conditions such as temperature, pH, and salinity. In order to determine the optimum temperature range of the bacterial growth, the strain was inoculated in LB medium and cultured at 4 ° C, 20 ° C, 30 ° C, 37 ° C, 42 ° C and 45 ° C. The strain was inoculated in a liquid medium and cultured in a water bath.

The optimum salinity range of the bacterial growth was adjusted to 0%, 1%, 2%, 3%, 4%, 5%, 6%, and 7% in LB medium using sodium chloride and the optimum pH range Was used by adjusting the pH of the LB medium to pH 1 from pH 3 to pH 11 using NaOH and HCl. The growth of the strain was confirmed by incubation for 5 days at the optimum growth temperature of each strain after the strain inoculation.

2.2. Coastal tidal flats and From the lobster  Growth Characteristics of Identified Strains by Environmental Conditions

2.2.1. Growth characteristics of strains identified in coastal tidal flats by environmental condition

end. EDB2 of Badge growth  Test result

In order to analyze the growth of the strain EDB2, five different media, marine agar (MA), Luria-Bertani (LB), nutrient agar (NA), R ₂ A and tryptic soy broth As a result of the analysis, it was found that all the tested medium grew well (Table 3).

Growth ability test of strain EDB-2 in various media Medium MA LB NA R ₂ A TSA EDB2 +++ +++ +++ +++ +++

I. Effect of temperature on the growth of strain EDB2

In order to investigate the temperature range in which EDB2 could grow, growth was examined at 4 ℃ to 45 ℃. The strain EDB2 was found to grow at 25 ° C to 42 ° C (Table 4).

Effect of temperature on growth of strain EDB-2 Temp (캜) 4 10 25 30 37 42 45 EDB2 - - +++ +++ +++ +++ -

All. Effect of pH on the growth of strain EDB2

In order to investigate the pH range in which EDB2 could grow, the growth was examined at pH 4 to pH 11. The strain EDB2 was shown to grow at pH 5 to pH 10 (Table 5).

Effect of temperature on growth of strain EDB-2 pH 4 5 6 7 8 9 10 11 EDB2 - +++ +++ +++ +++ +++ +++ -

la. Effect of salinity on the growth of strain EDB2

Growth of EDB2 strain was observed in medium containing 0-12% Zobell sea salts in order to determine the range of salinity to which EDB2 strain could grow. The strain EDB2 grew without salinity, but did not grow at salinity of more than 10% (Table 6).

Effect of salinity on the growth of EDB-2 strain Salinity (%) 0 One 2 3 4 5 6 7 EDB2 +++ +++ +++ +++ +++ +++ +++ +++

2.2.2. From the lobster  Sympathetic Bacillus  The culture medium of the strain Usability  Test result

The growth performance of two Bacillus strains selected in the present invention was tested using MA (marine agar), NA (nutrient agar), TSA (tryptic soy agar), R ₂ A and LB (Luria-Bertani) EBW4 was not able to grow in LB, TSA, or NA medium, unlike Bacillus strain, which was derived from land or freshwater. Both strains were grown in MA medium containing a large amount of salinity, (Table 7).

Growth ability test in various media Strain MA LB TSA NA R ₂ A CBW4
+ +
+
-
+
EBW4 + -
-
+
+

3. Coastal tidal flats and From the lobster  Sympathetic New caution  Physiological and biochemical characteristics

3.1. Morphological, physiological and biochemical characterization of bacteria in coastal tidal flats

In order to examine the morphological and physiological characteristics of bacteria, strains were inoculated on LBSS solid medium supplemented with sea salts and cultured at 25 ℃. First, the shape, color, and size of single colonies formed by 5-7 days of culture were observed. The shape and size of the strain were observed using optical microscope and scanning electron microscope. In the electron microscopic observation, the cultured bacteria were fixed with 4% glutaraldehyde for 2 hours and then washed with 50 mM phosphate buffer (pH 7.2).

The cells were fixed in 2% osmium tetroxide solution for 1 hour and dehydrated with 40, 50, 60, 70, 80, 90, 100% ethanol and lyophilized. After that, gold coating was performed using an ion sputter and observed with an electron microscope. The motility of the strain was confirmed by semisolid medium containing 0.4% agar. Oxidase - related oxidase was analyzed by colony color change using Oxidase Reagent Kit (BioM ㅹ rieux). Catelase was determined to be bubbles by adding 3% and hydrogen peroxide to the cells.

