KR20100134343A - Alage lysing bacteria rhodococcus sp. kbr-5 - Google Patents

Abstract

PURPOSE: A Rhodococcus sp. KBR-5 with abilities of suppressing algae growth and decomposing algae is provided to effectively resolve environmental problem by eutrophication. CONSTITUTION: A Rhodococcus sp. KBR-5(KCTC 11509BP) controls decomposition and growth of growth. The algae are microcystris, anabaena or oscillatoria. An algaecide contains Rhodococcus sp. KBR-5 culture liquid or supernatant thereof as an active ingredient.

Description

Algae lysing bacteria Rhodococcus sp. KBR-5}

The present invention relates to Rhodococcus KBR-5 [KCTC 11509BP] having algal growth control and resolution.

As population growth and urbanization progress, there are many algal blooms caused by severe eutrophication both domestically and globally. The damage caused by algal blooms and their ripple effects by algae eutrophication are significant. First, in terms of economic and industrial aspects, Korea is part of a water shortage nation, along with Denmark and Poland. In 2011, about 4 billion tons of water shortage is expected [Water Resources Corporation, Water Resources Mid-Long Term Plan, 2006]. Therefore, it is very important from the economic and industrial point of view that there is no green algae and to ensure clean and high quality surface water. In the socio-cultural aspect, the occurrence of hydration of cyanobacteria in eutrophication waters causes visual discomfort and recreational activity due to pigmentation or green algae on the surface of the water, and dead dead fish. In addition, it causes a health loss to the human body and livestock by secondary metabolites of cyanobacteria, and may cause discomfort due to the development of taste. From an ecological point of view, the destruction of ecosystems or habitat migration, population changes, and food loss are caused by ecosystem destruction. In particular, Microcystis aeruginosa produces hepatoxin microcystin. Microcystine is fatal to humans as well as to wild animals and livestock, and is not removed by current water treatment methods, causing many problems worldwide.

Recently, the occurrence of green algae caused by southern algae is frequently occurring in dam reservoirs or rivers, which are domestic sources of water, and therefore, there is an urgent need to develop technologies to control green algae generation. Until now, various methods for preventing and controlling green algae caused by eutrophication have been proposed and implemented [Table 1]. However, these methods have considerable technical and economic limitations and may cause secondary environmental pollution.

TABLE 1

Existing green algae control technology Characteristic Chemicals Algae algae using copper sulfate, copper, calcium and chlorine Algae Adsorbent Adsorption using adsorptive properties such as siliceous porous body, loess, copper sulfate Flocculant Prevents eutrophication by removing phosphorus using aluminum sulfate filtration Filtration using granular materials such as activated carbon and charcoal Biological restoration technology Eco-cleaning technology using natural microorganisms and natural enemies such as predators Dissolved Air Pressurization Removal of Volatile Organics and Trace Organic Contaminants by Pressurized Dissolution of Air

