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Decomposition of Organic chlorine Compounds in the soil by Effective Microorganisms
* Graduate Student of Hachinohe Institute of Technology ° Graduate Student of Hachinohe Institute of Technology °° Professor of Unversity of The Ryukyus °°° Professor of Hachinohe Institute of Technology

In Japan the new law on soil pollution was enforced in February of 2003. It shows how to legally treat the harmful chemical compounds in soil such as organic chlorine substances. The new soil purifying methods are currently being proposed. In this study, the model of polluted soil was created by incorporating three Organic chlorine Compounds (trichloroethylene, 1, 1-dichloroethylene, tetrachloroethylene) in the soil. Additionally, the experiment on decomposing the organic chlorine substances contained in the model was conducted by utilizing Effective Microorganisms.This model was incubated at 37 degrees C. for seven days to activate the microorganisms. The amount of residue of organic chlorine substances was measured by GC- MS. The result showed that the decomposition rate of trichloroethylene was 96% and that of 1, 1-dichloroethylene was 92% in the model. The possible application of bio-remediation system utilizing effective microorganisms will be described in this paper.

Organic chlorine Compounds including trichloroethylene have been used as a solvent for washing clothes and electronic parts for many years. Some experiments have been tried for solving many problems that were caused of Organic chlorine Compounds exposed to soil and water system. However, the cost of such method tends to expensive because the contamination area is wider than the expectation that had been examined. Then the other method that is based on biological way, bioremediation has been considered.

Authors have obtained the predicted result about disassembly of the organic chlorine Compounds in soil by using Effective Microorganisms (the authorized material for organic farming in Japan, product name as EM) which are the material developed for agriculture1). Effective Microorganisms was developed in the 1970’s, in order to solve many obstacles which were environmental pollution, destruction, etc. by using of agricultural chemicals and chemical fertilizer. Manufacturing and introduction of the product has started from the 1980’s in Japan. Then it has been manufactured in the more than fifty countries over of the world. The price of liquid microorganisms "EM" in an advanced nation is 20 US dollars per 1 litter. A user can cultivate this microorganisms based on a certain prescription. Therefore the cost of using this material could be reduced with scale merit.

Effective Microorganism "EM" was developed as a soil improvement agent for agriculture. And the effect which shows up in many fields has made clear not only agriculture but stock raising, fishery, environment, etc. by subsequent research. Regarding this product, the composition is shown in Table 1, and in Photograph 1. “EM” is Effective microorganisms, group of beneficial, naturally occurring, nonpathogenic, and food-grade microorganisms comprised mainly of lactic acid bacteria, yeast and photosynthetic bacteria, including aerobic and anaerobic microorganisms.

Table1. The composition of “EM”
Family of microorganismNameID No.
Lactic acid BacteriaLactobacillus plantarum
Lactbacillus casei
YeastSaccharomyces cerevisiae
Candia utilis
Photosynthetic BacteriaRhodopseudomonas palustris
Rhodobacter sphaeroides
Ray FungiStreptomyces cerevisiae
Streptomyces geiseus
FungiAspergillus oryzae
Mucor hiemalis
othersSome of species are replaced by those of similar characteristics

Microbes of EM

Photograph1. Microorganism of “EM”

2. Outline of Experiment

2.1 Materials and Reagents The materials and reagent which were used by this experiment are shown below. Standard soil Trichloroethylene, 1,1- dichloroethylene and tetrachloroethylene Effective microorganisms (EM-1)

2.2 Production of sample model I (with Effective Microorganisms)

2.2.1 Adjustment of soil
  • The container made from aluminum alloy was prepared. Then 27kg of standard soil was distributed equally 3kg and 9 samples were prepared.
2.2.2 Adjustment of Effective-Microorganisms diluted solution
  • The original solution of Effective-Microorganisms (EM) was diluted so that it might become 1%, 3%, 5% with refining water.

2.2.3 Adjustment of a reagent
  • Trichloroethylene, 1,1-dichloroethylene and tetrachloroethylene were diluted with refining water so that it might become 0.35mg / L, 0.005 mg / L, and 0.15 mg/L. with a micro pipette, respectively.

