KR20090126917A - Novel geobacillus thermodenitrificance sg-01 having nitrates-eliminating function and method using the same - Google Patents

Abstract

PURPOSE: A Geovacillus thermodenitrificance SG-01 strain having a function of removing nitrate is provided to produce fermentation compost and use as additive for increasing denitration. CONSTITUTION: A nitrate removal agent, compost fermentation agent, and additive for waste water treating apparatus contains the Geobacillus thermodenitrificance SG-01(KCCM-10777P) or its culture as an active ingredient. The Geobacillus thermodenitrificance SG-01(KCCM-10777P) is isolated from porcine feces compost. The Geobacillus thermodenitrificance SG-01 optimally grows at 50°C-65°C and pH 6-9.

Description

Novel Geobacillus thermodenitrificance SG-01 having Nitrates-eliminating function and method using the same

The present invention relates to a novel microbial strain having a nitrate removal function and a method of using the same, and more particularly, to a new Geobacillus dimodinitripicus SG-01 strain having excellent nitrate removal function, and denitrification using the same. It is related with the method of using for uses, such as an additive, in the process to make it into.

Recently, livestock wastewater has been recognized as the main culprit of odor generation and environmental pollution, and various complaints have occurred, and the dumping of ocean dumping for disposal is prohibited by local governments. They are composted and disposed of through a residential facility.

However, the nitrogen component represented by ammonia in livestock wastewater is recognized as a serious pollutant due to its own toxicity and there is no way to completely solve the above problems. Therefore, wastewater treatment technology to remove nitrogen is essential for the improvement of livestock environment as well as for the protection of water resources.

In general, a technique for removing nitrogen components is divided into biological treatment and physicochemical treatment. Especially, wastewater containing high concentrations of organic matter and nitrogen components such as livestock wastewater is widely used in biological treatment methods. Nitrogen contained in the waste water induces nitrification to oxidize ammonia to nitrite under aerobic conditions, or to nitrate to nitric acid, and denitrification to convert nitrous or nitrate to nitrogen under anoxic conditions. Nitrogen may be removed. The denitrification process is carried out by several heterotrophic microorganisms that reduce nitrous acid or nitric acid produced in ammonia. In particular, the removal of nitrate is the main target for nitrogen treatment in livestock wastewater, which is converted to ammonia by the oxidation process immediately after the discharge from the barn. This ammonia is a major source of odors and it is necessary to technically remove these pollutants through the removal of nitrogen.

Also, wastewater containing high concentration of ammonia nitrogen, such as livestock wastewater, is known to cause nitrite accumulation rather than normal nitrification process, which is known to impede the action of nitrobacter. Therefore, immediate denitrification process is important. Can be. Ammonia oxidizing bacteria and nitrite oxidizing bacteria which act on nitrification process are selected and used by many bacteria such as nitrosomonas and nitrobacter, but the microorganisms having nitrate removal function are relatively small and require continuous development.

Geobacillus dimodinitriphysis has been reported by Manachini et al. (2000), Nazina et al. (2001) and Thaddeus and Hubert (2006), and in Korea, geobacillus dimodinitriphyte including arabinose isomerase activity. Cons strains (Republic of Korea Patent Registration 10-0821377) has only been reported, nitrate removal function has not been studied yet.

Therefore, in order to solve the above problems, the present inventors confirmed that a specific microbial strain isolated from pig manure compost has a nitrate removal function, and as a result of identifying and identifying this strain, three new geobacillus genera having excellent nitrate removal function were identified. To identify the strain, one of them is named as Geobacillus thermodenitrificance SG-01 (Geobacillus thermodenitrificance SG-01) strain, to evaluate the optimum temperature, optimal growth curve, optimal pH, flame resistance, nitrate removal function The present invention was completed by confirming the presence of the present invention.

It is an object of the present invention to provide a novel strain of Geobacillus dimodinitripicus SG-01 which has excellent nitrate removal function and is resistant to high temperature and flame resistance.

In addition, the present invention provides a use of a new strain of Geobacillus dimodini tripicus SG-01 or a culture of this strain, as an additive having excellent nitrate removal function, a compost fermenter, an additive for wastewater treatment apparatus, and the like. There is a purpose.