Reduction techniques using conventional chemicals (Gibson et al., The algicidal effect of copper on a green and a blue-green alga and some ecological implications), and copper sulfate [Parker et al., Potassium salts inhibit growth of the Cyanobacteria Microcystis spp. In pond water and defined media: inplications for control of Microcystin-producing aquatic blooms.Appl.Environ.Microbiol.vol. 63, p 2324-2329, 1997]. Algae, which are not specific and are affected by these chemicals, have the disadvantage that they can affect ecosystem components, including toxin compounds, causing ecosystem disturbances. Therefore, recently, attention has been focused on biological treatment methods using natural phenomena in ecosystems. Studies have been reported to control eutrophication by inhibiting growth of cyanobacteria using phages, strains, zooplankton, protozoa, etc. [Hendry et al., Biological control of cyanobacteria: principles and possibilities. Hydrobiologia. vol. 395/396, p 161-172, 1999]. In the case of cyano phage, there is an advantage that it acts specifically for cyanobacteria, while there is a disadvantage that the phage is separately used for each of many kinds of cyanobacteria. In addition, studies on control of algae using algae bacteria have been reported [Aakano et al., Algicidal effect of the bacterium Alcaligenes denitrificans on Microcystis spp .. Aquatic Microbial Ecology. vol. 22, p 111-117, 2000]. Algae control has also been proposed through the growth inhibition chemicals (allelopathy) produced by large aquatic and terrestrial plants. Algae control using barley straw by many foreign researchers [Gibson et al., Journal of Applied Phycology, Barley straw as an inhibitor of algal growth 2: Laboratory studies. vol. 2, p 241-248, 1990; Pillinger et al., Journal of Applied Phycology, Barley straw as an inhibitor of algal growth III: the role of fungal decomposition. vol. 4, p 353-355, 1992; Barrett et al., Field and laboratory experiments on the effects of barley straw on algae. BCPC monograph No. 59: Comparing glasshouse and field pesticide performance II. p 191-200, 1994], Phenolic and benzoic acids derived from pine needles act as growth inhibitory chemicals in algal growth [Olmsted et al., Relative effects of known plant inhibitors or species from first two stages of oldfield succession. Southwestern Nat., Vol. 15, p 165-173, 1970; Rice et al., E Bot. Bull. Acad. Sinica., Effects of decaying rice straw on growth and nitrogen fixation of a bluegreen alga. vol. 21, p 111-117, 1980] have been reported. However, there is a disadvantage in that it takes 6 months to 1 year or more to confirm the effect of algae suppression, and it is difficult to apply directly to the appeal of severe change in precipitation [Everall et al., Water Research, The identification and significance of chemicals released from decomposing barley straw during reservoir algal control. vol. 31, p 614-620, 1997. In addition, their use may cause ecosystem disturbances, such as reduced biodiversity in the aquatic ecosystem and microbial community changes.

Therefore, recently, methods of using natural enemies or algae bacteria having excellent algae control ability have been actively studied.

In view of the above, the present inventors have isolated a new strain Rhodococcus sp. KBR-5 [KCTC 11509BP] from Rhodococcus sp. The present invention was completed by confirming the growth inhibition and resolution of algae.

Accordingly, an object of the present invention is to provide a KBR-5 [KCTC 11509BP] genus Rhodococcus having algae growth control and resolution.

In addition, the present invention is Rhodococcus sp. KBR-5) [KCTC 11509BP] It is another object to provide an algae containing a culture medium or a culture supernatant as an active ingredient.

In addition, the present invention is Rhodococcus sp. It is another object to provide an algae containing KBM-5) [KCTC 11509BP] produced as an active ingredient, a low molecular substance of the algae component.

The present invention is a genus of Rhodococcus sp. Rhodococcus sp. Can be inhibited and degraded algae growth. KBR-5) [KCTC 11509BP].

In addition, the present invention is Rhodococcus sp. KBR-5) [KCTC 11509BP] Another feature is a culture agent, a culture supernatant or an algae containing a low molecular substance of the algae component produced by the strain as an active ingredient.

KBR-5 genus Rhodococcus sp. KBR-5) [KCTC 11509BP] can effectively inhibit algae growth and decompose algae, so using this strain in an algae eutrophication is more economical and environmentally friendly than conventional methods.

Therefore, it is expected to be very useful for the restoration of aquatic ecosystems contaminated with eutrophication as well as preservation of biodiversity of water systems. In addition, it is possible to expect economic cost savings in water management due to green algae.

Hereinafter, the present invention will be described in detail.

The present invention KBR-5 ( Rhodococcus sp. KBR-5) [KCTC 11509BP].

As the algae, cyanobacteria such as Microcystis , Anabaena , Oscillatoria , etc. (cyanobacteria, cyanobacteria, blue-green algae) are preferable.

Referring to the process for identifying and identifying new strains according to the present invention as follows.

By microsis Samples were taken from the surface of the appeal (0-30 cm) between 12 and 15:00 from the time the green algae occurred. Water temperature, pH, transparency, DO, light intensity, turbidity, etc. were measured using a water quality analyzer. Water temperature and pH were measured on-site using a YSI meter [63/100 FT, YSI Inc, Yellow Springs, USA] immediately after harvesting and the samples taken were refrigerated.