The physiological and biochemical characteristics of other isolates were investigated by using various API kits (BioM ㅹ rieux) and using API 20E, APL 20NE, API 50CHB, and API ZYM kit simultaneously. At this time, the strain was cultured in LBSS solid medium at 25 ° C for 2 days to 3 days, and the grown cells were suspended in a sterilized 3% sea salt solution and performed according to the manufacturer's instructions. Nitrate reduction, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphtol-AS-Bi-phosphohydrolase, glucose acidification, gelatin liquefaction, arginine dihydrolase, urease, β-glucosidase, β -galactosidase, alkaline phosphatase, lipase (C14), etc., and indole production. We also investigated the assimilation of carbon sources such as mannitol, gluconate, malate, citrate, glucose, arabinose, mammose, N-acetyl-glucosamine, maltose, caprate, adipate and phenyl-acetate.

FIG. 8 shows the results of biochemical characterization of EDB2 by API 20E, FIG. 9 shows the results of biochemical characterization of EDB2 by API 20 NE, and FIG. 10 shows the biochemical characteristics of EDB2 by API CHB / The results of the analysis are shown. FIG. 11 also shows the results of biochemical characterization of EDB2 by API ZYM.

end. API  By 20 E EDB2  Biochemical characterization results

Biochemical characteristics of strain EDB2 were analyzed by using 20 substrates of API 20 E kit. As a result, strain EDB2 had urease and urea was used. The ability to produce acetoin by fermenting sugar through Voges-Proskauer test . No oxidation or fermentation reaction was observed when the nine substrates (amygdalin, arabinose, glucose, inositol, mannitol, melibiose, rhamnose, sorbitol, sucrose) ).

I. API  20 NE On by EDB2  Biochemical characterization results

The biochemical characteristics of strain EDB2 were analyzed by using 20 substrates of API 20 NE kit. As a result, strain EDB2 had nitrate reduction ability and showed β-galactosidase-positive reaction when PNPG was used as a substrate. Mannitol and malate were available (Fig. 9).

All. Biochemical characterization of EDB2 by API CHB / E

Biochemical characteristics of strain EDB2 were analyzed using 48 substrates of API CHB / E kit. As a result, strain EDB2 showed positive reaction with sorbitol and esculin, and negative reaction with other substrates (FIG. 10).

la. Biochemical characterization of EDB2 by API ZYM

As a result of measuring the biochemical properties of the strain EDB2, 20 kinds of enzyme activities of the API ZYM kit were measured. As a result, the strain EDB2 has alkaline phosphatase, esterase lipase, leucine arylamidase, trypsin, acid phosphatase, α-glucosidase and β-glucosidase enzyme (Fig. 11).

3.2. From the lobster  Morphological, physiological and biochemical characterization of identified strains

For the morphological and physiological characteristics of bacteria, strains were inoculated on MA (marine agar) solid medium and cultured at 25 ℃. First, the shape, color, and size of single colonies formed by 5-7 days of culture were observed. The shape and size of the strain were observed using optical microscope and scanning electron microscope. During the electron microscopic observation, the cultured bacteria were fixed with 4% glutaraldehyde for 2 hours and then washed with 50 mM phosphate buffer (pH 7.2). The cells were fixed in 2% osmium tetroxide solution for 1 hour and dehydrated with 40, 50, 60, 70, 80, 90, 100% ethanol and lyophilized. After that, gold coating was performed using an ion sputter and observed with an electron microscope.

The motility of the strain was confirmed by semisolid medium containing 0.4% agar. Oxidase - related oxidase was analyzed by colony color change using Oxidase Reagent Kit (BioM ㅹ rieux). Catelase was determined to be bubbles by adding 3% and hydrogen peroxide to the cells.

