WO2015016238A1

WO2015016238A1 - Electron transfer system and application for same

Info

Publication number: WO2015016238A1
Application number: PCT/JP2014/070002
Authority: WO
Inventors: 新太片山; 春芳章; 冬冬張; 智リン李; 大典鈴木
Original assignee: 国立大学法人名古屋大学
Priority date: 2013-07-31
Filing date: 2014-07-30
Publication date: 2015-02-05
Also published as: JPWO2015016238A1; JP6410188B2

Abstract

The problem addressed by the present invention is to provide a novel system suitable for cleaning a polluted environment. An electron transfer system is constructed using a metal humic acid complex as an electron transfer medium combined with an electron donor means and organisms as an electron acceptor.

Description

Electronic transmission system and its use

The present invention relates to an electron transmission system using an organic / inorganic composite. Specifically, the present invention relates to an electron transmission system using a composite of metal and humic acid as an electron transmission medium and its use (application to environmental purification, etc.). This application claims priority based on Japanese Patent Application No. 2013-158394 filed on Jul. 31, 2013, the entire contents of which are incorporated by reference.

Bioremediation is attracting attention as a technology for purifying contaminated environments (contaminated soil, groundwater, etc.). Bioremediation is a biostimulation that promotes the degradation of pollutants by increasing the growth and activity of microorganisms in the environment, and a bio-aug that attempts to decompose pollutants by adding and adding microorganisms that can degrade pollutants. It is roughly divided into In general, biostimulation is simpler and cheaper and has been put into practical use, and there are many reports of research and development for practical use.

In biostimulation, a decomposition accelerator is often used. For example, as a countermeasure against groundwater contamination of aliphatic chlorinated solvents such as trichlorethylene, electron donors (lactic acid-based, higher fatty acid-based, etc.) as various decomposition accelerators are sold by various companies. In addition, polylactic acid esters, higher fatty acid esters, lower fatty acid esters, etc. are added as promoters of microbial activity to contaminated sites where persistent organic pollutants such as dioxins and polychlorinated biphenyls have accumulated. A method for improving the degradation activity of microorganisms against pollutants has also been proposed (see, for example, Patent Documents 1 to 3). On the other hand, a method has been proposed in which decomposition is promoted by adding an electron transfer substance when decomposition does not proceed only by addition of an electron donor. For example, there is an example in which a water-soluble electron transfer substance having a quinone skeleton is added to a microorganism culture apparatus to promote the respiratory growth of the microorganism, and the hardly degradable organic pollutant is decomposed by the microorganism (for example, see Non-Patent Documents 1 and 2). ).

JP 2008-188478 A JP 2008-296176 A JP 2009-1212949 A

When water-soluble electron mediators such as those mentioned above are used as promoters of microbial degradation against soil and groundwater contamination, they are persistent substances that spread and remain in the groundwater environment. It will cause. In addition, since it is water-soluble, it is easy to inject, but it is necessary to inject repeatedly, which increases secondary contamination. Therefore, injection of an electron transfer material has not been carried out even at a contaminated site where there is a shortage of electron transfer materials (sites where purified microorganisms exist and microbial degradation is not promoted even if an electron donor is injected).

The main object of the present invention is to provide a new system or method suitable for the purification of a polluted environment under the above background. Another object is to provide an application of such a system or method.

Humic substances are a group of organic compounds that are abundant in soil, natural water, and various terrestrial and aquatic environments (References 1-3). Due to the high potential of humic substances, the effects of humic substances on anaerobic bioremediation using electron transport systems have been studied over the past 20 years (reference documents 4, 5). Most of the research on the redox transmission properties of humic substances concentrated on water-soluble humic substances that can be reduced by microorganisms, focusing on enhancing the ability of microorganisms to reduce iron oxides (references). 5-7). However, humic substances are present in a particulate form (solid humic substance) rather than dissolved in water in the natural environment. However, little is known about the ability of microorganisms to transfer electrons to solid humic substances (or microorganisms that accept electrons from a solid phase electron transfer material).