2.2.4 Addition of Effective Microorganism and Organic chlorine Compounds to the soil.
  • Three types of diluted solution of EM were sprinkled to three groups of soil models, and 9 pieces of sample model were produced. In this case, the diluted solution of 1liter was sprinkled to 3kg of soil so that Effective Microorganisms might spread uniformly. Furthermore, soil was fully mixed with drying. Then, three types of diluted solution of Organic chlorine Compounds, trichloroethylene, 1,1-dichloroethylene and tetrachloroethylene were mixed to the soil.

2.3 Production of Sample Model II (without Effective Microorganisms)
Three types of diluted solution of Organic chlorine compounds, trichloroethylene, 1,1- dichloroethylene and tetrachloroethylene were mixed to the soil without EM. 2.4 Decomposition of Organic Chlorine Compounds in Sample Model Sample model I and II were put into the 1,000ml Erlenmeyer flask, the lid was carried out with the silicone rubber cork, respectively so that Organic chlorine Compounds might not volatilize. In addition to such process, each flask was covered with a lab film for prevention of volatilizing surely. Then it cultivated for seven days at 37 degrees C. by the incubator so that activity of the microbe in a sample model might be promoted. After this process, the treatment for getting extract from a sample model by two kinds of solutions, methanol and Dichloromethane 1:3, was carried out. In this process, each sample model was treated for 3 hours with 2 kinds of solvents. Furthermore, filtration by filter paper was carried out, and it was concentrated by decompression evaporator. After the process, the total volume of the extract became approximately 1/10 comparison with non-concentrated.

2.5 Measurement of Organic chlorine Compounds in Sample Model
Gas chromatography (GC-MS) was used for the quantitative and the qualitative analysis of trichloroethylene, 1,1-Dichloroethylene, and tetrachloroethylene, which is contain in the sample model.

2.6 Screening of Composition of Microorganisms in Sample Model
In order to screen the composition of the microbe in the sample model, a part of that was immerse in sterile physiology salt solution and kept in a room at 28 degrees C. for 24 hours. Then, the supernatant fluid liquid was applied to three kinds of agar medium, (1)YG, (2)BtB, and (3)BHI. The medium was kept in the incubator at 37 degrees C. for five days. Then the colony of microorganisms that grew in the medium was observed by microscope.

2.7 Screening of Fluorescent Pseudomonas and Bacillus
Effective Microorganisms is mixed culture solutions, such as a lactic acid bacteria, yeast, and photosynthesis bacteria, as shown in Table 1. These kinds of microbes don’t decompose Organic chlorine Compounds directly. However, it was reported by the research of Ueda and Higa that Effective Microorganisms increases the activity of other microbes which exist in environment in the natural state2). Then the separation was done in order to investigate the activity of Pseudomonas genus and Bacillus genus that are known for decomposition Organic chlorine Compounds and live in the nature widely. A part of sample model was kept in the room at 28 degrees C. for 24 hours, after being under a sterilization physiology salt solution. Then, the supernatant fluid liquid was applied to the medium for cultivation of Pseudomonas and Bacillus. After the process, the medium were cultivated at 37 degrees C. by the incubator for 48 hours. And the microbe colony which grew was observed under the microscope, and the gene of a microbe was identified with 16rsRNA analysis equipment.

2.8 Decomposition of Organic chlorine Compounds by Fluorescent Pseudomonas and Bacillus Fluorescent Pseudomonas and Bacillus that separated from the sample model were cultivated at 37 degrees C. on YG medium for 24 hours. Then, it is decomposed into farmate, glyoxylic acid, and dichloroacetic acid, and, finally is decomposed into carbon dioxide. On the other hand, the course by which chloral is generated from trichloroethylene also exists. Then, in order to specific existence of the substance originating in trichloroethylene, extract of the sample model was analyzed by GC-MS by using the same compounds as the quality of a standard.

3. Results and Discussion
3.1 Results of Experiment regarding Sample Model I and II The rate of decomposition of the Organic chlorine Compounds in sample model I is shown in Table 2. The rate of decomposition of trichloroethylene was 96%, and 1,1-dichloroethylene was 92%. On the other hand, the rate of decomposition tetrachloroethylene was 2%. The m/z ratio was shown at 137 and it proofs the existence of chloral which is middle substance of Organic chlorine Compounds which the enzyme of microbe breaks in the process of decomposition3). The rate of decomposition of the Organic chlorine Compounds in sample model II is shown in Table 3. And the rate of decomposition of trichloroethylene was 22% , and 1,1- dichloroethylene was 15%. On the other hand, the rate of decomposition of tetrachloroethylene was 0%. Therefore, it was suggested that the soil could also decompose Organic chlorine Compounds without adding Effective Microorganisms. Regarding this matter, further examination is required for the consideration of a possibility. For example, Organic chlorine Compounds bond to soil according to the effect which is accompanied with the soil grain structure, and the possibility of other microbes unknown.
The rate of decomposition in sample model I
Name of Compoundconcentration (mg/L)daysrate (%)
1,1- dichloroethylene0.005792

