JP2008290026A - Cultivation composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cultivation composition which rapidly creates anaerobic conditions and preferentially proliferate bacteria concerning the reductive dechlorination reaction of organochlorine compounds. <P>SOLUTION: The cultivation composition is employed for being added to a substance to be treated for purifying the substance to be treated contaminated by organochlorine compounds with anaerobic bacteria having decomposition activity of the organochlorine compounds, and contains methanol, citric acid and a skim milk powder as main components. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a culture composition that is added to a substance to be treated in order to purify the substance to be treated contaminated with an organic chlorine compound by an anaerobic bacterium having an activity of decomposing the organic chlorine compound.

Contamination of soil and groundwater with organochlorine compounds such as tetrachloroethylene, trichlorethylene, 1,1,1-trichloroethane, and dichloroethylene has become a serious problem. For example, tetrachlorethylene is used in a large amount as a solvent for dry cleaning, and trichlorethylene is used in a large amount in the semiconductor industry as a degreasing detergent. These substances are volatile and appropriate treatment such as incineration is not performed. Caused contamination.
These organochlorine compounds are hardly soluble and hardly decomposable substances. Therefore, development of environmental purification technology for decomposing organic chlorinated compounds such as chlorinated organic compounds contained in soil or the like and purifying the contaminated environment is strongly desired.

Conventionally, as a purification technique for contamination by, for example, tetrachlorethylene, physical / chemical methods such as aeration, heating, solidification of contaminated soil, or adsorption / catalytic oxidation of contaminated groundwater have been employed.
However, in recent years, bioremediation technology that removes contamination by decomposing and detoxifying pollutants by utilizing microorganisms as one of the environmental purification methods is expected as a fundamental repair technology (for example, patent literature) 1, 2). Bioremediation is a technology that biologically decomposes harmful organic compounds and converts them into harmless substances such as carbon dioxide, methane, water, and inorganic salts.

Patent Document 1 discloses a method for purifying contaminants with an organic chlorine compound such as tetratrichloroethylene. In the presence of a reducing agent, reductive dehalogenation is promoted by a combination of a chemical reaction and a microorganism.
Patent Document 2 describes a biological repair method using anaerobic bacteria and aerobic bacteria, and additives used at that time. The additive includes propionate as an electron donor as a substance that promotes reductive dehalogenation of anaerobic bacteria, and lactose as a substance that creates an anaerobic state. Lactose is an effective nutrient source for aerobic microorganisms.

  In addition, Patent Document 3 describes a technique of adding an additive serving as a nutrient source for microorganisms to contaminated soil or the like in order to reductively decompose organochlorine compounds by the action of microorganisms. Such additives include organic acids and proteins.

  Non-patent document 1 discloses that bacteria involved in an anaerobic microbial dechlorination reaction using methanol as an electron donor are isolated and cultured, and anaerobic microbial dechlorination reaction of tetrachloroethylene using hydrogen, vitamin B12, and acetic acid as nutrients. It is described that is possible.

  Non-Patent Document 2 describes that when a microbial population that decomposes an organic chlorine compound such as tetrachloroethylene into dichloroethylene or vinyl chloride was analyzed, a kind of lactic acid bacteria was identified as a bacterium involved in the decomposition. is there.

JP 2003-71431 A Japanese Patent No. 3538643 JP 2006-142140 A Xavier et al, "Characterization of an H2-Utilizing Enrichment Culture That Reductively Dechlorinates Tetrachloroethene to Vinyl Chloride and Ethene in the Absence of Methanogenesis and Acetogenesis", Applied and Environmental Microbiology, Nov.1995, Vol.61, p.3928-3933 Masahiro Mizumoto, “Research and Development on Anaerobic Decomposition of Chlorinated Ethylene”, Organized by New Energy Industrial Technology Development Organization, Bioindustry Association, Biodegradation / Processing Mechanism Analysis and Control Technology Development Project 2005 Results Abstracts of Lecture Meeting September 26, 2005, p24-26

  In the technique described in Patent Document 1, reductive dehalogenation by a combination of a chemical reaction and a microorganism is promoted in the presence of a reducing agent. However, a purification technique using a chemical reaction has no reaction specificity, so Depending on the soil, there may be an unpredictable side reaction. Therefore, it is difficult to carry out purification with good reproducibility. Furthermore, in order to mix the reducing agent with the soil at the site, civil engineering work is required, which is not economical.