The humic substances that exist universally in nature have the property of forming a complex by coordinating with metals present in soil, water and sediment. It has been reported that physicochemical coprecipitation occurs between humic substances and iron oxides (Reference Document 16). Our research group also found that humin, a humic fraction that is insoluble under any pH conditions, contains a fairly high concentration of iron (reference 15). This iron-rich insoluble humin showed stable electron transfer capacity for microbial reductive dechlorination of pentachlorophenol (PCP), while humic acid (HA) from the same soil source was dissolved The electron transfer activity was unstable. Focusing on this fact and the fact that the iron content of humic acid was lower than that of humin containing a large amount of iron, water-insoluble iron humic acid that is an organic / inorganic composite by mixing humic acid and iron compound It was decided to prepare a complex and investigate its properties. As a result, it was found that the iron humic acid complex promotes the dechlorination activity of aromatic chlorine compounds by microorganisms as well as Humin. That is, it has been clarified that the iron humic acid complex functions as an electron transfer medium that improves microbial activity for purifying contaminated organic substances. Since the iron humic acid complex is insoluble in water, it usually does not flow out from the input site even when it is input into soil or groundwater. Moreover, the iron humic acid complex is made of an organic compound existing in the natural environment and does not cause environmental pollution. That is, it can be said to be a safe and hygienic material that has an environmental purification effect.

Here, the function of the iron humic acid complex as an electron transfer medium that improves the microbial activity of purifying contaminated organic matter is that an electron transfer system using microorganisms that can receive electrons from the iron humic acid complex can be constructed. Means. The electron transfer system is not only effective as a means of bioremediation (particularly biostimulation) as described above, but also used for microbial fuel cells, microbial electroculture (bringing microorganisms using electrical energy), etc. it can.

On the other hand, considering the ionic characteristics, the same effect can be achieved even if a complex with humic acid is formed using not only iron but also transition metals such as manganese and cobalt, and metals such as magnesium, zinc and selenium. Can do. If the metal of the iron humic acid complex is changed to a metal other than iron, an electron transfer medium having a different redox potential is obtained. If the metal humic acid complex having a specific redox potential thus obtained is used, it can be expected that a specific microorganism can be selectively cultured. That is, it can be said that the electron transfer system is useful as a new microorganism culture technique. Thus, the above-mentioned electron transmission system has a wide application range (use) and its value is extremely high. It should be noted that the complex formation of iron and humic acid, which is an essential micronutrient for all living organisms, and one of the most abundant metals in soil and sediment, has been extensively studied, and insoluble iron humic acid complexes have been introduced into plants. Although it has been reported that it becomes an iron supply source or a heavy metal adsorbent (reference documents 17 to 19), it is not known that it functions as an electron transfer medium and can mediate the transfer of electrons to microorganisms.
The invention shown below is mainly based on the above knowledge or result.
[1] An electron transfer system comprising a metal humic acid complex as an electron transfer medium, electron donating means, and a microorganism as an electron acceptor.
[2] The electron transfer system according to [1], wherein the metal constituting the metal humic acid complex is a transition metal, magnesium, zinc, or selenium.
[3] The electron transfer system according to [2], wherein the transition metal is manganese, iron, or cobalt.
[4] The electron transfer system according to [1], wherein the metal constituting the metal humic acid complex is a metal having a plurality of redox potentials.
[5] The electron donating means comprises a combination of a reducing organic compound and a reducing microorganism that receives electrons from the reducing organic compound and reduces the metal humic acid complex. ] The electronic transmission system as described in any one of.
[6] The reducing organic compound is one or more organic compounds selected from the group consisting of sugars, amino acids, organic acids, organic acid salts, alcohols, aromatic compounds, and biodegradable plastics. ] The electronic transmission system of description.
[7] The electron transfer system according to any one of [1] to [4], wherein the electron donating means is a cathode electrode.
[8] The electron transfer system according to [7], wherein electrons are supplied from the reducing organic compound to the anode electrode electrically connected to the cathode electrode through a microorganism.
[9] A combination of a reducing organic compound and a reducing microorganism that reduces the metal humic acid complex by receiving electrons from the reducing organic compound with respect to the anode electrode electrically connected to the cathode electrode. The electron transmission system according to [7], wherein electrons are supplied by the electron donating means.
[10] The electron transfer system according to any one of [1] to [9], wherein the microorganism as an electron acceptor is a dehalogenated microorganism, a bacterium having iron reducing ability, or a bacterium having nitrate reducing ability. .
[11] The electron transfer system according to any one of [1] to [9], wherein the microorganism as an electron acceptor is a microorganism that can exchange electrons with an extracellular solid phase.
[12] The microorganism as an electron acceptor is selected from the group consisting of Geobacter, Schwanella, Dehalobacter, Desulfitobacterium, Desulfromonas, Clostridium, Bacteroides, Desulfobrybacteria, Cedi The electron transfer system according to any one of [1] to [9], wherein the electron transfer system is one or more bacteria selected from the group consisting of a genus Bacterium and a sulfurospirillum bacterium.
[13] An environmental purification method comprising decomposing an organic halogen compound in a polluted environment by the electron transmission system according to [10].
[14] An environmental purification method comprising adding a metal humic acid complex to a contaminated environment.
[15] The environmental purification method according to [14], further comprising a step of adding a reducing organic compound as an electron donor to the contaminated environment.
[16] Further, as an electron donating means, a step of adding a reducing organic compound and a reducing microorganism that receives the electron donation from the reducing organic compound and reduces the metal humic acid complex to a contaminated environment, 14].
[17] The environmental purification method according to [14], further including a step of installing a cathode electrode as electron donating means in a contaminated environment.
[18] The environmental purification method according to any one of [14] to [17], further comprising a step of adding a dehalogenated microorganism as an electron acceptor to the contaminated environment.
[19] An environmental purification kit comprising a metal humic acid complex as an electron transfer medium and a reducing organic compound as an electron donor.
[20] A metal humic acid complex as an electron transfer medium, a reducing organic compound as an electron donating means, and a reducing microorganism that receives the electrons from the reducing organic compound and reduces the metal humic acid complex An environmental purification kit comprising:
[21] An environmental purification kit comprising a metal humic acid complex as an electron transmission medium and a cathode electrode.
[22] The kit for environmental purification according to any one of [19] to [21], further comprising a dehalogenated microorganism as an electron acceptor.
[23] A method for selectively culturing a microorganism, comprising culturing a microorganism-containing sample in the presence of a metal humic acid complex and an electron donor.
[24] An electron transfer medium comprising a metal humic acid complex.