The physiological and biochemical characteristics of other isolates were investigated by using various API kits (BioM ㅹ rieux) and using API 20E, APL 20NE, API 50CHB, and API ZYM kit simultaneously. At this time, the strain was cultured in MA solid medium at 25 ° C for 2 days to 3 days. The grown cells were suspended in a sterilized 3% sea salt solution and were performed according to the manufacturer's instructions. Nitrate reduction reaction, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphtol-AS-Bi-phosphohydrolase, glucose acidification, gelatin liquefaction, arginine dihydrolase, urease, β-glucosidase, β -galactosidase, alkaline phosphatase, lipase (C14), etc., and indole production. We also investigated the assimilation of carbon sources such as mannitol, gluconate, malate, citrate, glucose, arabinose, mammose, N-acetyl-glucosamine, maltose, caprate, adipate and phenyl-acetate.

3.2.1. Selection of lobsters Bacillus  Analysis of physiological biochemical characteristics of the strain

Biochemical and biochemical characteristics of Bacillus strains were analyzed using API 20NE kit. Both strains CBW4 and EBW4 had β-glucosidase and protease enzyme activity and could utilize maltose (Table 8).

Characterization of Bacillus strains using API 20NE kit Test Reactions / Enzymes CBW4 EBW4 NO ₃ reduction of nitrates to nitrites +/- +/- TRP İndole production - - GLU 산화 - - ADH arginine dihydrolase - - URE urease - - ESC hydrolysis (β-glucosidase) + + GEL hydrolysis (protease) + + PNPG β-galactosidase - w + GLU glucose assimilation W + w + ARA arabinose assimilation W + w + MNE mannose assimilation - w + MAN mannitol assimilation W + + NAG N-acetyl-glucosamine assimilation W + w + MAL maltose assimilation + + GNT gluconate assimilation W + W + CAP caprate assimilation - - ADI adipate assimilation W + w + MLT malate assimilation w + w + CIT citrate assimilation W + w + PAC phenyl-acetate assimilation W + w +

As a result of the test using API 20E kit, CBW4 showed high activity of arginine dihydrolase, lysine decarboxylase and gelatinase, while EBW4 had β-glucosidase and gelatinase activity (Table 9).

Characterization of Bacillus strains using API 20E kit Test Reactions / Enzymes CBW4 EBW4 OPNG Beta-galactosidase - + ADH Arginine dihydrolase + - LDC Lysine decarboxylase + - ODC Ornithine decarboxylase - - CIT Citrate utilization - - H ₂ S H2S production - - URE Urease - - TDA Tryptophane deaminase - - IND Indole production - - VP Acetone production - - GEL gelatinase + + GLU Glucose F / O - - MAN Mannitol F / O - - INO Inositol F / O - - SOR Sorbitol F / O - - RHA Rhamnose F / O - - SAC Sucrose F / O - - MEL Melibiose F / O - - AMY Amygdaline F / O - - ARA Arabinose F / O - -

As a result of the test using the API 50CHB / E kit, both CBW4 and EBW4 showed the ability to utilize glycerol, ribose, glucose, fructose, esculin, cellobiose, maltose, trehalose and starch. In addition, CBW4 was able to utilize arbutin, salicin, and lactose, while EBW4 was not available, but mannose, mannitol, α-methyl-D-glucoside, N-acethyl-glucosamine, melibiose, sucrose, raffinose, and D-turanose (Table 10).

Characterization of Bacillus strains using API 50CHB / E kit No . Substrate CBW4 EBW4 0 CONTROL - - One GLYcerol + + 2 ERYthritol - - 3 DARAbinose - - 4 LArbinose - - 5 RIBose + + 6 DXYLose - - 7 LXYLose - - 8 ADOnitol - - 9 beta -Methyl-D-Xyloside - - 10 GALzctose - + 11 GLUcose + + 12 FRUctose + + 13 MaNnosE - + 14 SorBosE - - 15 RHAmnose - - 16 DULCitol - - 17 INOsitol - - 18 MANnitol - + 19 SORbitol - - 20 alpha -Methyl-D-Mannoside w + - 21 alpha -Methyl-D-Glucoside w + + 22 N-Acethyl-Glucosamine w + + 23 AMYgdalin w + w + 24 ARButin + w + 25 Esculin + + 26 SALicin + w + 27 CELlobiose + + 28 MALtose + + 29 LACtose + - 30 MELibiose - + 31 Sucrose - + 32 TREhalose + + 33 INUlin - - 34 MeLeZitose - w + 35 RAFfinose - + 36 Starch + + 37 GLYcoGen w + + 38 XyLiTol - - 39 GENtiobiose - - 40 DTURanose - + 41 DLYXose - - 42 DTAGatose - - 43 DFUCose - - 44 LFUCose - - 45 DArabitoL - - 46 LArabitol - - 47 GlucoNaTe - w + 48 2-keto-Gluconate - W + 49 5-keto-Gluconate - -