  The additive described in Patent Document 2 includes propionate and lactose. However, since propionate shows an effect as an electron donor relatively slowly, it is difficult to quickly bring the contaminated soil into a reducing condition. There are also anaerobic bacteria that cannot use lactose. Therefore, the additive is not an additive that can be used anywhere.

  In the additive described in Patent Document 3, yeast extract, malt extract, meat extract, peptone, etc. are included as proteins, which are related to the purification of contaminated groundwater, for example, because they are similar to general medium components for culturing microorganisms. It is difficult to preferentially proliferate microorganisms.

  The technique described in Non-Patent Document 1 shows that dechlorination of tetrachloroethylene is possible using methanol as the only carbon source. However, for example, groundwater is often an aerobic condition and is not suitable for use in the purification of contaminated groundwater.

  Non-Patent Document 2 does not describe a technique for preferentially growing bacteria involved in the reductive dechlorination reaction of organochlorine compounds.

  Accordingly, an object of the present invention is to provide a culture composition that quickly creates anaerobic conditions and preferentially grows bacteria involved in the reductive dechlorination reaction of organochlorine compounds.

  In order to achieve the above object, the culture composition according to the present invention is added to the substance to be treated in order to purify the substance to be treated contaminated with the organic chlorine compound by anaerobic bacteria having an activity of decomposing the organic chlorine compound. The first composition of the culture composition is that it contains methanol, citric acid, and nonfat dry milk as the main components.

  In general, the redox potential of a bacterial culture environment such as a medium affects the growth of bacteria. In particular, anaerobic bacteria grow well when the bacterial culture environment has a low redox potential, that is, in an anaerobic state. The culture composition of this structure can reduce the oxidation-reduction potential of the bacterial culture environment to an anaerobic state.

The said culture composition contains methanol, a citric acid, and skim milk powder as a main component.
Methanol becomes an electron donor that can be rapidly used by bacteria when the oxidation-reduction potential of the bacterial growth environment decreases and becomes anaerobic. Conventionally, propionic acid has been used as an electron donor (Patent Document 3), but in this case, it has been difficult to quickly create a reducing environment. However, by using methanol as an electron donor as in this configuration, the bacterial growth environment can be rapidly reduced to a reducing environment.

  Citric acid is an element of the TCA cycle, and in order to make the bacterial growth environment anaerobic, reduction energy (NADH) necessary for lowering the redox potential can be obtained.

  Nonfat dry milk is a nutrient for growing bacteria, such as lactic acid bacteria, indirectly involved in the decomposition of organochlorine compounds.

  By adding the culture composition of this configuration to the bacterial culture environment, the oxidation-reduction potential of the bacterial growth environment is lowered to rapidly anaerobic, and organochlorine compounds, which are hardly degradable harmful substances, are reductively removed. Anaerobic bacteria that can be directly degraded by the chlorine reaction and bacteria that are indirectly involved in the degradation of organochlorine compounds can be grown in preference to other bacteria.

  And as a result of quickly adjusting the culture environment to anaerobic conditions, by allowing the growth and metabolism of anaerobic bacteria, the substance to be treated can be shortened and the decomposition efficiency can be improved. Can be purified.

  The second characteristic configuration of the culture composition according to the present invention is that the concentration of methanol is 5 to 100 mM, the concentration of citric acid is 0.5 to 10 mM, and the concentration of skim milk powder is 0.0005 to 0.01%. It is in the point.

  According to said 2nd characteristic structure, by making each component of a culture composition into the said density | concentration range, the level at which the anaerobic bacterium which decomposes | disassembles an organic chlorine compound directly can be favorably grown by making the oxidation reduction potential of a bacterial growth environment into Can be set to

Embodiments of the present invention will be described below with reference to the drawings.
This invention is a culture composition added to the said to-be-processed substance in order to purify the to-be-processed substance contaminated with the organochlorine compound with the anaerobic bacteria which have the decomposition activity of the said organochlorine compound.
The said culture composition contains methanol, a citric acid, and skim milk powder as a main component.