Effect of iron humic acid complex (Fe-HA) on PCP dechlorination reaction of PCP-phenol dechlorinated humin culture and effect of iron humic acid complex on adsorption of PCP and phenol. (a) The PCP-phenol dechlorination humin culture supplemented with the insoluble iron humic acid complex showed stable PCP (△) -phenol (○) dechlorination activity. The arrow indicates that 5% of the PCP-phenol dechlorinated humin culture was inoculated into fresh medium supplemented with PCP. (b) Dechlorination is not observed in the absence of iron humic acid complex or when not inoculated with PCP-phenol dechlorinated humin cultures. (c) PCP dechlorination reaction is not observed under the condition that only iron or Aldrich humic acid sodium salt (AHA) is added as a control. (d) The concentrations of PCP (Δ) and phenol (◯) in the inorganic medium with and without iron humic acid complex (2.5 g L ⁻¹ ) added. Since there is no difference in concentration between the two medium conditions, it can be seen that there is almost no adsorption. Effect of each treatment on microbial reductive dechlorination by iron humic acid complex. (A) Iron humic acid complex treated with MgCl ₂ , (b) Iron humic acid complex treated with NaOAc (sodium acetate), (c) Iron humic acid complex treated with NaOH (sodium hydroxide), ( d) Iron humic acid complex treated with Na ₄ P ₂ O ₇ (sodium pyrophosphate). Δ: PCP, ○: phenol. The arrow indicates that 5% of the PCP-phenol dechlorinated humin culture was inoculated into fresh medium supplemented with PCP. In sodium pyrophosphate, the microbial reductive dechlorination reaction did not occur in the second inoculation, indicating that the sodium pyrophosphate extract fraction has an electron transfer function. Promoting effect of iron humic acid complex on microbial reduction of iron (III) oxide. Electron accepting ability (EAC) of iron humic acid complex. Microbial and chemical reductions were analyzed and evaluated. (a) Microbial (Sewanella putrefaciens CN-32) reduction. (b) chemical (H ₂ / Pd) reduction. Fourier transform infrared absorption spectrum. (a) Iron humic acid complex treated with Na ₄ P ₂ O ₇ and iron humic acid complex. (b) Humic acid (AHA) and control iron samples. Cyclic voltammogram (CV). (a) Iron humic acid complex and background sample before and after reduction at a potential of -500 mV (vs. standard hydrogen electrode). (b) Control iron sample and control HA sample. (c) Iron humic acid complex treated with Na ₄ P ₂ O ₇ before and after electroreduction using a potential of -500 mV (vs standard hydrogen electrode). Inorganic medium was analyzed as electrolyte and background sample. The example of the specific aspect of the electronic transmission system of this invention. Electrons are exchanged through the metal humic acid complex (M-HA). Microorganism B as an electron acceptor dehalogenates an organic halogen compound. In (a), the microorganism A receives electrons from an organic compound and reduces M-HA. In (b), a cathode electrode is used as an electron donating means. In (c), electrons are supplied from the microorganism A 'to the anode electrode. In (d), a microorganism A ″ using M-HA as a final electron acceptor is used, and electrons are transferred using M-HA also on the anode side. Promoting effect of iron humic acid complex on microbial reduction of nitrate.