According to a feature of the present invention for achieving the above object, the present invention is a new geobacillus dimodinitripicus SG-01 (Geobacillus thermodenitrificance SG-01) strain having a nitrate or nitrite removal function (Accession Number: KCCM-10777P ).

In another aspect, the present invention provides a culture of a new geobacillus thermomoditrificance SG-01 strain (Accession Number: KCCM-10777P) having a nitrate or nitrite removal function.

In addition, the present invention provides a compost fermentation agent containing a strain of Geobacillus dimodini tripicus SG-01 or the strain culture as an active ingredient.

In order to achieve another object, the present invention provides a nitrate scavenger comprising the Geobacillus dimodinitripicus SG-01 strain or the strain culture as an active ingredient.

Furthermore, the present invention provides an additive for a wastewater treatment apparatus which contains the S.O. strain SG-01 strain or the strain culture as an active ingredient.

Hereinafter, the present invention will be described in detail.

At this time, if there is no other definition in the technical terminology and scientific terminology used has a meaning commonly understood by those of ordinary skill in the art.

In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

The present invention provides a new strain of Geobacillus sp. Specifically, the present invention provides a new strain of genus Geobacillus sp. Isolated from pig manure compost, preferably, strain Geobacillus dimodini Tripicus SG-01 isolated from pig manure compost.

The new Geobacillus sp. Strain of the present invention is named Geobacillus thermodenitrificance SG-01, and the Geobacillus demodinitriphilus SG-01 strain according to the present invention. On September 07, 2006, Korea was deposited with the Korean Culture Center of Microorganisms, an international depository, and was granted accession number KCCM-10777P.

In addition, the present invention provides a method for separating the Geobacillus sp. Strain from pig manure compost. The inventors of the present invention isolated and identified a specific microbial strain, Geobacillus dimodinitripicus SG-01 from the manure compost of pigs, and cultured this strain to obtain a culture.

In addition, due to the excellent nitrate removal function of the Geobacillus dimodini tripicus SG-01 strain of the present invention provides a use for nitrate remover, compost fermentation agent, additives of waste water treatment apparatus and the like. The present invention is preferably used as an additive containing a new geobacillus dimodinitripicus SG-01 or a culture of this strain as an active ingredient, specifically, when cultivating crops that need to remove nitrate, such as livestock wastewater, nitrate removal It is preferable to use as a nitrate remover, a compost fermentation agent, an additive of the waste water treatment apparatus.

As described above, the Geobacillus dimodini tripicus SG-01 strain of the present invention is a high temperature fermentation compost and liquid manure, or biological sludge discharged from the livestock wastewater treatment process and treatment process of food factories, chemical plants, etc. It can be usefully used in a wide variety of processes, such as being used as an additive to enhance the denitrification in the purification of organic sludge and the like.

 In addition, the new Geobacillus dimodini tripicus SG-01 strain according to the present invention can be used to remove wastewater nitrates and nitrites for the purification and deodorization of wastewater, so as to add a compost fermentation agent, livestock manure treatment agent, additives of wastewater treatment equipment, etc. It can be widely used for cultivating crops that require nitrate removal.

Hereinafter, the present invention will be described in more detail with reference to specific examples.

However, the following examples are only for exemplifying the present invention, and the content of the present invention is not limited to the following examples, and various changes or modifications can be made within the spirit and scope of the present invention. It is obvious to those who have.

<Example 1> Isolation and Identification of Strains of the genus Geobacillus from Porcine Manure

The present invention, in order to separate a specific Geobacillus sp. Microorganism from pig manure compost, smear the pig manure compost samples on LB agar plate medium and incubated at 60 ℃ for 24 hours to primarily five strains Selected.

Using the strains selected above, strains based on color, odor, colony form, and gram-positive, but the fastest growth were isolated. This strain was again activated by passage 3-4 times, incubated for 24 hours at 37 ° C. using LB medium, and then preserved by glycerol stock and used for the next experiment.

