JP6196137B2 - Methods for purifying contaminated soil or water - Google Patents

Description

  The present invention relates to a method for purifying soil or water contaminated with inorganic compounds that are generally difficult to biodegrade and biologically degradable, and more specifically, the organic compounds that are difficult to biodegrade. The present invention relates to a method for decomposing or immobilizing inorganic compounds (hereinafter sometimes referred to as harmful substances) harmful to organisms and organisms using specific microorganisms.

  In recent years, oils such as heavy oil and drilling oil, halogenated hydrocarbons such as trihalomethane, dioxins, PCBs, benzene, phenols and other biodegradable organic compounds, arsenic, chromium, lead, mercury, uranium, etc. Environmental pollution of soil, groundwater, sediments, etc. due to inorganic compounds harmful to living organisms has become obvious and has become a problem. Moreover, when the said hazardous | toxic substance is contained in industrial wastewater etc., advanced treatment is calculated | required from a viewpoint of environmental pollution prevention.

  As a method for purifying soil contaminated with biologically indegradable organic compounds or inorganic compounds harmful to living organisms, technologies (Patent Document 1 and Patent Document 2) that decompose and detoxify harmful substances using specific microorganisms are available. Proposed. In this way, the method of reducing the concentration of environmental pollutants using the activity of microorganisms is called bioremediation, and it is possible to purify the environment by adding specific microorganisms or nutrient sources of microorganisms to the environment to be purified. Therefore, it is attracting attention as a low-cost purification method with low environmental impact.

  In addition, a purification technique using a specific microorganism is also applied to wastewater treatment, and a decomposition technique for a biologically indegradable organic compound (Patent Document 3) and a heavy metal precipitation separation technique (Patent Document 4) have been proposed.

JP-A-7-123976 JP 2008-246269 A Japanese Patent Publication WO2005-001066 Special table 2002-533218 gazette JP-A-7-236897 JP 2003-250529 A

Lovley, DR, & Lonergan, DJ (1990). Anaerobic Oxidation of Toluene, Phenol, and p-Cresol by the Dissimilatory Iron-Reducing Organism, GS-15. Applied and environmental microbiology, 56 (6), 1858-64. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=184522&tool=pmcentrez&rendertype=abstract Sung, Y., Fletcher, KE, Ritalahti, KM, Apkarian, RP, Ramos-hernndez, N., Robert, A., Mesbah, NM, et al. (2006). Novel Metal-Reducing and Bacterium Geobacter lovleyi sp. nov. Strain SZ, a Novel Metal-Reducing and Tetrachloroethene-Dechlorinating Bacterium †. Society. doi: 10.1128 / AEM.72.4.2775 Amos, BK, Sung, Y., Fletcher, KE, Gentry, TJ, Wu, W.-M., Criddle, CS, Zhou, J., et al. (2007). Detection and quantification of Geobacter lovleyi strain SZ: implications for bioremediation at tetrachloroethene- and uranium-impacted sites.Applied and environmental microbiology, 73 (21), 6898-904.doi: 10.1128 / AEM.01218-07 Taylor, P., Anderson, RT, & Lovley, DR (2010) .Naphthalene and Benzene Degradation under Fe (III)-Reducing Conditions in Petroleum-Contaminated Aquifers Naphthalene and Benzene Degradation under Fe (III) -Reducing Conditions in Petroleum-Contaminated Aquifers. Bioremediation Journal, (February 2012), 37-41.

  Here, the most important issue in the environmental purification technology is how to adapt a specific microorganism that decomposes and renders harmful substances harmful as a priority species in the environment to be purified.

  In general, when specific microorganisms that decompose and detoxify harmful substances originally live in the environment to be purified, the environment is purified by supplying nutrient sources, oxygen, etc. for those microorganisms. Since the growth of other microorganisms is also activated, it is difficult to make the necessary microorganisms a priority species of the environment simply by supplying nutrient sources, oxygen and the like.