1. Electronic transmission system The 1st aspect of the present invention is related with an electronic transmission system. The electron transfer system of the present invention utilizes a microorganism functioning as an electron acceptor (hereinafter referred to as “electron accepting microorganism” for convenience of explanation), and is the maximum in that a metal humic acid complex is used as an electron transfer medium. There are features. The electron transfer system of the present invention includes a metal humic acid complex as an electron transfer medium, an electron donating means, and a microorganism (electron accepting microorganism) as an electron acceptor, and electrons from the electron donating means are metal humic acid complexes. To the electron-accepting microorganisms. As described above, the electron transfer system of the present invention transfers electrons outside the cell and is distinguished from the electron transfer system in the cell.

(1) Metal Humic Acid Complex Humic substances that are one of the components of soil and sediment are classified into three types, humic acid, fulvic acid, and humin, based on solubility in alkali and acid. Humic acid is also called humic acid. Humic acid is roughly classified into natural humic acid (natural humic acid) and regenerated humic acid (regenerated humic acid) produced industrially by oxidative degradation of natural materials. Humic acid is a component that is precipitated by acid extraction after soil or sediment is alkali extracted. That is, it is a polymer organic acid that is soluble in an alkaline aqueous solution and insoluble in an acidic aqueous solution. Humic acid has many aromatic rings in its basic skeleton, and carboxyl groups and hydroxyl groups are bonded to the aromatic rings. Due to such structural features, humic acid exhibits a buffering action, a surface active action, an ion retention action and the like. Note that fulvic acid is a high-molecular organic acid that is soluble in both alkaline and acidic aqueous solutions, and humic acid is a humic substance that is insoluble in both alkaline and acidic aqueous solutions.

In the present invention, a complex of humic acid and metal (referred to as “metal humic acid complex” in the present invention) is used as an electron transfer medium. The metal constituting the metal humic acid complex is not particularly limited as long as the complex exhibits electron transfer properties. For example, transition metals such as manganese, iron, and cobalt, magnesium, zinc, and selenium can be employed. A preferred metal is iron. If a metal having a plurality of oxidation-reduction potentials (for example, transition metals of manganese, iron, and cobalt) is used, an electron transfer system that exhibits a plurality of oxidation-reduction potentials can be constructed. Such an electron transfer system has an advantage that it can be controlled to a target oxidation-reduction potential, and is suitable for use in selective culture of microorganisms. In this specification, “selective culture” includes (i) isolation culture of specific microorganisms (particularly difficult-to-cultivate microorganisms), (ii) maintenance culture of specific microorganisms (particularly difficult-to-cultivate microorganisms), and (iii) microbial populations Accumulation of specific microorganisms (especially difficult-to-culture microorganisms) from (iv), and (iv) maintenance culture of microorganisms containing specific bacteria. The metal humic acid complex includes, for example, humic acid and an aqueous metal salt solution (in the case where the metal is iron, iron sulfate (II), iron chloride (II), iron citrate (III), etc. can be used)). After mixing, the pH of the solution is set to a weakly alkaline to neutral to weakly acidic range and maintained at a predetermined temperature (for example, 20 ° C. to 40 ° C.) for a predetermined period (for example, 1 day to 2 weeks), and the precipitate is recovered. Obtainable.

