Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2000-03-24
2002-12-24
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S262000, C435S253600, C435S267000, C435S290100, C435S289100
Reexamination Certificate
active
06498028
ABSTRACT:
TECHNICAL FIELD
This invention relates to a technique of enriching decomposing bacteria which can be used to treat soil polluted by organochlorine compounds, such as agricultural chemicals, particularly to prevent contamination of river water and groundwater caused by agricultural chemicals in soil, and a technique of isolating the decomposing bacteria by utilizing the enriching technique, and more particularly to techniques of these kinds for enriching and isolating decomposing bacteria for decomposing organochlorine agricultural chemicals difficult to decompose.
BACKGROUND ART
To maintain today's agricultural production, agricultural chemicals cannot be dispensed with, and to conserve flora in golf courses or the like as well, agricultural chemicals are used in large quantities. On the other hand, there is a concern that agricultural chemicals work as contaminants to have undesirable effects on the environment, especially to be a pollution source of river water and groundwater. An organochlorine agricultural chemical pentachloronitrobenzene (“PCNB”) used for killing bacteria causing soil disease is pointed out to be one of contaminants causing such a pollution. PCNB is an organochlorine compound very difficult to decompose, and as matters stand, a method of efficiently disposing PCNB remaining in soil and the like has not been proposed.
To restore soil contaminated by organochlorine compounds, such as agricultural chemicals, with decomposing bacteria has been considered to be a useful technique. This technique makes use of decomposing bacteria, among microbes inhabiting in soil in enormous numbers, which are capable of decomposing organic compounds serving as functional skeletons in agricultural chemicals and the like, thereby rendering the organic compounds harmless or eliminating the same from the environment. Therefore, it is possible to eliminate contaminants, such as agricultural chemicals or the like, from the environment by collecting bacteria which are capable of decomposing organochlorine compounds and exploiting such capabilities of the decomposing bacteria.
The method of selective enrichment/isolation of a specific kind of bacteria from various soil-inhabiting bacteria includes a soil percolation technique in which a column or the like is filled with soil containing inhabiting decomposing bacteria to form an enrichment soil layer, and an inorganic salt medium, which contains only organochlorine compounds, such as agricultural chemicals, as solo carbon and nitrogen sources, is continuously circulated through the enrichment soil layer, whereby a specific kind of decomposing bacteria, that is, decomposing bacteria which is capable of using the carbon or nitrogen source contained in the inorganic salt medium for assimilation or co-metabolism is selectively enriched for isolation. Actually, however, the above conventional soil percolation technique generally takes a long time period of one half to one year to enrich and isolate decomposing bacteria, and depending on the kind of an organic compound, there are cases where no suitable decomposing bacteria can be enriched by the method. The fact that such a long time period is required is a large impediment encountered in putting to practical use the river water or groundwater pollution control technique using decomposing bacteria.
The present invention has been made under these circumstances, and an object thereof is to improve the conventional soil percolation technique to thereby provide a method of enriching and isolating decomposing bacteria capable of decomposing an organochlorine agricultural chemical PCNB which is difficult to decompose, in a short time period, and to provide decomposing bacteria for efficiently disposing of PCNB.
DISCLOSURE OF THE INVENTION
To attain the object, the present inventors have improved the conventional soil percolation technique in the following respects, and thereby established the technique which is capable of largely enhancing the speeds of enrichment and isolation of decomposing bacteria. The gist of the improvement is that a porous material having an infinite number of micropores is fragmented to pieces of approximately several mm to ten and several mm in size such that the porous material can be handled with ease and at the same time has a large effective surface area, and then the fragmented porous material is mixed into an enrichment soil layer as an artificial microhabitat. According to this technique, decomposing bacteria can be effectively enriched and isolated over a time period of approximately three weeks to three months, although the required time period is slightly different depending on the kind of a contaminant and the kind of bacteria decomposing the contaminant. The inventors have already filed a patent application concerning the technique (Japanese Patent Application No. Hei
9-30176
).
According to the present invention, by using the improved soil percolation technique, a specific kind of decomposing bacteria were selectively enriched and isolated by continuously circulating an inorganic salt medium containing an organochlorine agricultural chemical PCNB as only sources of carbon and nitrogen to the enrichment soil layer. As a result, aerobic bacteria could be obtained in a very short time period which effectively carries out complete decomposition of PCNB. Out of the enriched and isolated decomposing bacteria, three strains which have a high PCNB-decomposing activity were examined for identification, and it was found that the three strains belong to aerobic bacteria named
Burkholderia cepacia.
As far as the inventors know, decomposing bacteria for decomposing PCNB are mostly anaerobic bacteria, and
Burkholderia cepacia
obtained by the improved soil percolation technique (
Burkholderia cepacia
KTYY97, National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology, 1-3, Higashi 1-chrome, Tsukuba-shi, Ibaraki 305-8566, Japan, Receipt No. FERM BP-6721, Received May 18, 1998, hereinafter referred to as “the present decomposing bacteria”) provides possibility of quite novel uses since this bacteria are aerobic and completely decomposes or degrades PCNB.
It is found that if a loopful—using an inoculating needle—of the present decomposing bacteria is added to 30 ml of an inorganic salt medium containing PCNB in a concentration of 3 to 4 mg/liter, it is possible to completely decompose PCNB in approximately five days such that all chlorine atoms bonded to each PCNB molecule are removed from the molecule. Further, the study of the present inventors revealed that the present decomposing bacteria are capable of decomposing even an organochlorine agricultural chemical CNP which has been conventionally considered to be very difficult to decompose. Aerobic bacteria, such as the present decomposing bacteria, are less restricted in the manner of handling the same, unlike anaerobic bacteria, and hence only by applying the present decomposing bacteria to a soil contaminated by PCNB, it is possible to effectively decompose the PCNB, which makes it possible to prevent river water and groundwater pollution.
The present decomposing bacteria can be enriched and isolated by the improved soil percolation technique proposed by the present inventors. This method comprises mixing a soil containing an organochlorine agricultural chemical PCNB with a fragmented porous material having an infinite number of micropores and at the same time a greater adsorptivity for adsorbing PCNB than the soil to form an enrichment soil layer, and circulating through the enrichment soil layer an inorganic salt medium containing a carbon and nitrogen source, the carbon and nitrogen source being formed by only PCNB, thereby enriching the decomposing bacteria in the fragmented porous material.
The soil containing the organochlorine agricultural chemical PCNB is preferably a soil continuously using PCNB as an agricultural chemical. The present decomposing bacteria are hardly populated in ordinary soils, and on the other hand, is relatively thickly populated in a soil usi
Takagi Kazuhiro
Yoshioka Yuuichi
National Institute for Agro-Environmental Science Independent Ad
Patten Patricia
Rader & Fishman & Grauer, PLLC
Weber Jon P.
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