  In addition, when specific microorganisms used for environmental purification do not live in the environment to be purified, the microorganisms for purification are separately cultured and added to the environment to be purified, thereby decomposing and detoxifying harmful substances. However, microorganisms introduced from outside in this way are difficult to survive and multiply by breaking into the ecological niche formed by existing microbial species, and the number of individuals gradually decreases. Decomposition of the target object becomes difficult. Moreover, even if it is adapted to a new environment at the beginning, the number of solids is decreased due to changes in various environmental factors, and the function of decomposition and purification is lowered.

  As a method of adapting specific microorganisms used for environmental purification to the environment to be purified as a priority species, sterilize microorganisms originally inhabiting the environment to be purified, add specific microorganisms after reducing the number of individuals Although methods (Patent Document 5 and Patent Document 6) have also been proposed, it is difficult to establish a specific microorganism as a preferred species in the environment to be purified even if this method is used. Even if the microorganisms settle in the environment to be purified, it is difficult to maintain the decomposition activity of harmful substances at a high level for a long period of time. The environment cannot be obtained.

  The present invention has been made in view of the above problems, and in the environmental purification technology using specific microorganisms, the activity of microorganisms that decompose or immobilize harmful substances is maintained at a high level for a long period of time, and stably in the environment. The purpose is to provide a method for decomposing or immobilizing harmful substances to obtain a clean environment.

  The present inventors have focused on microorganisms having an electron transfer protein in the outer cell membrane as microorganisms that decompose or immobilize harmful substances.

  Microorganisms that have electron transport proteins in the outer membrane can perform "extracellular electron transfer" directly to extracellular materials, and can use water-insoluble iron and manganese oxides as energy sources. it can. Microorganisms having an electron transfer protein in the outer membrane have the ability to proceed with a wide variety of organic decomposition and purification reactions. Microorganisms that do not have an electron transfer protein in the outer membrane perform “extracellular electron transfer” via a free-diffusing electron mediator, but the rate is extremely slow. It is presumed that the difference in the “extracellular electron transfer” speed affects the resolution of the hardly-decomposable organic matter.

 Here, specific examples of the microorganism having an electron transfer protein in the extracellular membrane include bacteria such as Geobacter sp. And Shewanella sp. These bacteria are: It is known that various hardly decomposable organic substances are decomposed by the metabolic reaction to fix inorganic substances.

  For example, in Patent Document 4, bacteria belonging to the genus Geobacter and Chewanella have the ability to biologically reduce metals such as arsenic, chromium, lead, mercury, uranium, and precipitate these metals as water-insoluble metal species. And a technique for separating it from wastewater.

  Regarding the decomposition of organic matter, it was confirmed that Geobacter metallireducence GS-15 strain oxidizes toluene and phenol to carbon dioxide (Non-patent Document 1), and Geobacter lovely reductively decomposes tetrachloroethylene and trichlorethylene (Non-patent Document 2). ing.

  In addition, the presence of Geobacter bacteria as indigenous bacteria at contaminated sites has been confirmed in field studies at various contaminated sites (Non-patent Document 3), and aromatic compounds such as benzene in the aquifer of contaminated sites. When the oxidation reaction proceeds, it is also reported that the occupancy rate of Geobacter bacteria is high in the indigenous microorganism (Non-patent Document 4).

  In order to solve the above-mentioned problems, the present inventors conducted extensive research on microorganisms having an electron transfer protein in the extracellular membrane represented by Geobacter. As a result, the resolution of persistent organic substances is extremely strongly expressed during the growth of the microorganisms. And the present invention has been completed based on the findings.

That is, the purification method of contaminated soil or water according to the present invention is a soil or purification process of water contaminated by organic compounds and / or inorganic compounds, in soil or water to be purified, the cell outer membrane A method for purifying contaminated soil or water by decomposing or immobilizing pollutants by growing microorganisms having electron transfer proteins on the surface, and in the soil or water to be purified, A means for growing microorganisms having a function of sterilizing at least a part of microorganisms living in soil or water to be purified, and activating microorganisms having electron transfer proteins in the extracellular membrane to promote growth A method for purifying contaminated soil or water comprising the step of adding a growth agent for the purpose to the soil or water to be purified, the method comprising: An electron transfer material containing at least one selected from flavins or quinones, and an organic solvent or an organic salt for increasing the solubility of the electron transfer material And at least one of them .