It is possible to use a composite of two or more kinds of metal and humic acid, or to use two or more kinds of metal humic acid formed using different metals.

In the present invention, the metal humic acid complex functions as an electron transfer medium. More specifically, an electron is received to change from an oxidized form to a reduced form, and an electron is supplied to change from a reduced form to an oxidized form. In the present invention, the electrons for converting the metal humic acid complex from the oxidized form to the reduced form are generated by the electron donating means described in detail below.

(2) Electron donating means The electron donating means used in the present invention will be described with reference to FIG. FIG. 7 shows four modes. For convenience of explanation, in FIG. 7, a dehalogenated microorganism (microorganism B) is used as the electron-accepting microorganism.

In the first embodiment (corresponding to FIG. 7 (a)), as an electron donating means, a reducing organic compound and a reducing microorganism (Fig. 7) that receives an electron donation from the reducing organic compound and reduces the metal humic acid complex. Among them, a combination with microorganism A) is used. In this embodiment, a reducing organic compound is used as the electron donor. A reducing organic compound is an organic compound that generates electrons upon oxidation, such as sugar (3 to 7 carbon), amino acid, organic acid (lactic acid, fumaric acid, pyruvic acid, succinic acid, malic acid, Formic acid, acetic acid, citric acid, higher fatty acids, etc.) or organic acid salts (lactate, fumarate, pyruvate, succinate, malate, formate, acetate, citrate, higher fatty acid, etc.) , Alcohols (lower alcohols such as ethanol, propanol and butanol to higher alcohols such as palmitol), aromatic compounds (phenol, polyphenol, phenolic acid and salts thereof), biodegradable plastics (polyhydroxybutyric acid (PHB), Polycaprolactone (PCL), polylactic acid (PLA), polylactic acid-based resin (see, for example, JP-A-2011-104551), Starch-based resin) and the like are used. The reducing microorganism converts the oxidized metal humic acid complex into a reduced metal humic acid complex. Typically, microorganisms that breathe using the metal humic acid complex as the final electron acceptor are used. Examples of such microorganisms include Schwanella bacteria and Geobacter bacteria that can exchange electrons with the extracellular solid phase.

In the second mode (corresponding to FIG. 7B), a cathode electrode is used as the electron donating means. That is, the oxidized metal humic acid complex is directly electrochemically reduced using an electrode (cathode). The material of the electrode is not particularly limited. Examples of electrode materials are C, Au, Pt, and Ti.

On the other hand, if a microorganism using the electrode as a final electron acceptor (microorganism A ′ in the figure) is used, electrons obtained by respiration of the microorganism can be supplied to the cathode electrode (FIG. 7C). Corresponding third aspect). In the third aspect, electrons are supplied from the reducing organic compound to the anode electrode that is electrically connected to the cathode electrode via the microorganism. Examples of microorganisms that use an electrode as the final electron acceptor include Schwanella bacteria and Geobacter bacteria that can exchange electrons with an extracellular solid phase.

Furthermore, if a microorganism (microorganism A ″ in the figure) using the metal humic acid complex as a final electron acceptor is used, electrons can be exchanged using the metal humic acid complex also on the anode side (FIG. 7th (d) corresponding 4th aspect). In this fourth aspect, the reducing organic compound and the reducing microorganism that reduces the metal humic acid complex by receiving electrons from the reducing organic compound with respect to the anode electrode that is electrically connected to the cathode electrode. Electrons are supplied by the electron donating means comprising the combination. Examples of microorganisms that use the metal humic acid complex as the final electron acceptor include Schwanella bacteria and Geobacter bacteria that can exchange electrons with the extracellular solid phase.

The third mode (FIG. 7 (c)) and the fourth mode (FIG. 7 (d)) take out microorganisms from organic compounds and flow them through an electric circuit, and are so-called “microbial fuel cells”. become.

It should be noted that chemical reduction (for example, reduction with hydrogen gas / palladium catalyst or sodium borohydride) can be used as an electron donating means. However, the above-described microbial reduction (first aspect) and electrochemical reduction (second aspect to fourth aspect) are preferable from the viewpoint of efficiency and introduction into the groundwater environment.