In order to observe the characteristics of the isolated bacteria was carried out by scanning electron microscopy (SEM) using Karnorsky's method. Strains were simply treated with 2% paraformaldehyde overnight at 4 ° C. The sample was washed three times with 0.05 M sodium cacodylate buffer (pH 7.2) three times for 10 minutes, and then washed with distilled water after adding 1% osmium tetraoxide. And dehydrated with 30%, 50%, 70%, 80%, 90%, 100% ethanol. The dried sample was treated with 100% hexamethyldisilazane for 15 minutes and dried again at 50 ° C. Then, gold-coating was observed by scanning electron microscopy.

Specifically, as shown in the scanning electron micrograph of Figure 1, the three strains isolated in the present invention was confirmed to be Gram-positive bacillus, such as Bacillus, Geobacillus. Three strains were found to be 99.9% similar to the Geobacillus thermodenitrificance among the Geobacilli sp. Strains by API 50 CHB kit analysis (Biomeriu, France). The present inventors evaluated the isolated strain as a predominant strain on pig manure compost by the fact that Geobacillus dimodinitripicus was found in compost at 60 ° C by Hatsu et al.

The results are shown in Table 1 below.

Results of Sugar Fermentation Experiments of Giobacilli strains using API 50 CHL Kit No Biochemical Results No Biochemical Results 0 Control - 25 Esulin + One Glycerol + 26 Salicin + 2 Erythritol - 27 Cellobiose + 3 D Arabinose - 28 Maltose + 4 L Arabinose - 29 Lactose - 5 Ribose + 30 Melibiose - 6 D Xylose + 31 Sucrose - 7 L Xylose - 32 Trehalose + 8 Adonitol - 33 Inulin - 9 Beta Methyl-D-Xylose - 34 Melezitose + 10 Galactose - 35 Raffinose + 11 Glucose + 36 Starch + 12 Fructose + 37 Glycogen + 13 Mannose + 38 Xylitol - 14 Sorbose - 39 Gentiobiose - 15 Rhamnose - 40 D Turanose - 16 Dulcitol - 41 D Lyxose - 17 Inositol - 42 D tagatose - 18 Mannitol + 43 D Fucose - 19 Sorbitol - 44 L Fucose - 20 Alpha-Methyl-D-Mannoside - 45 D Arabitol - 21 Alpha-Methyl-D-Glucoside - 46 L Arabitol - 22 N-Acethyl-Glucosamine - 47 Gluconate - 23 Amygdalin - 48 2-Keto-Gluconate - 24 Arbutin - 49 5-Keto-Gluconate -

In addition, in order to compare the 16S rDNA sequences of the strains of the genus Geobacillus sp. 9F primer (5'-AGAGTTTGATCCTGGCTCAG-3 ') using a 926R primer and amplified by 30 cycles PCR (polymerase chain reaction) at 55 ℃ As a result of analyzing the 16S rDNA nucleotide sequence using a program called BLAST on a PC computer, as shown in SEQ ID NO: 1, 962 nucleotide sequences of the genus Geobacillus sp. Strain were 99% homologous to the geobacillus dimodinitripicus strain. (similarity) was again found to be the highest (see Sequence Listing in Attachment).

SG-01 was named as Geobacillus thermodenitrificance SG-01 and was used in subsequent experiments.

From the above results, the Geobacillus dimodini tripicus SG-01 strain according to the present invention was deposited on September 07, 2006 to the Korean Culture Center of Microorganisms, an international depositing institution, and deposited accession number KCCM-10777P. You are given a call.

<Example 2> Optimum growth temperature investigation of Geobacillus dimodini tripicus SG-01 strain

In order to investigate the optimum growth temperature of the Geobacillus dimodinitripicus SG-01 strain of the present invention, inoculated into 300 ml Erlenmeyer flask using LB liquid medium to 37, 50, 55, 60, 65, 70 ℃ Placed in a adjusted incubator and incubated for 24 hours.

In addition, in order to measure the total number of viable bacteria of the genus Geobacillus sp. Strain, the culture solution was taken at a price of 6 hours, serially diluted 10-fold with 0.85% NaCl solution, and plated by 0.1 mL aliquots into LB agar plates. . The smeared flat plate was incubated at 37 ° C. for 48 hours, and the colony forming unit was measured therefrom.