In this specification, a to-be-processed substance shows the soil, groundwater, sludge, incinerated ash, etc. which were contaminated with organochlorine compounds, such as tetrachloroethylene, for example.
Examples of the organic chlorine compounds include tetrachloroethylene, trichloroethylene, 1,1,1-trichloroethane, dichloroethylene, dioxins such as polychlorinated dibenzodioxins (PCDD) and polychlorinated dibenzofurans (PCDF), and the like.

  Representative examples of anaerobic bacteria having the ability to resolve organochlorine compounds include the genus Dehalococcoides, Methanobacterium, Methanosarcina, Methanolobus, and Acetobacteria. Bacteria such as genus Acetobacterium, genus Desulfobacterium, genus desulfomonile, genus Dehalospirillum, genus Dehalobacter, genus Dehalobacterium, genus Clostridium Is exemplified.

As described above, it has been clarified that one type of lactic acid bacterium is present as a bacterium involved in the decomposition in the microbial population that decomposes organic chlorine compounds. That is, it is considered that the lactic acid bacteria are indirectly involved in the decomposition of the organic chlorine compound.
Examples of the lactic acid bacteria include the genus Lactobacillus, the genus Bifidobacterium, the genus Lactococcus, the genus Pediococcus, and the genus Leuconostoc.

  Other bacteria that are considered to be indirectly involved in the degradation of organochlorine compounds include Pseudomonas aeruginosa, and other bacteria include Pseudomonas sp., Burkoderia sp., Rhodococcus sp. Rhodococcus sp.) And the like.

  Therefore, anaerobic bacteria that are directly involved in the reductive dechlorination reaction of organochlorine compounds and lactic acid bacteria that are indirectly involved in the degradation of organochlorine compounds are important bacteria for the reductive dechlorination reaction of organochlorine compounds. I understand that.

The culture composition of the present invention preferentially grows a group of bacteria that are directly and indirectly involved in the degradation of organochlorine compounds.
In general, the redox potential of a bacterial culture environment such as a medium affects the growth of bacteria. Anaerobic bacteria grow well when they are in an anaerobic state where the redox potential of the bacterial culture environment is low. The culture composition of the present invention reduces the redox potential of the bacterial culture environment.
As described above, the culture composition of the present invention contains methanol, citric acid, and nonfat dry milk as main components.

Methanol becomes an electron donor that can be rapidly used by bacteria when the oxidation-reduction potential of the bacterial growth environment decreases and becomes anaerobic. By using methanol as an electron donor, the bacterial growth environment can be rapidly reduced to a reducing environment.
In addition, aerobic bacteria (for example, Pseudomonas sp.), Facultative anaerobic bacteria (for example, Klebsiella sp.), Obligate anaerobic bacteria (for example, Clostridium sp., Methanosarcina ( Methanosarcina sp.)) Is known to produce vitamin B12 with methanol. Vitamin B12 is an essential nutrient for the growth of, for example, dehalococcides. Therefore, the methanol added to the culture composition can synthesize the essential nutrients of dehalococcides not only as an electron donor that can be rapidly utilized by bacteria, but also in soil bacteria existing in the bacterial growth environment. it can.

  Citric acid is an element of the TCA cycle, and in order to make the bacterial growth environment anaerobic, reduction energy (NADH) necessary for lowering the redox potential can be obtained.

Nonfat dry milk is a nutrient for growing bacteria involved in the decomposition of organochlorine compounds, such as lactic acid bacteria.
Non-fat dry milk is, for example, dried from powdered milk after removing almost all moisture from the raw milk. The raw milk is not particularly limited, but milk obtained from various mammals such as cows and humans is used. The manufacturing process of skim milk powder should just be a normal process. For example, raw milk is concentrated by heating under reduced pressure, and then spray-dried. In skim milk powder used in the culture composition of the present invention, it is not necessary to adjust the amount of specific components.
The composition of skim milk powder is, for example, about 3 g of protein 34.0 g, fat 1.0 g, carbohydrate 53.3 g, ash 7.9 g and vitamin B group 2 mg, Ca, Na, K, Mg, P, etc. Contains minerals. Some variation is allowed depending on the type of raw milk.