(3) Microorganism as an electron acceptor (microorganism B in FIG. 7)
In the electron transfer system of the present invention, microorganisms are used as electron acceptors. “Microorganism as an electron acceptor” refers to a microorganism that receives electrons from the outside and uses the received electrons for growth, proliferation, function, and the like. Microorganisms as electron acceptors include, for example, Geobacter bacteria, Schwanella bacteria, Dehalobacter bacteria, Desulfitobacterium bacteria, Desulfromonas bacteria, Clostridium bacteria, Bacteroides bacteria, Desulfofibrio bacteria, Cedimenti Bacterium bacteria and sulfurospirillum bacteria are used. Two or more kinds of microorganisms may be used in combination. Moreover, you may decide to use the microorganism group thru | or microbial community containing one or more types of microorganisms which function as an electron acceptor. The microorganism group or microbial community here may contain a microorganism that does not function as an electron acceptor.

Preferably, a microorganism that can exchange electrons with an extracellular solid phase is used. According to the microorganism, respiration using electrons directly received from outside the cell becomes possible. Examples of such microorganisms include the genus Shuwanella and the genus Geobacter.

On the other hand, if a dehalogenated microorganism is employed, an electron transmission system suitable for dehalogenation of the environment such as soil, groundwater, river water, lake water, seawater and the like can be obtained. Examples of dehalogenated microorganisms are Desulfomonile tiedjei DCB-1 strain, Dehalospirillum Dehalospirillum [Sulfurospirillum] multivorans, Dehalobacter restrictus PER-K23 strain (Dehalobacter restrictus PER-K23), Dehalobacter restrictusTETEA strain (Dehalobacter restrictus TEA), Dehalobacter sp. FTH1 strain (Dehalobacter sp. FTH1) Desulfitobacterium . PCE1 strain (Desulfitobacterium sp. PCE1), Desulfitobacterium sp. PCE-S strain (Desulfitobacterium sp. PCE-S), Desulfitobacterium flapieri TCE1 strain (Desulfitobacterium frappieri TCE1), Desulfitobacterium sp. Y51 Strain (Desulfitobacterium sp. Y51) Desulfuromonas chloroethenica TT4B (Desulfuromonas chloroethenica TT4B), Clostridium bifermentans DPH-1 (Clostridium bifermentans DPH-1), Dehalococcoides イ maccartyi 195 (Dehalococcoides maccartyi 195), Dehalococcoide GT Strain (Dehalococcoides sp. GT), Dehalococcoides sp. VS strain (Dehalococcoides sp. VS) (Damborsky, J. (1999): Tetrachloroethene-dehalogenating bacteria, Folia Microbiologica, Vol.44, pp.247-262 .; Holliger, C., Whlfarth, G. and Diekert, G. (1999): Reductive dechlorination in the energy metabolism of anaerobic bacteria, FEMS Microbiology Reviews, Vol.22, pp.383-398 .; Gerritse, J. Renard, V. Pedro Gomes, TM, Lawson, PA, Collins, MD and Gottschal, JC (1996): Desulfitobacterium sp. Strain PCE1, tetrachloroethene or ortho-chlorinated phenols. Archives of Microbiology, Vol.165, pp.132-140 .; Suyama, A., Iwakiri, R., Kai, K., Tokunaga, T., Sera, N. and Furukawa, K . (2001): Isolation and characterization of Desulfitobacterium sp. Strain Y51 capable of efficient dehalogenation of tetrachloroethene and polychloroethanes, Bioscience, Biotechnology, and Biochemistry, Vol.65, pp.1474-1481 .; Chang, YC, Hatsu, Jung, K., Yoo, YS and Takamizawa, K. (2000) Isolation and characterization of a tetrachloroethylene dechlorinating bacterium, ClostridiumtribifermentansmentDPH-1. Journal of Bioscience and Bioengineering, Vol.89, pp.489-491-491. Gatell, X., Chien, Y., Gossett, JM and Zinder, SH (1997): Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene, Science, Vol.276, pp.1568-1571 .; Maymo-Gatell, X ., Anguish, T. and Zinder, SH (1999): Reductive dec hlorination of chlorinated ethenes and 1, 2-dichloroethane by "Dehalococcoides ethenogenes" 195, Applied and Environmental Microbiology, Vol.65, pp.3108-3113 .; Youlboong Sung, 1, Kirsti M. Ritalahti, 1 Robertah and Frank E. Loffler1, (2006): Appl. Environ. Microbiol., vol. 72, pp. 1980-1987; Muller, J. A., B. M. Rosner, G. von Abendroth, G. Meshulam-Simon , P. L. McCarty, and A. M. Spormann: Molecular identification of the catabolic vinyl chloride reductase from Dehalococcoides sp. Strain VS and its environmental distribution (2005): Appl. Environ.9 -50 .; Yoshida, N. Ye, L., Baba, D. and Katayama, A .: A Novel Dehalobacter sp. Is involved in extensive 4,5,6,7-tetrachlorophthalide (fthalide) dechlorination (2009): Appl . Environ. Microbiol., 75, 2400-2405; Luijten, MLGC, GCDe Weert, J., Smidt, H., Boschker, HTS, De Vos, WM, Schraa, G. and Stams, AJM (2003): Description of Sulfurospirillum halorespirans sp. nov., an anaerobic, tetrachloroethene-respiring bacterium, Vol.53, pp.787-793.).