The contaminated soil or water purification method according to the present invention is characterized in that the organic solvent or organic salt for increasing the solubility of the electron transfer material is an aqueous acetic acid solution.

In addition, the method for purifying contaminated soil or water according to the present invention includes a step of sterilizing at least a part of microorganisms inhabiting the soil or water to be purified, and an electron transfer protein in an outer membrane. The step of adding a growth agent for activating microorganisms and promoting growth to soil or water to be purified is repeatedly performed.

  Here, the disinfectant added in the step of disinfecting at least a part of the microorganisms inhabiting the soil or water to be purified is an acid capable of reducing the pH of the soil or water to be purified to 3.0 or less. It is preferable that the step of disabling the disinfectant is addition of a pH neutralizing agent.

  In addition, the proliferation agent for activating the microorganism having an electron transfer protein in the extracellular membrane and promoting proliferation includes at least one selected from an electron transfer material, an electron acceptor, an electron donor, or a nutrient, By including the electron transfer material, the contaminated soil or water can be efficiently purified. At this time, since the electron transfer material has poor solubility in water, the growth agent includes at least one of an organic solvent or an organic salt for increasing the solubility of the electron transfer material.

  Further, the electron transfer material is at least one selected from flavins or quinones, an organic solvent or an organic salt for increasing the solubility of the electron transfer material is an aqueous acetic acid solution, and the electron acceptor is trivalent iron. Or at least one selected from metal compounds consisting of tetravalent manganese or fumarate, and the electron donor includes at least one selected from acetate, formate, and glucose. This is a preferable mode for activating a microorganism having an electron transfer protein.

  Among the microorganisms having an electron transfer protein in the extracellular membrane, the most preferable microorganism for purifying contaminated soil or water is a Geobacter bacterium.

  According to the method for purifying contaminated soil or water of the present invention, microorganisms having an electron transfer protein in the extracellular membrane exhibit extremely strong organic matter resolution and metal reducing ability during growth. By creating the growth state of the microorganism in water, it becomes possible to efficiently decompose or immobilize harmful substances.

  In addition to the microorganism, a proliferation agent added to activate microorganisms having an electron transfer protein in the extracellular membrane by sterilizing at least a part of microorganisms living in the soil or water to be purified. The ratio used becomes small, and it becomes possible to easily create a growth state of a microorganism having an electron transfer protein in the extracellular membrane in soil or water to be purified.

  In addition, the process of sterilizing at least some of the microorganisms inhabiting the soil or water to be purified, the process of adding a fungicide to the soil or water to be purified, and the added disinfectant after a certain period of time are invalidated By adopting the step, the microorganisms that live in the soil or water to be purified can be easily sterilized.

  In addition, when a microorganism having an electron transfer protein in the outer membrane does not live in the soil or water to be purified, a microorganism having an electron transfer protein can be added to the separately cultured outer membrane. Thus, it is possible to reliably produce a growth state of the microorganism in the soil or water to be purified.

  Furthermore, in the present invention, a step of sterilizing at least a part of microorganisms that live in the soil or water to be purified, and a growth agent for microorganisms having an electron transfer protein in the extracellular membrane are added to the soil or water to be purified. The step of performing is repeated. In general, when the number of individuals in a closed environment increases to some extent, microorganisms stop growing and the number of individuals no longer increases. However, in the present invention, microorganisms that live in the soil or water to be purified are repeatedly sterilized. Therefore, the number of microorganisms having an electron transfer protein in the outer membrane also decreases each time, and the growth state of the microorganisms can be created for a long period without the saturation of the number of individuals continuing. In other words, the excellent organic matter resolving power and metal reducing ability of microorganisms with electron transfer proteins in the extracellular membrane are maintained, and toxic substances in the soil or water to be purified can be decomposed or immobilized stably over a long period of time. It becomes possible.

  Here, the disinfectant added in the step of disinfecting at least a part of the microorganisms inhabiting the soil or water to be purified is an acid that can reduce the pH of the soil or water to be purified to 3.0 or less. If the step of disabling the disinfectant is the addition of a pH neutralizing agent, disinfection of microorganisms living in the soil or water to be purified becomes easier.