As a result of enzymatic activity test using API ZYM kit, both strains CBW4 and EBW4 showed alkaline phosphatase, leucine arylamidase and α-chymotrypsin activity. In addition, CBW4 has very high acid phosphatase activity and β-glucosidase activity. EBW4 had an esterase activity that was not detected in the CBW4 strain (Table 11).

Characterization of Bacillus strains using API ZYM kit NO . Enzyme Assayed For CBW4 EBW4 -One One Control 2 Alkaline phosphatase 4+ 4+ 3 Esterase (C4) One- 3+ 4 Esterase Lipase (C8) One- 2- 5 Lipase (C14) 0- 0- 6 Leucine arylamidase 4+ 3+ 7 Valine arylamidase 2- 2- 8 Crystine arylamidase One- One- 9 Trypsin 2- 0- 10 alpha-chymotrypsin 3+ 3+ 11 Acid phospatase 5+ 0- 12 Naphtol-AS-B1-phosphohydrolase One- 0- 13 alpha -galactosidase 0- 0- 14 β-galactosidase 0- 2- 15 β-glucuronidase 0- 0- 16 α-glucosidase 2- 2- 17 β-glucosidase 4+ 0- 18 N-acetyl-β-glucosaminidase 0- 0- 19 α-mannosedase 0- 0- 20 alpha-fucosidase 0- 0-

4. Application of the strain identified in the coastal tidal flats as a cleaning agent for organic matter

Fig. 12 shows the results of pure culture of strain EDB-2, which can be used in the solid and liquid media, and Fig. 13 shows the results of the endosulfan degradation test of the strain EDB2 by HPLC.

4.1. Polymerase chain reaction activity test

Bacillus strain, CBW4 (Accession No .: KACC91722P) and EBW4 (Accession No .: KACC91749P) isolated in the present invention to LB solid medium containing cellulose, starch, casein, and Tween 80, Cellulase, amylase, protease, and lipase activity in the degradation of these macromolecules were investigated. The cellulolytic activity of the strain was determined after 30 days of incubation at 30 ° C for 3 days, followed by 30 minutes of staining with congo red, followed by discoloration with 1M NaCl for 5 minutes, staining with iodine for 30 minutes and discoloration with 1M NaCl for 5 minutes As shown in Fig.

The casein and Tween 80 were cultured for 2 days and 3 days, respectively, and their resolution was confirmed by a transparent ring observed with naked eyes without performing any staining process. Alkaline protease activity was determined in 100 mM Tris-HCl buffer (pH 10.0) using Hammerstan casein (Merck) at a concentration of 0.5% (Joo et al., 2002). One Unit of Enzyme Activity is defined as the amount of enzyme that releases 1 g of tyrosine per minute at 50 캜 under standard assay conditions.

4.1.1 EDB2 Polymer organic matter resolution test

end. Polymer organic matter decomposition test of EDB2

As a result of the organic degradation test with strain EDB2, the strain EDB2 was found to have excellent lipid and carbohydrate degrading ability and high activity against nucleic acid degrading enzyme (Table 12).

Degradation test of organic matter by polymer EDB-2 Salinity  (%) Starch
(One%) DNase CM /
Cellulose Casein
(One%) Tween 20 40 60 80 EDB2 +++ +++ - - + +++ +++ ++

I. Assessment of organoleptic resolution and pure microbial screening for pure cultured single microorganisms

Using a single strain isolated through concentration culture, the resolving ability was examined in a BM medium supplemented with 10 ppm of endosulfan as a single carbon source and an energy source. Among them, strains with excellent resolution were selected. EDB2 was very well grown in solid medium and liquid medium containing endosulfan as a single carbon source and energy source, and each strain isolated from the endosulfurization concentrate culture was very well grown. (Figs. 12 and 13).

