JP4704901B2 - Novel strains with aromatic hydrocarbon degradability under anaerobic conditions and uses thereof - Google Patents

Description

  The present invention relates to a microorganism having an ability to degrade aromatic hydrocarbons under anaerobic conditions and an environmental purification method using the microorganism.

  In recent years, environmental pollution of oil, soil, ocean, groundwater, etc. has increased particularly in recent years. Petroleum components are toxic to living organisms, so if oil spills into the environment due to leaks from refineries, tanker stranding, etc., not only humans but also animals such as animals, fish, birds, seaweed, shellfish, etc. Effect. In particular, volatile aromatic hydrocarbons represented by benzene, toluene, ethylbenzene, and xylene are highly toxic among petroleum components, and because they are volatile, they diffuse from the atmosphere and have some water solubility. There is also a danger of diffusion from groundwater, which is a big problem. In addition, wastewater discharged from paint factories and the like contains a large amount of volatile aromatic hydrocarbons used as raw materials, and the treatment thereof is also a big problem.

  Up to now, methods for volatilizing and recovering volatile aromatic hydrocarbons have been mainly used in the treatment site in an actual polluted environment. However, the cost is high, or the recovered hydrocarbon is recovered in some way. There were problems such as having to process again, and it was not an effective means. On the other hand, in recent years, the application of environmental remediation using microorganisms, so-called bioremediation, has come to be discussed. So far, many volatile aromatic hydrocarbon-degrading bacteria have been isolated and their degradation mechanisms have been analyzed. Many of these are aerobic bacteria that require oxygen for volatile aromatic hydrocarbon degradation. In actual pollution sites, oxygen is consumed by the aerobic bacteria decomposing pollutants in the early stage, and it is often anaerobic. The number of volatile aromatic hydrocarbon-degrading anaerobic bacteria isolated is less than that of aerobic bacteria, and only one case has been reported so far. 2). For this reason, the development of microorganisms having high anaerobic aromatic hydrocarbon resolving ability has been awaited in the process of bioremediation or wastewater treatment.

Coates, JD, Chakraborty, R., Lack, JG, O'Connor, SM, Cole, KA, Bender, KS, Achenbach, LA (2001) Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas.Nature 411: 1039-1043 Chakraborty, R., Coates, J.D. (2005) Hydroxylation and carboxylation-Two crucial steps of anaerobic benzene degradation by Dechloromonas strain RCB Appl.Environ. Microbiol. 71: 5427-5432.

  An object of the present invention is to provide means for quickly purifying an anaerobic environment contaminated with volatile aromatic hydrocarbons by using a microorganism having an ability to decompose aromatic hydrocarbons under anaerobic conditions.

In order to solve the above-mentioned problems, the present inventor added benzene labeled with a stable isotope [ ¹³ C] to gasoline-contaminated groundwater and cultured anaerobically. The nucleic acid of benzene-degrading bacteria takes in heavy carbon [ ¹³ C] derived from ¹³ C-labeled benzene, so the specific gravity is larger than the nucleic acid of bacteria that do not degrade benzene, so physically separate the nucleic acids of benzene-degrading bacteria and non-degrading bacteria be able to. As a result of analyzing the nucleic acid of benzene-degrading bacteria, the present inventors have found a strain whose number of bacteria increases with benzene degradation and completed the present invention.

  That is, the present invention provides the following (1) to (4).

(1) A microorganism belonging to the genus Azoarcus having the ability to decompose aromatic hydrocarbons under anaerobic conditions.

(2) The microorganism according to (1), which can grow using benzene as a single carbon source and can decompose benzene under anaerobic conditions.

(3) The microorganism according to (1) or (2), wherein the base sequence of DNA corresponding to 16S rRNA is 90% or more homologous to the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

(4) An environmental purification method comprising purifying an environment contaminated with volatile aromatic hydrocarbons by the microorganism according to any one of (1) to (3).

  The present invention provides a novel microorganism having volatile aromatic hydrocarbon resolution. This microorganism can efficiently purify the anaerobic environment contaminated with volatile aromatic hydrocarbons such as benzene.

  Hereinafter, the present invention will be described in detail.

The microorganism of the present invention belongs to the genus Azoarcus and has the ability to decompose volatile aromatic hydrocarbons under anaerobic conditions. The microorganisms of the present invention include microorganisms that can grow using volatile aromatic hydrocarbons such as benzene as a single carbon source and can decompose benzene under anaerobic conditions. Examples of such microorganisms include DN11 strain and AN9 strain isolated by the present inventors.

  These strains isolate microorganisms with benzene-degrading ability from groundwater contaminated with gasoline and, at the same time, analyze microorganisms considered to be dominant in the soil at the gene level by using the DGGE method. It is a novel strain obtained by selecting microorganisms having a homology of 99% or more with the gene sequence obtained in the above from the isolated microorganisms.

A part of the base sequence of DNA encoding 16S rRNA of these strains was determined and identified by NCBI program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). As a result, these strains were found to belong to the genus Azoarcus. However, these strains are recognized as new strains because they show different properties in benzene degradation under anaerobic conditions than known strains belonging to Azoarcus . Azoarcus sp. DN11 ( Azoarcus sp. -It was named SP AN9 strain ( Azoarcus sp. AN9 strain).

  Azo Arcus SP DN11 strain (hereinafter referred to as “DN11 strain”) and Azo Arcus SP AN9 strain (hereinafter referred to as “AN9 strain”) are deposited with the following deposit number at the Patent Microorganism Depositary Center for Product Evaluation Technology. Has been.