From the sample taken by the above method in the eutrophicated water system, microcistis, a green algae-producing race, is incubated with BG11 (pH 7.5) medium continuously at 120 μE / m ² / s. Microorganisms coexisting with cultured microcistis are separated by microbial method using MPN method using various nutrient media (LB, RA, NA, 1/5 or 1/20 diluted LB). Pure separation. Microorganism cultured supernatant and culture supernatant in 2% microcystis eruginosa UTEX 2388 culture medium and microcistis eruginosa NIES 843 culture solution were treated to 2% concentration, and then cultured for 1 to 3 days. Microorganisms with excellent growth inhibitory activity and unalterable resolution after several passages are finally selected.

Among the selected strains, microorganisms with excellent inhibition of growth and stable resolution of cyanobacterial eruzinosa were identified as 16s rDNA sequencing method, which was identified as Rhodococose. KBR-5 ( Rhodococcus sp. KBR-5). The new strain was deposited on 12 May 2009 at the Korea Institute of Bioscience and Biotechnology, and the accession number was assigned to KCTC 11509BP.

In order to confirm the cyanobacterial resolution of the strain, the new strain culture medium and the culture supernatant were mixed and cultured in cyanobacteria microsis eruginosa UTEX 2388 and microcistis eruginosa NIES 843 to confirm that the growth of cyanobacteria is inhibited and degraded. It was. In addition, it was confirmed that the low molecular weight (molecular weight 2,000 ~ 8,000) showing the algae function of the various metabolites they produce shows the growth inhibition and resolution of cyanobacteria.

The new strain of the present invention is a bacterium that coexists with the green algae-causing species in eutrophication system, and its effect is excellent even with the culture supernatant without bacteria, as well as its activity against cyanobacterial eruzinosa selectively inhibits growth. It is possible to decompose microcistis eruginosa, and eco-friendly control method with no ecosystem disturbance because there is no secondary environmental pollution such as reduction of biodiversity in water ecosystem by minimizing damage to other aquatic organisms due to species selectivity. As an advantage.

In addition, the strain has the advantage of excellent growth inhibition and resolution for other green algae causing species such as Anabanna and Oscillatoria in addition to microcistis eruginosa.

Thus, KBR-5 ( Rhodococcus sp. KBR-5) [KCTC 11509BP] is an effective and selective biological regulator of toxic cyanobacterial microsis erusinosa, with no secondary environmental pollution and no biological disturbance in water ecosystems in controlling cyanobacteria. It can be expected to be faster and faster than other tidal control methods, and its marketability is very small and simple.

Therefore, the present invention is the genus Rhodococcus sp. KBR-5) [KCTC 11509BP] It includes not only the culture or culture supernatant but also the algae containing the small molecule of the algae component which these strains produce as an active ingredient.

In addition, the new strain of the present invention can increase the cleanliness of eutrophicated appeal, thereby reducing the economic cost of water supply management such as filter paper closure in water management and increasing the value of visual clean appeal, thereby increasing the social added value and economic and social ㆍ It is expected to be very useful culturally.

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

Example 1; Isolation of Microorganisms

After sampling the upper layer (water surface 30 cm) from the area where the green algae occurred, isolate the green algae dominant species algae (targeting microcistis), and then BG11 (pH 7.5) medium [NaNO ₃ 17645 μM, K ₂ HPO ₄ 223.9 μM, MgSO ₄ 7H ₂ O 304.3 μM, Na ₂ CO _{3 200} μM, CaCl ₂ 243.3 μM, Ferric citrate 24.5 μM, Citric acid 31.2 μM, EDTA 3.4 μM, Microelement (H ₃ BO ₃ 5.72 g, MnCl ₂ 4H ₂ O 3.62 g, ZnSO ₄ 7H ₂ O 0.444 g, Na ₂ MoO ₄ 2H ₂ O 0.782 g, CuSO ₄ 5H ₂ O 0.158 g, Co (NO ₃ ) ₂ 6H ₂ O 0.0988 g per liter) 1 ml per liter] Were continuously irradiated at 120 μ E / m ² / s and microorganisms with stable microbial community were isolated and cultured through several passages. In order to isolate microorganisms in stable microorganisms, microcistis cultures or mucilage (mucilage) surrounding microcistis, vortex the cyanobacterial solution for 30 minutes, and then various nutrient media (LB, R2A). , NA and 1/5 or 1/20 diluted LB) were diluted and plated and incubated at 37 ° C. for 24 to 48 hours. The resulting colonies were passaged three or four times in the same medium to isolate strains that inhibited growth and degraded algae.