For HPLC analysis, endosulfan was treated at 5 ppm concentration and EDB2 - induced endosulfan degradation was measured. It showed about 85% endosulfan resolution in 7 days after incubation. The strain EDB2, which has the highest endosulfan resolution, was selected and used for identification of physiological, biochemical and genetic characteristics.

4.1.2 Selection from Lagoons Bacillus  Results of Polymerase Enzyme Activity Test of Strain

Both the CBW4 and EBW4 strains showed DNAse activity and protease activity by degrading casein. CBW4 had an enzymatic activity to degrade strach, but EBW4 did not have starch resolution. In addition, both CBW4 and EBW4 were shown to degrade most of the tween, indicating lipolytic enzyme activity.

Therefore, in the present invention, two Bacillus strains isolated from the nematode can be used to purify the tidal environment contaminated with organics, because of their excellent protein, carbohydrate and lipid decomposition (Table 13).

Polymerase Enzyme Activity Test Strain Starch  One% DNAse CM / cellulose Casein  One% Tween 20 40 60 80 CBW4 +
+
-
+
+
+
+
+
EBW4 -
+ - +
- + + +

4.2. Application of a new strain to a tidal flat cleaning agent

As a result of 16S rRNA sequencing analysis, EDB2 was found to be a species contained in Rhodococcus . As mentioned earlier, the genus Rhodococcus has excellent ability to remove degradable materials that can not be readily degraded by other organisms, and is considered one of the most promising groups for biologically treating polluted environment. There are a wide range of compounds, including aliphatic and aromatic hydrocarbons, oxides, and halogen compounds, which are degraded by Rhodococcus spp .

Most of these compounds are complex synthetic molecules that are degradable and toxic. They are listed in the US Environmental Protection Agency (EPA) First Pollutant List (USEPA, 1996) and the first list of hazardous substances in the Toxic Substance and Disease Registry (ATSDR, 1997) . The importance of Rhodococcus in environmental biotechnology has been highly emphasized and much discussion has been made about the role of Rhodococcus as a biocatalyst in the degradation of pollutants. Therefore, Rhodococcus is considered to be suitable for environmental restoration, and it is believed that the strains or enzymes produced by them can be utilized.

As a new strain identified from the tidal flats of the present invention, Rhodococcus pyridinivorans EDB2, Accession No .: KACC91762P) and a new strain Bacillus ( Bacillus sp. . sp) CBW4 (Accession No: KACC91722P) and EBW4 (Accession No: KACC91749P) was inoculated in a liquid culture medium at the same rate at 25 ℃ to 42 ℃, 1-2 ilgan was incubated at pH5 to pH10, the result obtained in the step of culturing A part was dried with a liquid microbicide, the remaining part was aseptically dried, and the result obtained after the drying step was aseptically pulverized to prepare a powdery microbial agent. FIG. 14 shows a process for producing a microbial preparation using a composite strain derived from a tidal flat. Fig. 15 shows an example of a microbial preparation prepared using a mite-derived complex.

In the case of a liquid preparation, food coloring may be added as an excipient for color control, vitamin C may be added as an excipient for improving shelf life, and Tween 80 may be added as an excipient for activating the enzyme ability. In the case of a solid powder, rice bran, degreasing steel, molasses and the like are additionally added as an excipient for fermentation, and silicate or the like can be added as an excipient for improving shelf stability.

As a new strain isolated from the tidal flats in this way, Rhodococcus pyridinivorans EDB2, Accession No .: KACC91762P) and a new strain Bacillus ( Bacillus sp. . sp) CBW4 (Accession No: KACC91722P) and EBW4 (Accession No: KACC91749P) complex microbial preparation according to the culture is utilized very useful for restoring the coastal tidal contaminated with toxic substances such as endoseolpan as viewed organic resolution of .

Since the microorganism of the present invention has the ability to decompose DNA, agar, starch and tween, it is able to decompose compounds such as endosulfan by a wide range of biodegradability, and thus can efficiently pollute coastal tidal flats without causing secondary environmental pollution It will be useful to purify the environment.