As a result of investigating the optimum temperature condition of the genus Geobacilli strain, it was shown that the growth from 50 ℃ to 65 ℃ as shown in Figure 2, but did not grow at 37 ℃ or 70 ℃. The optimum growth temperature was found to reach the highest bacterial count when incubated at 55 ℃ ~ 60 ℃. After 12 hours, the optimum growth temperature was decreased over 65 ℃.

Example 3 Investigation of Growth Characteristics of Geobacilli D. Modinifolius SG-01 Strain

In order to investigate the growth characteristics of the Geobacillus dimodinitripicus SG-01 strain of the present invention, LB liquid medium was incubated for 72 hours at 55 ℃ without adjusting the pH in a 7 liter fermenter (Jar fermenter) .

In addition, in order to measure the total viable count of the strains of the genus Geobacilli, colony forming unit (colon forming unit) was measured by plating on a plate medium as in Example 2.

As a result of investigating the optimum growth conditions of the strains of the genus Geobacillus, as shown in Figure 3, after culturing for 12 hours it was found to reach the peak, that is, the peak phase to 5.7 × 10 ⁸ CFU / ml and then gradually decreases . At this time the pH was observed to maintain a constant 7.0.

<Example 4> Optimal pH investigation of Geobacillus dimodini tripicons SG-01 strain

The present invention, in order to investigate the optimal pH characteristics of S.O. strain SG-01 strain, first inoculated the cells in LB medium to 1% and incubated for 24 hours at 37 ℃ and then to pH 5.0-9.5 Adjusted citrate-Na ₂ HPO ₄ Optimal for growth by standing in the buffer (pH 5-6.8), Tris-HCl buffer (pH 7-8.0), glycine-NaOH buffer (pH 8.5-9.5) for 30 minutes and measuring the number of microorganisms remaining. pH was investigated.

As a result, as shown in Figure 4, the geobacillus dimodinitripicus SG-01 strain of the present invention did not grow below pH 6 and above pH 9, but can be utilized in the neutral region because it shows the maximum absorbance value at pH 7 Was judged.

<Example 5> Flame-proof investigation of Geobacillus dimodini tricyclics SG-01

In order to investigate the flame resistance of the Geobacillus dimodinitripicus SG-01 strain of the present invention, NaCl is added to LB broth about 0-4%, and the Geobacillus dimodini tripicus SG-01 strain is 1% each. Inoculation. The culture medium was collected after shaking for 24 hours and 48 hours while shaking the inoculated liquid medium at 150 rpm at 37 ° C., and the absorbance was measured at 600 nm using UV-spectrophotometer (U-2000, Hitachi, Japan) to determine growth. .

As a result, as shown in Figure 5, the genus Geobacillus strains of the present invention was shown that the growth is possible even in the presence of 3% NaCl, 1 ~ 2% was found to increase the growth rather. As a result, it was judged that it can be used for marine or wastewater.

 Example 6 Investigation of Nitrate Scavenging Ability of Geobacillus Demodinitripicus SG-01 Strain

In order to investigate the nitrate removal function of the geobacillus dimodinitripicus SG-01 strain of the present invention, the nitrate (nitrate, NaNO ₃ ) and nitrite (nitrite, NaNO ₂ ) were put in an Erlenmeyer flask containing LB liquid medium to 100 ppm. The bacteria were inoculated to 1% and cultured anaerobicly. Samples were taken from the Erlenmeyer flask at 6 hour intervals to determine the residual nitrate and dinitrate concentrations. Nitrate was measured with a slight modification of Donald and Nicholas (1957). After 160ul was collected and centrifuged, 100ul of the supernatant was transferred to another Eppendorf tube, and 50ul brucine-sulanilic acid solution was added thereto and allowed to stand for 5 minutes. 500 μl of sulfuric acid (80%) was added to the mixture and left in the dark for 10 minutes, and then 500 μl of distilled water was added to calculate the absorbance at 540 nm (Donald, DJ and Nicholas, AN: Determination of nitrate and nitrite, p 981-984.In Colowick, SP and Kaplan, NO (ed.), Methjods in enzymology, vol. 3. Academic Press, New York (1957). The nitrite concentration was sampled and centrifuged at 3,000 rpm for 20 minutes. 100ul supernatant was taken in a test tube, and color developing reagent (Sulfanylamide solution, Naphthylenediamine solution, 200ul each) was added and developed in the dark for 5 minutes. After adding 500ul of distilled water, the absorbance was measured at 540nm to calculate the concentration.