Example 2: Identification of Isolated Strains

Identification of the isolation strain isolated in Example 1 was cultured using the same medium, and genomic DNA was isolated using a genomic DNA isolation kit (Quiagen, Hilden, Germany). In order to amplify the variable region in 16S rDNA by PCR, two universal primers (9F, 536R) commonly used in 16s rDNA were used.

9F Primer: GAGTTTGATCCTGGCTCAG (SEQ ID NO: 1)

536R Primer: GTATTACCGCGGCTGCTG (SEQ ID NO: 2)

PCR conditions were denatured at 95 ° C. for 5 minutes, amplified DNA for 30 minutes at 95 ° C. for 1 minute, 56 ° C. for 30 seconds, and 72 ° C. for 30 seconds, and finally extended at 72 ° C. for 10 minutes to terminate the PCR reaction. .

About 500 bp of amplified DNA was isolated from the electrophoresis result and transformed using the cloning vector pGEM T-easy vector (Quiagen, Hilden, Germany).

The sequences of the variable regions in 16S rDNA were compared and analyzed using the BLAST Search Program (NCBI) (SEQ ID NO: 3). As a result of sequencing of 16S rDNA, it was confirmed that the new strain of the present invention had high homology with Rhodococcus genus through "The BLAST search" [Table 1], and the strain of the present invention was RKB of genus Rhodococcus. -5 ( Rhodococcus sp. It was named KBR-5) and was deposited on May 12, 2009 at the Korea Institute of Bioscience and Biotechnology, an international microorganism depositing institution, and was assigned accession number KCTC 11509BP.

16S rDNA sequencing results Strain Similarity (%) Proximity Microbial Name Access number KBR-5 349/350 (99%) Rhodococcus sp. gb | FJ015033.1 |

Example 3: Inhibition of growth of cyanobacteria on resolution of algae

Inoculate 30 ml of Cyanobacterial eruginosa UTEX 2388 and 30 ml of microcistis eruginosa NIES 843 in 100 ml of fresh BG11 medium, and 2% inoculation of Rhodococcus KBR-5 culture and culture supernatant for 3 days Mixed cultures. Culture conditions were incubated at 120 ℃ E / m ² / s, algae culture conditions.

Both culture and supernatant of the strain showed growth inhibition and resolution for microcistis eruginosa and inhibited growth and degradability of cyanobacteria in supernatant cultured supernatant with cells removed to avoid microbial community changes when applied to an environment where eutrophication occurred. Was investigated. As shown in FIG. 1, the microalgae microcistis eruginosa UTEX 2388 and microcistis eruginosa NIES 843 were visually observed in a mixed culture solution inoculated with a supernatant of KBR-5 culture supernatant of algae and Rhodococcus. Compared to. As a result, it was confirmed that green (chlorophyll a) peculiar to the bacterium was significantly reduced in both the isolated bacterial culture medium and the culture supernatant within 3 days after the mixed culture. In addition, growth inhibition and resolution of the microcistis eruginosa algae Anabaena variabilis NIES 23 were also confirmed in the same manner, and growth inhibition and resolution could be confirmed [FIG. 2].

The mixed culture solution inoculated with 2% culture supernatant of cyanobacteria and Rhodococcus KBR-5 and microcistis eruginosa monoculture were compared by counting optical microscopy [Fig. 3]. . As a result, it was observed that the numbers of microcistis eruginosa UTEX 2388, microcistis eruginosa NIES 843, and Anabae or NIES 23 decreased rapidly in culture medium on day 3 of inoculation. Particularly, in the case of microcistises, growth inhibition was observed from the 2nd day of the mixed culture, and on the 3rd day, the microcistiss were decomposed. KBR-5 genus mycobacteria according to the present invention was confirmed to have a growth inhibition and resolution for the microcistis and Ana banna, in particular it was found to have an excellent algae effect against the microcistis.