  By adding the culture composition of the present invention to the substance to be treated, the anaerobic state can be rapidly reduced to anaerobic state by reducing the redox potential of the bacterial growth environment, and the organochlorine compound can be decomposed by reductive dechlorination reaction. It is possible to grow bacterial bacteria and bacteria that are indirectly involved in the degradation of organochlorine compounds in preference to other bacteria.

  As a usage mode of the culture composition of the present invention, when the substance to be treated is a liquid such as contaminated groundwater, it can be used as a tablet or a liquid mode containing each component of the culture composition. On the other hand, when a to-be-processed substance is contaminated soil, it can be used as an aspect of the liquid containing each component of a culture composition. The above usage modes are examples, and the present invention is not limited to these modes.

  The following experiment examined the ability of dehalococcides to decompose trichlorethylene when the culture composition of the present invention was used.

  In the culture composition of the present invention, the presence or absence of each component and the concentration conditions were variously changed, added to groundwater (substance to be treated) contaminated with trichlorethylene, which is an organic chlorine compound, and the redox potential was measured.

<Presence or absence of addition of each component>
The contaminated groundwater is filled in a 15 mL plastic tube, and the three components of the culture composition are not added (sample No. 1), single (sample No. 2 to 4), or two types (sample No. 1). 5-7) What was mixed 3 types (sample No. 8: culture composition of this invention) was added to the said contaminated groundwater, respectively. The tube in this state was sealed and cultured at 20 ° C. for 2 weeks, and the redox potential was measured. The results are shown in Table 1. In addition, skim milk powder (made by Difco) was used for skim milk powder.

  From the results in Table 1, sample No. No. 1 and the sample No. When each component was added independently like 2-4, the oxidation-reduction potential did not show the value suitable for anaerobic bacteria to grow. On the other hand, sample no. In the case of the culture composition in which two kinds were mixed as in 6 to 7, and In the case of the culture composition in which three kinds were mixed as in 8, the redox potential was sufficiently lowered and showed a value suitable for the growth of anaerobic bacteria.

  Sample No. For 1-4, 8, PCR was performed to evaluate the abundance of the bacteria after culture. In this PCR, we tried to detect lactic acid bacteria as bacteria that are indirectly involved in the degradation of organochlorine compounds, and dehalococcides as anaerobic bacteria that can directly degrade organochlorine compounds by reductive dechlorination. .

  In the detection of lactic acid bacteria, `` Jens et al, '' Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophorisis '', APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2001, p2578-2585 The primer sequence and PCR amplification conditions were set based on the information described in “1.

  As primers, specific primers designed for the target site of 16S rDNA of lactic acid bacteria, that is, forward primer Lac1 (sequence recognition number 1) and reverse primer Lac2 (sequence recognition number 2) were used.

  The composition of the PCR reaction solution was 25 pmol primer (Lac1, Lac2), 0.2 mM dNTP, reaction buffer, 20 mM tetramethylammonium chloride, 25 μg BSA, 2.5 U rTaq polymerase (manufactured by Amersham Pharmacia Biotech), 1 μL template DNA solution To make a total volume of 50 μL.

  The PCR reaction was performed using a GeneAmp2400 thermal cycler (manufactured by PerkinElmer). PCR amplification conditions were set such that after initial denaturation at 94 ° C. for 2 minutes, 35 cycles of 94 ° C. for 30 seconds-61 ° C. for 1 minute-68 ° C. for 1 minute were performed, and finally, an extension reaction was performed at 68 ° C. for 7 minutes.

  After the PCR reaction, the PCR product was electrophoresed on a 1.5% agarose gel. After completion of electrophoresis, the gel was stained with ethidium bromide to evaluate the presence or absence of a PCR product derived from lactic acid bacteria (Table 2).

  In the detection of dehalococcides, "Theo et al," Development of a real-time PCR method for quantification of the three genera Deharobacter, Dehalococcoides, and Desulfitobacterium in microbial communities. ", Journal of Microbiological Methods, 57 (2004) 369 -378 ”, primer sequences and PCR amplification conditions were set.

As primers, specific primers designed for the target site of dehalococcides 16S rRNA, ie, forward primer Dco728F (sequence recognition number 3) and reverse primer Dco944R (sequence recognition number 4) were used.
The composition of the PCR reaction solution was 5 μL of QuantiTect SYBR Green PCR Master Mix (manufactured by Qiagen), 0.8 μL primer (Dco728F, Dco944R), 2.4 μL diluted water, 1 μL template DNA solution were mixed to make the total volume 10 μL. .