Agricultural Biotechnology Research Institute KACC91762P 20121122 Agricultural Biotechnology Research Institute KACC91749P 20121026 Agricultural Biotechnology Research Institute KACC91722P 20120608

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Bacillus spp., Identified from lugworm and microbial cleaning          agent <130> P201212064 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1378 <212> RNA <213> Bacillus sp. EBW4 <400> 1 tcgagcgaat ggattgagag cttgctctta tgaagttagc ggcggacggg tgagtaacac 60 gtgggtaacc tgcccataag actgggataa ctccgggaaa ccggggctaa taccggataa 120 tattttgaac tgcatggttc gaaattgaaa ggcggcttcg gctgtcactt atggatggac 180 ccgcgtcgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat gcgtagccga 240 cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300 gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360 aaggctttcg ggtcgtaaaa ctctgttgtt agggaagaac aagtgctagt tgaataagct 420 ggcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta 480 atacgtaggt ggcaagcgtt atccggaatt attgggcgta aagcgcgcgc aggtggtttc 540 ttaagtctga tgtgaaagcc cacggctcaa ccgtggaggg tcattggaaa ctgggagact 600 tgagtgcaga agaggaaagt ggaattccat gtgtagcggt gaaatgcgta gagatatgga 660 ggaacaccag tggcgaaggc gactttctgg tctgtaactg acactgaggc gcgaaagcgt 720 ggggagcaaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg 780 ttagagggtt tccgcccttt agtgctgaag ttaacgcatt aagcactccg cctggggag 840 acggccgcaa ggctgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900 tggtttaatt cgatgcaacg cgaagaacct tacctactct tgacatccag agaattcgct 960 agagatagct tagtgccttc gggagctctg agacaggtgc tgcatggctg tcgtcagctc 1020 gtgttgtgaa atgttgggtt aagtcccgca acgagcgcaa cccttatcct tacttgccag 1080 cgggtcatgc cgggaacttt agggagactg ccggtgataa accggaggaa ggtggggacg 1140 acgtcaagtc atcatggccc ttacgagtag ggctacacac gtgctacaat ggcgagtaca 1200 gagggttgcg aagccgcgag gtggagctaa tctcagaaag ctcgtcgtag tccggattgg 1260 agtctgcaac tcgactccat gaagtcggaa tcgctagtaa tcgtggatca gaatgccacg 1320 gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt gggctgca 1378 <210> 2 <211> 1374 <212> RNA <213> Bacillus sp. CBW4 <400> 2 tcgagcgaat gatgaggagc ttgctcctct gatttagcgg cggacgggtg agtaacacgt 60 gggtaatctg cctgtaagac ggggataact ccgggaaacc ggggctaata ccggataaca 120 agagaagaag catttcttct ttttgaaagt cggtttcggc tgacacttac agatgagccc 180 gcggcgcatt agctagttgg tgaggtaacg gctcaccaag gcgacgatgc gtagccgacc 240 tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc 300 agtagggaat cttcggcaat gggcgaaagc ctgaccgagc aacgccgcgt gagcgatgaa 360 ggccttcggg tcgtaaagct ctgttgttag agaagaacaa gtacgagagt aactgctcgt 420 accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 480 cgtaggtggc aagcgttatc cggaattatt gggcgtaaag cgcgcgcagg cggtctctta 540 agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg ggagacttga 600 gtgcaggaga gaaaagtgga attccacgtg tagcggtgaa atgcgtagag atgtggagga 660 acaccagtgg cgaaggcggc tttttggcct gtaactgacg ctgaggcgcg aaagcgtggg 720 gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaggtgttg 780 gggggttcca ccctcagtgc tgaagttaac acattaagca ctccgcctgg ggagtacgac 840 cgcaaggttg aaactcaaag gaattgacgg gggcccgcac aagcagtgga gcatgtggtt 900 taattcgaag caacgcgaag aacccttacc aggtcttgac atcctctgac cactctagag 960 atagagcttt ccccttcggg ggacagagtg acaggtggtg catggttgtc gtcagctcgt 1020 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgacctta gttgccagca 1080 ttcagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaaagg 1200 gttgcgaagc cgcgaggcca agccaatccc aaaaagccat tctcagttcg gattgtaggc 1260 tgcaactcgc ctacatgaag ccggaattgc tagtaatcgc ggatcagcat gccgcggtga 1320 atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgt aaca 1374 <210> 3 <211> 1429 <212> RNA <213> Rhodococcus pyridinivorans EDB2 <400> 3 cggggggtgg cgcgtgctac catgcagtcg aacgatgaag cccagcttgc tgggtggatt 60 agtggcgaac gggtgagtaa cacgtgggtg atctgccctg cactctggga taagcctggg 120 aaactgggtc taataccgga tatgacctcg ggatgcatgt tctggggtgg aaagtttttc 180 ggtgcaggat gagcccgcgg cctatcagct tgttggtggg gtaatggcct accaaggcga 240 cgacgggtag ccggcctgag agggcgaccg gccacactgg gactgagaca cggcccagac 300 tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga tgcagcgacg 360 ccgcgtgagg gatgacggcc ttcgggttgt aaacctcttt cacccatgac gaagcgcaag 420 tgacggtagt gggagaagaa gcaccggcca actacgtgcc agcagccgcg gtaatacgta 480 gggtgcgagc gttgtccgga attactgggc gtaaagagct cgtaggcggt ttgtcgcgtc 540 gtctgtgaaa tcccgcagct caactgcggg cttgcaggcg atacgggcag actcgagtac 600 tgcaggggag actggaattc ctggtgtagc ggtgaaatgc gcagatatca ggaggaacac 660 cggtggcgaa ggcgggtctc tgggcagtaa ctgacgctga ggagcgaaag cgtgggtagc 720 gaacaggatt agataccctg gtagtccacg ccgtaaacgg tgggcgctag gtgtgggttt 780 ccttccacgg gatccgtgcc gtagccaacg cattaagcgc cacgcctggg gagtacggcc 840 gcaaggctaa aactcaaagg aattgacggg ggcccgcaca agcggcggag catgtggatt 900 aattcgatgc aacgcgaaga accttacctg ggtttgacat gtaccggacg actgcagaga 960 tgtggtttcc cttgtgggcc ggtagacagg tggtgcatgg ctgtcgtcag ctcgtgtcgt 1020 gagatgttgg gttaagtccc gcaacgagcg caacccttgt cctgtgttgc cagcacgtga 1080 tggtggggac tcgcaggaga ctgccggggt caactcggag gaaggtgggg acgacgtcaa 1140 gtcatcatgc cccttatgtc cagggcttca cacatgctac aatggtcggt acagagggct 1200 gcgataccgt gaggtggagc gaatccctta aagccggtct cagttcggat cggggtctgc 1260 aactcgaccc cgtgaagtcg gagtcgctag taatcgcaga tcagcaacgc tgcggtgaat 1320 acgttcccgg gccttgtaca caccgcccgt cacgtcatga aagtcggtaa cacccgaagc 1380 cggtggccta accccttgtg ggagggagcc gtcgaatgtg atcgtaccc 1429