Example 4: Inhibition of growth of blue algae and resolution of new strain producing low molecular weight material

The molecular weight of the substance showing the algae effect in the metabolite produced by KBR-5 of the new strain Rhodococose according to the present invention was confirmed through a dialysis experiment. It can be presumed that this algicidal effect is caused by the metabolite produced by KBR-5 of the new strain Rhodococose of the present invention with reference to the results of the microaltisian cell culture supernatant. Therefore, dialysis experiment was performed to confirm the molecular weight of the metabolite having the algal effect produced by the new strain Rhodococos genus. KBR-5 genus Rhodococcus was shaken for 30 hours at 30 ° C. and 150 rpm using LB liquid medium. After removing the cells by centrifugation for 10 minutes at 12,000 rpm, using a 0.22 ㎛ diameter filter to obtain a culture supernatant. Using the dialysis membranes of different molecular weights, the algal effect was compared in the mixed culture solution inoculated with 2% of the culture supernatant obtained from the Cyanobacterial eruzinosa UTEX 2388 culture medium and the microcistis eruginosa UTEX 2388 single culture medium. As a result, as shown in Figure 4, it was confirmed that among the metabolites produced by KBR-5 of the new strain Rhodococose low molecular weight material having a molecular weight of 2,000 and 8,000 has a killing effect against microcistis eruginosa UTEX 2388 .

KBR-5, a genus of Rhodococcus mycobacteria according to the present invention, has a low treatment concentration and an excellent algae effect in which growth inhibition and degradation occur in a fast time with respect to microcistis eruginosa, and thus the algae control agent and water system in the green algae generation area. It can be used as a biological material for purification of polluted environment.

In addition, compared to the treatment time of 6 months or more of the existing technology, the effect of the two days in the case of KBR-5 in the Rhodococos, 2% lower concentration to maximize the killing effect.

In addition, the low molecular weight between 2,000 and 8,000 molecular weight produced by KBR-5 in the new strain Rhodococose shows excellent algae effect, so it can be used as a bio-material for environmentally-friendly pollution and environmental purification that eliminates secondary environmental pollution, and can be commercialized. .

Therefore, it is expected to have a great effect on the industrialization of low-cost, eco-friendly and efficient green algae control biomaterials.

1 is a photograph showing the growth inhibition of microcystis aeruginosa of the new strain Rhodococcus sp. KBR-5 (KCTC 11509BP) genus Rhodococcus .

Figure 2 is a photograph showing the growth inhibition and resolution of Anabaena v Anabilis NIES 23 of KBR-5 [KCTC 11509BP] genus Rhodococcus.

Figure 3 is a graph showing a change in the number of cyanobacteria cells after mixing and cultured KBR-5 of the genus cyanobacteria Rhodococos.

Figure 4 is a photograph showing the growth inhibition and resolution of the low molecular weight (molecular weight 2,000 ~ 8,000) produced by KBR-5 [KCTC 11509BP] genus Rhodococcus.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Alage lysing bacteria Rhodococcus sp. KBR-5 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 9F primer <400> 1 gagtttgatc ctggctcag 19 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 536R primer <400> 2 gtattaccgc ggctgctg 18 <210> 3 <211> 350 <212> DNA <213> Rhodococcus sp. KBR-5 <400> 3 cggcgtcgct gcatcaggct ttcgcccatt gtgcaatatt ccccactgct gcctcccgta 60 ggagtctggg ccgtgtctca gtcccagtgt ggccggtcac cctctcaggt cggctacccg 120 tcgtcgcctt ggtaggccat taccccacca acaagctgat aggccgcggg cccatcctgc 180 accgataaat ctttccacca cccaccatgc gataggaggt catatccggt attagaccca 240 gtttcccagg cttatcccga agtgcagggc aaatcaccca cgtgttactc acccgttcgc 300 cgctcgtgta ccccgaaagg ccttaccgct cgacttgcat gtgttaagca 350

Claims (3)

Rhodococcus sp. KBR-5 (KCTC 11509BP), characterized by breaking down algae or controlling growth. [4] The genus Rhodococcus sp. KBR-5 [KCTC 11509BP] according to claim 1, wherein the algae is Microcystis , Anabaena or Oscillatoria . ]. An algae comprising a culture solution or a culture supernatant of KBR-5 [KCTC 11509BP] in Rhodococcus of claim 1 as an active ingredient.

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