PCR reaction and electrophoresis were performed by the same method as that used for the PCR detection of lactic acid bacteria described above, except for PCR amplification conditions. PCR amplification conditions were set such that after initial denaturation at 94 ° C for 15 minutes, 50 cycles of 94 ° C for 30 seconds-58 ° C for 20 seconds-72 ° C for 30 seconds were performed.
After completion of the PCR reaction and electrophoresis, the presence or absence of a PCR product derived from dehalococcides was evaluated (Table 2).

  Sample No. At 3 (only citric acid added), dehalococcides grow but lactic acid bacteria do not. Sample No. 2 (only methanol added), sample no. 4 (only skim milk powder added) grows lactic acid bacteria and dehalococcides, but the oxidation-reduction potential of the bacterial growth environment (+192 mV and +28 mV, respectively) is a culture composition mainly composed of methanol, citric acid and skim milk powder. Compared to the oxidation-reduction potential (−96 mV) of the product (sample No. 8) (see Table 1).

<Degradation evaluation of trichlorethylene>
Sample No. About 1, 5-8, it was evaluated how much trichlorethylene is decomposed | disassembled. The culture conditions were the same as described above except that the culture period was 3 weeks. The results are shown in Table 4. The trichlorethylene concentration was measured by the headspace GC / MS method according to the analysis method of JIS K 0125 5.2.

  Sample No. Even if the redox potential is sufficiently lowered by mixing two types of components as in 6 to 7 (−60 mV and −42 mV, respectively, see Table 1), the trichlorethylene concentration is a sample in which each component is not added. No. About the same as 1. Therefore, sample no. In 6 and 7, although the redox potential is sufficiently lowered and shows a value suitable for the growth of anaerobic bacteria (dehalococcides), trichloroethylene is hardly decomposed.

  On the other hand, sample no. In the case of the culture composition in which three kinds of each component were mixed as shown in FIG. 8, it was found that the culture composition was reduced to about 1/20 as compared with the other samples (sample Nos. 1, 5 to 7). In this case, the oxidation-reduction potential is sufficiently lowered to show a value suitable for the growth of anaerobic bacteria (dehalococcides), and trichlorethylene is sufficiently decomposed.

  That is, it is suggested that, for example, other bacteria exist as factors affecting the reduction in redox potential, the growth of dehalococcides, and the decomposition of organochlorine compounds. The other bacterium is a bacterium that is indirectly involved in the decomposition of the organochlorine compound.

  As described above, by using a culture composition (sample No. 8) containing methanol, citric acid, and nonfat dry milk as main components, trichlorethylene is finally decomposed into ethylene, and contaminated groundwater, which is a substance to be treated. Is purified.

<Concentration conditions for each component>
In a culture composition in which three components of methanol, citric acid, and nonfat dry milk were mixed, the concentration conditions of each component were variously changed and added to the contaminated groundwater, and the redox potential was measured. The culture conditions were the same as in the experiment described above. The results are shown in Table 4.

  From the results in Table 4, when the concentration of methanol is 5 to 100 mM, the concentration of citric acid is 0.5 to 10 mM, and the concentration of skim milk powder is 0.0005 to 0.01%, it is suitable for anaerobic bacteria to grow. It has been found that the level of redox potential decreases to a sufficient extent. When the concentration of methanol is 10 to 50 mM, the concentration of citric acid is 1 to 5 mM, and the concentration of skim milk powder is 0.001 to 0.05%, the level of the redox potential is lowered to a particularly preferable level.

  Thus, when the level of redox potential decreases, the bacterial growth environment becomes anaerobic, and anaerobic bacteria that can decompose organochlorine compounds by reductive dechlorination are proliferated over other bacteria. It becomes possible.

[Another embodiment]
In the above-described embodiment, the culture composition of the present invention is added to the material to be treated in order to purify the material to be treated contaminated with the organic chlorine compound by the anaerobic bacteria having the activity of decomposing the organic chlorine compound.
The culture composition of the present invention is not limited to such an embodiment. For example, as described below, the culture composition can be used to easily create anaerobic culture conditions and quickly detect desired anaerobic bacteria.