Claims (11)

Rhodococcus pyridinivorans EDB2 (Accession No .: KACC91762P) with salinity tolerance and endosulfan resolution identified in coastal tidal flats;
A strain CBW4 (accession number: KACC91722P) in Bacillus sp. Having a salinity tolerance identified from the nematode and having the ability to decompose any organic substance selected from protein, carbohydrate, lipid and surfactant and a strain in Bacillus sp. EBW4 (accession number: KACC91749P) in a culture medium and culturing at 25-30 DEG C for 1-2 days.
The microbial purification agent for tidal flat purification according to claim 1, characterized in that any one of an excipient for color control or an excipient for improving shelf life or an excipient for enzyme capacity activation is additionally added to the cultured liquid product &Lt; / RTI &gt;
The method for producing a microbial cleaning agent for tidal flat according to claim 2, comprising aseptically drying the product obtained in the culturing step.
The method according to claim 3, further comprising aseptically pulverizing the resultant product obtained after the drying step.
Rhodococcus pyridinivorans EDB2 (Accession No .: KACC91762P) with salinity tolerance and endosulfan resolution identified in coastal tidal flats;
A strain CBW4 (accession number: KACC91722P) in Bacillus sp. Having a salinity tolerance identified from the nematode and having the ability to decompose any organic substance selected from protein, carbohydrate, lipid and surfactant and a strain in Bacillus sp. A microbial purification agent for purification of tidal flats containing a complex culture of EBW4 (Accession No .: KACC91749P) as an active ingredient. delete delete delete delete delete delete

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