  In addition, a proliferation agent for activating a microorganism having an electron transfer protein in the outer membrane and promoting proliferation includes at least one selected from an electron transfer material, an electron acceptor, an electron donor, or a nutrient, By including the transfer material, an environment in which a microorganism having an electron transfer protein in the outer cell membrane is easier to grow than other microorganisms.

  Further, at least one selected from flavins or quinones as an electron transfer material, an acetic acid aqueous solution as an organic solvent or organic salt for increasing the solubility of the electron transfer material, and trivalent iron or tetravalent manganese as an electron acceptor. When at least one selected from the following metal compounds or fumarate is used as the electron donor, at least one selected from acetate, formate, and glucose, the growth of microorganisms having an electron transfer protein in the outer membrane Is very preferable, and can promote decomposition or immobilization of harmful substances in soil or water to be purified.

  The microorganism having an electron transfer protein in the extracellular membrane is a bacterium belonging to the genus Geobacter, so that excellent contaminated soil or water corresponding to a wide variety of biodegradable organic substances and inorganic substances harmful to living organisms can be obtained. It becomes a purification method.

  The most important point in the contaminated soil or water purification method of the present invention is to create a growth state of microorganisms having an electron transfer protein in the extracellular membrane in the soil or water to be purified. As a result, the excellent organic matter resolving power and metal reducing ability of microorganisms having an electron transfer protein in the extracellular membrane are maximized, and an unprecedented environmental purification effect can be obtained.

  In order to create a growth state of microorganisms having an electron transfer protein in the extracellular membrane in the soil or water to be purified, the present invention sterilizes at least a part of the microorganisms living in the soil or water to be purified. A step (hereinafter also referred to as a sterilization step) and a step of activating a microorganism having an electron transfer protein in the extracellular membrane and adding a proliferation agent for promoting proliferation (hereinafter referred to as a proliferation agent addition step) There is also. The sterilization step is performed to reduce the number of microorganisms other than those having electron transfer proteins in the outer membrane, but at the same time, the number of microorganisms having electron transfer proteins in the native outer membrane may be reduced. When a microorganism having an electron transfer protein is activated in the extracellular membrane in a state where the absolute number of microorganisms is small and a proliferation agent for promoting growth is allowed to act, the ratio of the proliferation agent to other microorganisms decreases, It is effectively used for the growth of microorganisms having electron transfer proteins in the outer membrane.

  The sterilization method in the sterilization step is not particularly limited, and a known method such as heat treatment, ultraviolet treatment, ozone treatment, treatment with a bactericide can be adopted, but a growth state of microorganisms having an electron transfer protein in the extracellular membrane can be created. Therefore, it is necessary to adopt a method in which the soil or water to be purified does not become an unsuitable environment for the growth of microorganisms. In consideration of workability, economy, and effectiveness of sterilization, it is preferable to add a sterilizing agent to the soil or water to be purified, and to invalidate the added sterilizing agent after a certain period of time. When considering disinfecting disinfectants, it is difficult to use organic disinfectants, and the pH of oxidants such as hypochlorite and soil or water to be purified may be 3.0 or less. It is preferable to use an acid that can be used. The oxidizing agent is a reducing agent, and the acid can be easily invalidated by neutralization with an alkali, and the sterilizing power can also be reduced by diluting with a large amount of water. Since the optimal pH for growth of Geobacter, a type of microorganism that has an electron transfer protein in the outer cell membrane, is neutral, neutralization is essential when sterilized with acid, such as neutral to weakly alkaline buffers. It is preferable to add a pH neutralizing agent.

  In addition, a proliferation agent for activating a microorganism having an electron transfer protein in the outer membrane and promoting proliferation is at least one selected from an electron transfer material, an electron acceptor, an electron donor, or a nutrient. More specifically, examples of the electron transfer material include flavins such as riboflavin and flavin mononucleotide, or quinones such as menaquinone and anthraquinone-2,6-disulfonic acid, and examples of the electron acceptor include trivalent iron and tetravalent manganese. As the metal compound, fumarate, and electron donor, acetate, formate, glucose, and yeast extract can be used as nutrients. Here, each substance constituting the above-mentioned growth agent may be added to the soil or water to be purified at different timings, or several substances may be added simultaneously, or all may be added simultaneously. It doesn't matter. When several substances are added at the same time, a one-part preparation may be added, or individual preparations may be added simultaneously. In addition, the purification effect can be enhanced by appropriately setting the ratio of each substance constituting the proliferation agent in accordance with the environment to be purified and harmful substances.