  For example, in a machine tool used in a factory or the like, cutting oil is used for the purpose of cooling the cutting tool, cutting performance, and preventing the temperature of the workpiece / machine from rising. In the case of water-soluble cutting oil, bacteria such as bacteria are generated and bad odor is likely to occur. The substance causing bad odor is hydrogen sulfide, and this hydrogen sulfide is generated by sulfate ions being reduced under anaerobic conditions by sulfate-reducing bacteria which are anaerobic bacteria.

  Sulfate-reducing bacteria are contained in the water that dilutes the water-soluble cutting oil. This diluting water is usually not sterilized tap water but unsterilized industrial water or well water. Therefore, in order to prevent the generation of hydrogen sulfide that causes bad odor, it is necessary to check whether the dilution water contains sulfate-reducing bacteria and to use dilution water that does not contain sulfate-reducing bacteria. There is.

In order to test whether the dilution water contains sulfate-reducing bacteria, the dilution water is anaerobically cultured and detection of the sulfate-reducing bacteria is attempted.
In general, the following methods are known for anaerobic culture.
In order to culture anaerobic bacteria that cannot survive in the presence of oxygen, it is necessary to remove oxygen from the culture environment. To anaerobic culture medium, add liver fragment to liver broth to increase the reducing power of the medium itself, liver broth, sodium thioglycolate and cysteine are added, and oxygen in the medium is removed by autooxidation of SH group There is a thioglycolate medium.
As methods for removing oxygen from the culture environment, there are a method of physically removing oxygen and a method of chemically removing oxygen.
As a method of physically removing oxygen, there are a method of discharging oxygen in a container by a pump, or a method of culturing bacteria on the bottom of a thick medium.
As a method of chemically removing oxygen, a gas pack method in which a chemical agent and water are reacted to generate hydrogen gas, and hydrogen gas generated by the action of the catalyst and oxygen in the container are reacted to form water. A steel wool method in which metal absorbs oxygen, a method of culturing with bacteria having high oxygen absorption ability, and the like are known.

  However, it is difficult to easily determine whether or not sulfate-reducing bacteria are contained by performing the above-described anaerobic culture and anaerobically culturing the diluted water at a site such as a factory.

  If the culture composition containing methanol, citric acid, and skim milk powder of the present invention as main components is used, as shown in the above-described embodiment, the oxidation-reduction potential of the bacterial growth environment is lowered to quickly make the anaerobic state. Therefore, anaerobic culture can be easily performed.

<Culture of sulfate-reducing bacteria>
In the culture conditions shown in the embodiment described above, sulfate-reducing bacteria (Desulfovibrio sp.) Were used under the same conditions except that dilution water (industrial water) for diluting water-soluble cutting oil was used instead of contaminated groundwater. Culture was attempted. The components and concentrations of the culture composition used were also the sample No. used in the above-described embodiment. Same as 1-8. The presence or absence of generated odor and the color of the precipitate were evaluated according to the culture conditions. The results are shown in Table 5.

  As a result, sample no. The odor could be confirmed only with 8 (culture composition of the present invention). And since the color of a deposit is black, a deposit is iron sulfide. Therefore, it is recognized that the odor component is hydrogen sulfide. Therefore, sulfate-reducing bacteria were cultured under the culture conditions of this embodiment.

  As described above, by using the culture composition of the present invention, it is possible to easily determine whether or not a desired anaerobic bacterium is contained by simply performing anaerobic culture at a site such as a factory.

  The culture composition of the present invention can be used to purify a substance to be treated contaminated with an organic chlorine compound by an anaerobic bacterium having a decomposition activity of the organic chlorine compound.

Claims (2)

A culture composition that is added to the substance to be treated in order to purify the substance to be treated contaminated by the organochlorine compound with an anaerobic bacterium having a decomposition activity of the organochlorine compound,
A culture composition comprising methanol, citric acid and nonfat dry milk as main components.   The culture composition according to claim 1, wherein the methanol concentration is 5 to 100 mM, the citric acid concentration is 0.5 to 10 mM, and the skim milk concentration is 0.0005 to 0.01%.