Azo Arcus SP DN11 strain: NITE P-153 (Deposit date: November 17, 2005, Identification: DN11)
AZO ARCUS SP9 AN9 stock: NITE P-152 (Deposit date: November 17, 2005, Identification: AN9)
The mycological properties of DN11 strain and AN9 strain are shown below.

A. Morphological properties Morphologically, DN11 and AN9 strains are almost common, and the cell shape is gonococcus and has no flagella. When cultured on an agar plate using dCGY medium (0.05% casamino acid, 0.01% yeast extract, 0.05% glycerol), the color of the colony is colorless to pale yellow and transparent. Furthermore, the substrate specificity of both strains is as shown in Table 1.

B. DNA sequence corresponding to 16S rRNA (hereinafter referred to as “16S rRNA gene”)
The nucleotide sequences of 16S rRNA gene of DN11 strain and AN9 strain are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

  The base sequence was determined according to Current Protocols in Molecular Biology (eds.) (Greene Publishing Associates and Wiley-Interscience, N.Y. (1987)). DNA sequencing was performed using an automated DNA sequencer (ABI 3730; Perkin Elmer, Inc., USA).

  Microorganisms of the present invention include DN11 strain and AN9 strain, as well as microorganisms that exhibit certain similarities with these strains. The microorganism having a certain similarity refers to, for example, a microorganism in which the base sequence of 16S rRNA gene used for classification of microorganisms is similar to the above two strains. Specifically, a microorganism in which the base sequence of the 16S rRNA gene is 90% or more, preferably 97% or more homologous to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, is a microorganism exhibiting a certain similarity to the above strain include.

  Such a microorganism having a certain similarity to the above strain can be isolated from the groundwater contaminated with volatile aromatic hydrocarbons by using the base sequences described in SEQ ID NOs: 1 and 2 as an index.

  In order to grow the microorganism of the present invention, a usual culture method can be mentioned. Culturing can be performed under anaerobic or aerobic conditions. The microorganism of the present invention is inoculated into a BSM medium containing an organic substance, an inorganic salt, a nitrogen source, and other nutrient sources, and cultured by, for example, a stationary culture method or a shaking culture method.

  The temperature condition in the culture is set in the range of the growth temperature of the microorganism to be used, preferably in the range of the optimum growth temperature. For example, it can be set to 20 to 30 ° C, preferably 25 ° C. In addition, what is necessary is just to set the pH of a culture medium in the range of 6.5-7.5.

  Substances added to the medium as inorganic salts include phosphates, magnesium salts, calcium salts, iron salts, and other trace metal salts as necessary. The nitrogen source is not particularly limited as long as it can be assimilated by the microorganism of the present invention, and examples thereof include peptone, cascitone, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, and various amino acids. These nitrogen sources may be used alone or in combination of two or more. Furthermore, an appropriate amount of vitamins or the like may be added as a nutrient source for promoting the growth of the microorganism of the present invention.

  The culture time varies depending on the amount and type of nutrient source, but is usually 7 days or longer, preferably 3 to 14 days.

The microorganism of the present invention can be used for purification of an anaerobic environment contaminated with volatile aromatic hydrocarbons. The “environment” here includes soil, ocean, groundwater, and drainage. Purification of the environment is performed by spraying a culture solution of microorganisms cultured under the above conditions or a dry powder obtained by freeze-drying microorganisms to a contaminated environment. At this time, dry powder and inorganic salts that assist in growth may be mixed and granulated, and the powdered and granular preparations may be dispersed in a contaminated environment. The amount of microorganisms used in the process, depending on the soil and sea water pollution, etc., can be arbitrarily determined, there usually if culture contaminated ground water 1 m ³ or contaminated soil 100 m ² 1L, in dry cell About 5g. Furthermore, the contaminated waste water is purified by mixing the culture solution or the dried microbial cells with the contaminated waste water and culturing for about 7 to 50 days under anaerobic conditions.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

  30 ml each of BSM medium (Table 2) was dispensed into a 50 ml glass vial to inoculate DN11 strain. Benzene was added to a final concentration of 15 μM, and nitrate was added to a final concentration of 2 mM, followed by culturing at 25 ° C. for 28 days. During the culture period, a small amount of gas is recovered from the gas phase in the vial as needed, and this is analyzed directly by GC, and the residual ratio relative to the initial concentration is calculated by measuring the benzene concentration in the aqueous phase in the vial. did. For comparison, the benzene concentration was measured in the same manner when the DN11 strain was not inoculated and when nitrate was not added. In addition to the measurement of the benzene concentration, the total bacterial count (TDC) in the culture solution was measured with a fluorescence microscope. These results are shown in FIG.

  As shown in FIG. 1, the decrease in benzene was not observed even after 28 days in the uninoculated and non-nitrate-added vials, but the nitrate-added vial inoculated with the DN11 strain was degraded by 60% in 14 days from the inoculation . On the 14th day, the same amount of benzene as that initially added was added again, and when the cultivation was continued, the benzene concentration decreased to about 76%. In addition, a significant increase in the number of bacteria was observed only in vials where a decrease in benzene was observed. When nitrate concentration in the vial was examined by HPLC, a decrease in nitrate concentration was observed with a decrease in benzene. From the above results, it was clarified that the decrease of benzene and the growth of bacteria depend on nitrate reduction. It turns out that anaerobic benzene decomposition | disassembly can be performed by using the microorganisms of this invention.

It is a figure which shows the time-dependent change of the benzene residual rate when the microorganisms of this invention are added, and when not adding.

Claims (3)

Azo Arcus SP DN11 strain (Accession number NITE P-153). Azo Arcus SP AN9 strain (Accession number NITE P-152) An environmental purification method comprising purifying an environment contaminated with volatile aromatic hydrocarbons by the microorganism according to claim 1 or 2 .

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