  Among the substances constituting the proliferation agent, the flavins of the electron transfer material are particularly important because they have an action of promoting the electron transfer reaction of Geobacter. However, since flavins have low hydrophilicity, it is necessary to use an organic solvent or an organic salt for increasing the solubility in order to obtain a high-concentration solution to be added as a proliferating agent. A preferred solvent for increasing the solubility of flavins is an aqueous acetic acid solution.

  The disinfectant added in the disinfecting step, the disinfectant for disinfecting disinfectant, and each substance constituting the growth agent may be added to the soil or water to be purified simultaneously or separately. Absent. Moreover, when adding simultaneously, it is also possible to add what was made into one liquid formulation.

  Here, microorganisms having an electron transfer protein in the extracellular membrane, especially bacteria of the genus Geobacter, are known to live as indigenous bacteria in various environments, and soil or water to be purified in the present invention. In the unlikely event that a microorganism having an electron transfer protein in its outer membrane does not live in the soil or water to be purified, Add to soil or water to be purified. Even in this case, by performing the sterilization step and the growth agent addition step, it is possible to create a growth state of a microorganism having an electron transfer protein in the extracellular membrane in the soil or water to be purified. .

  The sterilization step and the growth agent addition step are preferably performed repeatedly. Thereby, it can be avoided that the number of microorganisms having an electron transfer protein in the extracellular membrane is saturated and the growth is stopped.

  In the present invention, microorganisms having electron transfer proteins in the outer membrane are used to decompose and purify harmful substances. It is possible to decompose and purify harmful inorganic substances.

  The most recommended embodiment of the present invention is to use an acetic acid aqueous solution as a disinfectant for disinfecting at least a part of microorganisms that inhabit the soil or water to be purified. Since the aqueous acetic acid solution serves as a solvent for flavins, it is added to the soil or water to be purified in a state where the flavins are dissolved. Acetic acid acts as a bactericidal agent and then acts as an electron donor when neutralized to acetate, and in the presence of acetate, the growth of geobacter is accelerated, giving priority to indigenous geobacter in the soil or water to be purified. The seed effect is also obtained. Thus, by using the acetic acid aqueous solution in the present invention, very efficient environmental purification becomes possible.

  Examples Hereinafter, the method for purifying contaminated soil or water according to the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

The following tests were conducted using sewage collected at a sewage treatment plant in Tokyo. The reagents used in the test are as follows. In addition, as a result of examining the collected sewage in advance, it was confirmed that Geobacter was inhabiting.
Reagent 1: 10% aqueous acetic acid solution in which 1 mmol / L riboflavin is dissolved Reagent 2: pH 7.5 HEPES buffer (HEPES = 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid)
Reagent 3: 500 mmol / L sodium fumarate aqueous solution Reagent 4: 1% yeast extract aqueous solution Reagent 5: 10 mmol / L benzene aqueous solution

<Example 1>
50 mL of Reagent 1 was added to 1 L of sewage to adjust the pH to 2.9, and after 30 minutes, 50 mL of Reagent 2, Reagent 3, and Reagent 4 were sequentially added. Thereafter, 60 mL of Reagent 5 was added to prepare Test Water 1 containing benzene 0.5 mmol / L (39 mg / L). The test water 1 was allowed to stand at 30 ° C. under anaerobic conditions, and the benzene concentration was measured after 2 days. The result was 0.11 mmol / L (decomposition rate 78%).

<Comparative Example 1>
50 mL of reagent 2 and 150 mL of sterilized ion-exchanged water were added to 1 L of sewage, and then 60 mL of reagent 5 was added to obtain test water 2 containing 0.5 mmol / L of benzene. The test water 2 was allowed to stand at 30 ° C. under anaerobic conditions, and the benzene concentration was measured after 2 days. The result was 0.45 mmol / L (decomposition rate 10%).