Table3. The rate of decomposition in sample model II
Name of Compoundconcentration (mg/L)daysrate (%)
1,1- dichloroethylene0.005792

3.2 Screening of Microbes in Sample Model I and II
Regarding the microbes that have the possibility of decomposing Organic chlorine Compound, Fluorescent Pseudomonas and Bacillus were screened from sample model I. On the other hands, only Bacillus was screened from sample model II. For such reasons, small amount but various kinds of microbes which have the ability to decompose trichloroethylene were incubated and screened by using Effective Microorganisms.

As for the reason, Effective Microorganism diversifies the aspect of microbes. Then the physiological compounds which were produced by the activity of microbes were exchanges. Furthermore the relationship between microbes was improved.

3.3 Decomposition rate of Organic chlorine Compounds by microbes screened from Sample Model I
The decomposition result of the Organic chlorine Compound by the microbe (Fluorescent Pseudomonas, Bacillus) which screened from sample model I is shown in Table 4 and 5. The rate of decomposition of each microbe was not shown as the same as the rate of table 2. Therefore, it is guessed that activities of various microbes affects decomposition of Organic chlorine Compounds in sample model I.

Table4. Rate of decomposition of Fluorescent Pseudomonas
Name of CompoundRate of decomposition (%)

Table5. Rate of decomposition of Bacillus
Name of CompoundRate of decomposition (%)

4. Conclusion
The microbe which merely increases Trichloroethylene as one energy source is not discovered until now. On the other hand, it is known for adding simultaneously a certain kind of microbe, and the organic matter used as the source of nutrition of the microbe in pollution soil, the decomposition reaction of Trichloroethylene will come with the enzyme of the microbe produced. For example, if a methane-utilizing bacteria and methanol of the source of nutrition are added in the polluted soil, it is known that methane monooxygenase which is a kind of enzyme will be secreted by activity of a microbe, and the decomposition reaction of trichloroethylene will progress as a result. About the method of utilizing the microbes which exist in soil in the natural state like this examination, some subjects which should be examined about the details are left behind. Regarding the activity of the microbe which exists in soil in the natural state, it is important to verify the quantity and its reaction velocity of the process in which an Organic chlorine Compounds is disassembled.

In this research, a possibility of promoting disassembly of the organic chlorine compound in the polluted soil was suggested with the activity of microbes which exist in soil in the natural state were activated by using Effective Microorganisms developed as soil improvement materials for agriculture were used. About decomposition of trichloroethylene in sample model I, there is generalization of the action as follows. Trichloroethylene is changed into chloral by the enzyme (monooxyganase) generated by activity of microbe. After that, chloral was decomposed into trichloroacetateacid or 2.2.2-trichloroethanol. In addition to the process, chloral changes to trichloroethyleneoxid in another process. Then, it changes to glyoxylic acid and dichloroacetate and, finally it is decomposed to carbon dioxide. Therefore, it is guessed that disassembly of the organic chlorine compound by the exam is the same as that of the case of methane-utilizing bacteria is used.

Many polluted area of Trichloroethylene has been reported these days. On the other hand, the bioremediation which used methane by reasons, such as a problem of cost, has not resulted in utilization. n the other hand, the system which had flexibility and relevancy may be able to be built by activating the microbe in soil by using Effective Microorganisms because this method does not need special equipments, such as sending air into soil.

As an example of the bioremediation due to Effective microorganisms, purification of the saline soils in Pakistan is mentioned. The example is shown in a photograph 2. This was done in paddy field in Pakistan4). Soil was improved by using of Effective Microorganisms and the quantity of a rice doubled. Although it has the purification capability by the microbe in the nature, it is clear that the present environmental pollution is over the purification capability.

We want to suggest to establish the system of bioremediation which has safety and low cost by Effective Microorganisms which consist of only natural microbes, without using any genetic engineering.
Saline Crops
Photograph2. Purification of the saline soils in Pakistan
(The lower crop was raised on the field treated with Effective Microorganisms.)

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