If a bacterium having an iron reducing ability (for example, a Geobacter bacterium) is used, the electron transfer system of the present invention can be used to promote iron reduction. Similarly, if a bacterium having nitrate reducing ability (for example, a Schwannella bacterium) is used, the electron transfer system of the present invention can be used to promote nitrate reduction.

When the electron transmission system of the present invention is used for environmental purification, organic acids and microorganisms (those that generate electrons by decomposing organic substances) existing in the environment to be purified can also be used as the electron donating means. In this aspect, the metal humic acid complex as the electron transfer medium and the dehalogenated microorganism as the electron acceptor are applied to the environment to be purified, and the electron transfer system of the present invention is constructed in the environment. It will be. However, it does not interfere with the combined use of electron donating means prepared separately, for example, reducing organic compounds as electron donors, or reducing microorganisms that reduce metal humic acid complexes by receiving electrons from reducing organic compounds Alternatively, both of these may be added to the environment. When the electron transfer system of the present invention is used for environmental purification, it is also one of preferred embodiments to use an electrode (cathode) as an electron donor. In this embodiment, the electrodes are placed in the environment and electrons are donated.

On the other hand, if the electron transfer system of the present invention is used to purify the environment where a dehalogenated microorganism that functions as an electron acceptor exists, the microorganism may be used as a dehalogenated microorganism as an electron acceptor. Good. In this aspect, the metal humic acid complex as the electron transfer medium and the electron donating means are applied to the environment to be purified, and the electron transfer system of the present invention is constructed in the environment (however, It does not prevent the combined use of microorganisms as electron acceptors prepared separately).

In addition, if both a substance that functions as an electron donating means (organic acid, microorganism, etc.) and a dehalogenated microorganism that functions as an electron acceptor exist in the environment, a metal humic acid complex as an electron transfer medium It is also possible to construct the electronic transmission system of the present invention in the environment using only

2. Applications of electronic transmission system (environmental purification method and environmental purification kit)
The second aspect of the present invention relates to an environmental purification method that is a typical application of the electron transmission system of the present invention. The environment purification method of the present invention uses the electronic transmission system of the present invention. More specifically, the electron transfer system of the present invention is constructed in a polluted environment, and organic halogen compounds that are pollutants are decomposed and removed by the action of dehalogenated microorganisms as electron acceptors. Examples of contaminated environments include soil, sediment, groundwater, river water, lake water, seawater, drainage / drainage (life drainage, industrial wastewater, industrial wastewater, etc.).

Organic halogen compound is a general term for organic compounds containing halogen atoms in the molecule. Organochlorine compounds such as pentachlorophenol (PCP), tetrachloroethylene, trichloroethylene, dichloroethylene, trichloroethane, vinyl chloride, carbon tetrachloride, chloroethane, methylene chloride, chloroform, dichloroethane, chloropropane, polychlorinated biphenyl (PCB), dioxins, poly Organic halogen compounds include organic bromine compounds such as brominated diphenyl ether (PBDE), bromodichloromethane, chlorodibromomethane, and bromoform, and organic fluorine compounds such as fluorocarbons, chlorofluorocarbons, and perfluorocarboxylic acids. Dioxins are a general term for polychlorinated dibenzopararadixin (PCDD) and polychlorinated dibenzofuran (PCDF). One of the PCBs, coplanar PCB (Co-PCB), is also classified as a dioxin.