<Comparative example 2>
100 mL of reagent 1 and reagent 2 previously mixed at a volume ratio of 1: 1 to 1 L of sewage was added, and then 50 mL each of reagent 3 and reagent 4 were sequentially added. Thereafter, 60 mL of reagent 5 was added to prepare test water 3 containing benzene at 0.5 mmol / L. The test water 3 was allowed to stand at 30 ° C. under anaerobic conditions, and the benzene concentration was measured after 2 days. The result was 0.27 mmol / L (decomposition rate 46%).

<Example 2>
25 mL of Reagent 1 was added to 500 mL of test water after measuring the benzene concentration in Example 1, and 25 mL each of Reagent 2, Reagent 3, and Reagent 4 were sequentially added after 30 minutes. Then, the reagent 5 was added suitably and it was set as the test water 4 containing 0.5 mmol / L of benzene. The test water 4 was allowed to stand at 30 ° C. under anaerobic conditions, and the benzene concentration was measured after 2 days. The result was 0.12 mmol / L (decomposition rate 76%).

<Comparative Example 3>
50 mL of reagent 1 and reagent 2 previously mixed at a volume ratio of 1: 1 was added to 500 mL of test water after measuring the benzene concentration in Example 1, and then 25 mL each of reagent 3 and reagent 4 were sequentially added. did. Then, the reagent 5 was added suitably and it was set as the test water 5 containing 0.5 mmol / L of benzene. The test water 5 was allowed to stand at 30 ° C. under anaerobic conditions, and the benzene concentration was measured after 2 days. The result was 0.30 mmol / L (decomposition rate 40%).

  As apparent from the test results, a good benzene decomposition effect was observed in Example 1 in which the sterilization step and the proliferation agent addition step of the present invention were performed. On the other hand, even if the sewage is inhabited by Geobacter, the benzene resolution is very low if it is kept in a neutral anaerobic state as in Comparative Example 1, and it is proliferated as in Comparative Example 2 which is the prior art. If only the agent addition step is performed, only about half the benzene resolution of the present invention can be obtained.

  Furthermore, as shown in Comparative Example 3, after a while after performing the sterilization step and the growth agent addition step, the initial benzene resolution cannot be obtained only by adding the growth agent again. The initial benzene resolution could be maintained by carrying out the process and the growth agent addition process together. This is because, in Comparative Example 3, the number of Geobacter individuals reached a saturation state and the benzene resolution was lowered because the growth state could not be maintained, whereas in Example 2, the sterilization process was performed again to perform the Geobacter individual. Once the number is reduced, the growth state of Geobacter can be created thereafter, and good benzene resolution is maintained.

  The method for purifying contaminated soil or water according to the present invention can be widely used for purifying soil, groundwater, sediment, wastewater, etc. contaminated with organic compounds that are hardly biodegradable or inorganic compounds harmful to living organisms. .

Claims (3)

An organic compound and / or contaminated with inorganic compounds soil or purification process of water in the soil or water to be purified, degradation or contamination by growing a microorganism having an electron transfer protein to the extracellular membrane A method for purifying contaminated soil or water to be immobilized , wherein the means for growing a microorganism having an electron transfer protein in an extracellular membrane in the soil or water to be purified is contained in the soil or water to be purified. Sterilizing at least a part of microorganisms that inhabit, and adding a growth agent for activating and promoting the growth of microorganisms having electron transfer proteins in the extracellular membrane to the soil or water to be purified A method for purifying contaminated soil or water, wherein the microorganism having an electron transfer protein in the outer membrane of the microorganism is a bacterium belonging to the genus Geobacter, and the growth agent is An electron transfer material that includes at least one selected from the Rabin acids or quinones, characterized in that it comprises at least one, the one of the organic solvent or organic salts to increase the solubility of the electronic transfer material contamination Soil or water purification methods. The method for purifying contaminated soil or water according to claim 1, wherein the organic solvent or organic salt for increasing the solubility of the electron transfer material is an aqueous acetic acid solution . A step of sterilizing at least a part of microorganisms inhabiting the soil or water to be purified, and a proliferation agent for activating the microorganisms having an electron transfer protein in the extracellular membrane to promote proliferation; The method for purifying contaminated soil or water according to claim 1 or 2, wherein the step of adding to soil or water is repeatedly performed .