As a result, as shown in Figure 6, the geobacillus dimodini tripicus SG-01 strain of the present invention is rapidly increased as the incubation time after 12 hours of nitrate removal rate of about 85.8% of the removal rate after 24 hours After 48 hours, the removal rate was 91.9%. The nitrite removal rate decreased rapidly with time, showing a removal rate of about 27% at 12 hours of culture, and a removal rate of about 99.1% after 24 hours, and almost nitrite was removed after 48 hours.

1 shows a scanning electron microscope (SEM) photograph of a Geobacillus thermomoditrificance SG-01 strain of the present invention.

Figure 2 is a graph showing the optimum growth temperature of the geobacillus dimodini tripicus SG-01 strain of the present invention in the range of 37 ~ 70 ℃.

Figure 3 is a graph showing the growth characteristics using the LB liquid medium of the Geobacillus dimodini tripicus SG-01 strain of the present invention.

4 is a graph showing the growth rate at each pH of the Geobacillus thermomoditrificance SG-01 strain of the present invention.

Figure 5 is a graph showing the flame resistance of the Geobacillus dimodini tripicus SG-01 strain of the present invention in the range of 0 ~ 4% NaCl.

6 is a graph showing the nitrate and nitrite removal function of the Geobacillus dimodini tripipans SG-01 strain of the present invention.

<110> Hankyong University Academic Cooperation Center <120> Novel Geobacillus thermodenitrificance SG-01 having          Nitrates-eliminating function and method using the same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 962 <212> DNA <213> Artificial Sequence <220> <223> Geobacillus thermodenitrificance SG-01 <400> 1 gtgcttatcg cgttagctgc agcactaaag ggtgtgaccc ctctaacact tagcactcat 60 cgtttacggc gtggactacc aggggtatct aatcctgttt gctccccacg ctttcgcgcc 120 tcagcgtcag ttgcaggcca gagagccgcc ttcgccactg gtgttcctcc acatctctac 180 gcatttcacc gctacacgtg gaattccgct ctcctctcct gcactcaagt cccccagttt 240 ccaatgaccc tccacagttg agccgtgggc tttcacatca gacttaaagg accgcctgcg 300 cgcgctttac gcccaataat tccggacaac gctcgccccc tacgtattac cgcggctgct 360 ggcacgtagt tagccggggc tttctcgtga ggtaccgtca ccgcgccgcc ttattcaaac 420 ggcgctcctt cgtccctcac aacagagctt tacgacccga aggccttctt cgctcacgcg 480 gcgtcgctcc gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg 540 agtctgggcc gtgtctcagt cccagtgtgg ccggtcaccc tctcaggccg gctacgcatc 600 gttgccttgg cgagccgtta cctcaccaac tagctaatgc gccgcgggcc catccgcaag 660 tgacagccga agccgccttt caaccaaaga ccatgcggtc tttggtgtta tccggtatta 720 gctccggttt cccggagtta ttccggtctt gcgggcaggt tgcccacgtg ttactcaccc 780 gtccgccgct gaccaaacaa gagcaagctc tcgttcggcc cgctcgactt gcatgtatta 840 ggcacgccgc cagcgttcgt cctgagccag gatcaaactc taatcactaa gtgaattcgc 900 ggccgcctgc aggtcgacca tatgggagag ctcccaaacg cgttggatgc atagcttgag 960 tt 962  

Claims (5)

Geobacillus thermodenitrificance SG-01 strain with access to nitrate or nitrite removal (Accession No .: KCCM-10777P). Geobacillus dimodinitripicus SG-01 (Accession No.:KCCM-10777P) strain culture with nitrate or nitrite removal function. A nitrate scavenger comprising the Geobacillus dimodinitripicus SG-01 strain of claim 1 or the strain culture of claim 2 as an active ingredient. A compost fermentation agent comprising the Geobacillus dimodinitripicus SG-01 strain of claim 1 or the strain culture of claim 2 as an active ingredient. An additive for a wastewater treatment apparatus comprising the Geobacillus dimodinitripicus SG-01 strain of claim 1 or the strain culture of claim 2 as an active ingredient.

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