Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1995-05-02
2002-05-28
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S243000, C435S261000, C435S262500, C435S264000, C435S270000, C435S822000
Reexamination Certificate
active
06395533
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for recovering microorganisms from soil, and more specifically, it relates to a method for efficiently recovering microorganisms from soil with a good reproducibility by dispersing the soil containing the microorganisms in an organic acid buffer solution.
2. Related Background Art
In recent years, environmental pollution caused by hydrocarbons such as aromatic hydrocarbons, paraffins and naphthenes or organic chlorine compounds such as trichloroethylene has become a serious problem. Strongly desired are technologies to prevent further expansion of such serious environmental pollution and to purify and remedy the environment. Such technologies for soil environment remediation include physicochemical treatments, e.g., aeration, sun bleaching, vacuum vessel treatment, vacuum extraction, etc. However, these physicochemical treatments are not satisfactory for practical use, considering the operation cost, operability, energy consumption, treatment capacity, or that these treatments are to merely extract the pollutant and not to convert it to a harmless substance.
Therefore, much hope is laid on environment remediation utilizing microorganisms as a practical means in place of above physicochemical methods.
For example, in soil there have been found various kinds of microorganisms which can degrade soil-polluting, hardly degradable compounds such as aromatic hydrocarbons and organic chlorine compounds. Application of such microorganisms to the polluted soil to decompose the pollutants in the soil has been studied. Further, studies have been started on application of recombinant microorganisms having enhanced decomposing activity by genetic recombination techniques to soil.
To establish and popularize the environmental remediation methods utilizing microorganisms as a practical and socially effective technology, it is highly important to grasp growth and survival of the microorganism in the environment to which it is applied, as well as to develop various useful microorganisms.
In the agricultural production activity utilizing soil, it is also important to understand the activity, growth, propagation and survival of the original or introduced microorganisms in soil.
Many kinds of microorganisms are living in soil, but only small minority of these microorganisms have been identified, and most of them remain still unknown. In addition, for most of these microorganisms, even conditions necessary for their separation and culture have not been known. Isolation and identification of unknown microorganisms in soil are accompanied by many difficulties due to the specific environment, soil. This has been a main obstacle to the study of ecology of such microorganisms. Heretofore, contrivance and improvement in the separation and cultivation methods have been made to advance the ecological study of the soil microorganisms, but it is considered that there are many unknown microorganisms present in soil whose activity may be important in view of ecology. It is said that for only 0.1% or less of the total soil microorganisms, separation and cultivation methods have been established.
Therefore, it is one of the important tasks to grasp the ecology of the microorganisms which are hard to separate and cultivate, not only in the field of the applied technology such as soil remediation but also in fundamental science.
As a means for solving the above-mentioned problems, the following two methods are now under development in place of the conventional method problematically requiring a certain cultivation process to detect and count the microorganisms in soil:
(1) DNA analyzing method: a method in which DNA is isolated from the microbial cells and analyzed to study the microbial ecosystem in soil, and
(2) Microbial particle detection method: a method in which the microbial cells themselves are separated from soil particles, and their ecosystem is then directly analyzed by the use of a fine particle measuring instrument such as a flow cytometer.
In above method (1), to use DNA as a detection means, it is necessary to recover the DNA of the microbial cells from a certain environmental sample. As a technique for recovering the microbial DNA from the soil, there are two methods available, a cell (microbial cell) recovery method and a direct cell lysis method.
The cell recovery method has a problem in that the recovery of the microbial cells from the soil greatly varies, depending upon the soil type and the kind of objective microorganism. For example, when microbial cells are recovered from soil, the recovery from one soil sample may be 40% or more, while that from another soil sample may be as little as 10% or more. This method has an advantage that the origin of the recovered DNA is definite and its purity is high, but the method has simultaneously a problem in that the amount of the recovered DNA is very small. For example, the amount of DNA obtained from 100 &mgr;g of soil by the conventional method is at most about 100 &mgr;g, and in some cases, it is as small as 1 to 2 &mgr;g. In consequence, there will occur a problem that the amount of DNA is not sufficient for analysis or it is so small that particular care must be taken for its handling.
On the contrary, the direct cell-lysis method has a feature that DNA is recovered in an amount of 1 to 2 mg per 100 g of soil, which is 10 to 100 times higher than the above-mentioned cell recovery method. It has a problem, however, that the target DNA is often contaminated with free DNA of unknown origin in soil, for example, those derived from dead bacteria, filamentous fungi, protozoans, plants etc. Such contamination interferes with the measurement of the objective DNA.
Accordingly, in order to utilize DNA analysis method as a detection means for the soil microorganisms, it is indispensable to recover the DNA of the target microorganism from soil. The prior techniques have, however, advantages and disadvantages as described above, and there is strongly required an extraction method of DNA from soil which can provide DNA of satisfying purity in a high recovery.
Also in above method (2), where microbial cells themselves are analyzed by detecting fine particles, the microbial cells must be recovered from soil, as in the above-mentioned method (1). Thus, the instability of the recovery rate of microbial cells becomes a serious problem.
In general, when microbial cells are separated from soil, the soil sample is first dispersed in pure water or a pH buffer solution (usually a phosphate buffer of about 100 mM and around pH 7 is used). At this point, the microbial cells are also dispersed in a dispersing medium to some extent, but if the sample is allowed to stand, most of the microbial cells cohere and precipitate together with the soil particles. The degree of this cohesion-precipitation is determined compositely depending upon the type of soil (physical properties of the soil particles, e.g., particle diameter, charging state, ion exchange capacity and the like), the amount of electrolytes in the soil, the kind and the amount of microbial cells present in the soil. However, it is extremely difficult to foresee or control the degree of cohesion-precipitation of the dispersed soil which is the main cause of the variation for the recoveries of microbial cells.
Furthermore, recent advancements in microbiology have elucidated that a part of soil microorganisms (mostly bacteria) secrete a polymer-like substance to fix themselves to the soil particles or the like, and, in that state, they propagate and function. Since such microbial cells cannot be separated from the soil particles by a simple dispersing operation such as stirring, there is a problem in that the recovery of such microbial cells is low and resultant analysis does not reflect the actual microbial ecosystem.
As a solution of this problem, a method has been proposed in which the polymer-like substance is digested by using various enzymes, and then microbial cells are separated (Japanese Patent Application No. 5-307
Kawaguchi Masahiro
Kuriyama Akira
Sugawa Etsuko
Naff David M.
Ware Deborah K.
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