Pentachlorophenol (PCP) is a highly persistent chlorinated organic pollutant found extensively in soil, sediment and aquatic systems. In the past, PCP was widely used as a fungicide, herbicide and wood preservative, but its use was prohibited because it is highly toxic to the liver and kidneys and increases the risk of cancer (Reference 8). PCP is still classified as an important pollutant and is listed on the US and World Health Organization (WHO) list of drinking water standards (Refs. 9, 10).

In the environmental purification method of the present invention, necessary elements are added to the contaminated environment so that the above-described electron transmission system is constructed and functions in the contaminated environment. For example, the “addition” can be performed by charging, spreading, coating, mixing, or the like. Typically, a metal humic acid complex as an electron transfer medium, an electron donating means, and a dehalogenated microorganism as an electron acceptor are added to a contaminated environment, and the electron transfer system of the present invention is constructed in the contaminated environment. . However, as described above, when there is an electron donating means in the environment to be purified, only two elements of a metal humic acid complex as an electron transfer medium and a dehalogenated microorganism as an electron acceptor are added. You may decide. However, it does not interfere with the combined use of electron donating means prepared separately, for example, reducing organic compounds as electron donors, or reducing microorganisms that reduce metal humic acid complexes by receiving electrons from reducing organic compounds Alternatively, both of these may be added to the environment. On the other hand, when a dehalogenated microorganism that functions as an electron acceptor exists in the environment to be purified, a metal humic acid complex as an electron transfer medium and an electron donating means (for example, a reducing organic compound and a reducing organic compound). You may decide to add only two elements of the combination of the reducing microorganisms which receives an electron donation from a compound and reduces a metal humic acid complex. Furthermore, if there are substances or microorganisms that function as electron donors and dehalogenated microorganisms that function as electron acceptors in the environment to be purified, only the metal humic acid complex as an electron transfer medium is added. You may decide to do it.

In order to maintain the effect (that is, environmental purification) of the environmental purification method of the present invention or to improve efficiency, one or more elements may be replenished periodically or as needed. Normally, the electron donor in the environment decreases as the environment is purified by the environmental purification method of the present invention. Thus, the electron donor is a particularly desirable element to be replenished. In addition, dehalogenated microorganisms as electron acceptors are also susceptible to the influence of the surroundings, and thus can be said to be an element to be replenished.

The present invention further provides a kit (environment purification kit) used for the environment purification method. In one embodiment of the environmental purification kit of the present invention, a metal humic acid complex as an electron transfer medium and a reducing organic compound as an electron donor (for example, sugar, amino acid, organic acid, organic acid salt, alcohol, aroma Group compound, one or more organic compounds selected from the group consisting of reducing organic substances such as biodegradable plastics). In another aspect, the metal humic acid complex as an electron transfer medium, a reducing organic compound as an electron donating means, and the metal humic acid complex is reduced by receiving electrons from the reducing organic compound. And a combination of reducing microorganisms. The kit of the further embodiment includes a metal humic acid complex as an electron transfer medium and an electrode (cathode) as an electron donating means. A dehalogenated microorganism as an electron acceptor may be included in the kit of the present invention. In addition, various substances useful for maintaining and growing dehalogenated microorganisms as electron acceptors (minerals, vitamins, carbon sources, reducing agents, etc.), various substances useful for maintaining quality (eg preservatives), etc. It may be included in the purification kit. By using the environmental purification kit, the environmental purification method of the present invention can be easily carried out. In the environmental purification kit, each element is usually applied to the purification target simultaneously or sequentially. Preferably, all of the kit elements are applied simultaneously. “Simultaneous” here does not require strict simultaneity. Therefore, not only when each element is applied under the condition that there is no time difference, such as applying each element after being mixed, but after applying one element, another element is applied immediately. Application under conditions without a substantial time difference is also included in the concept of “simultaneous” herein.

3. Applications of electron transfer system (selective culture method for microorganisms)
The present invention provides a selective culture method of microorganisms as a further aspect. The method of the present invention can be used to accumulate or isolate specific microorganisms from a sample containing microorganisms. It can also be used to stably culture and maintain a specific microorganism or a group of microorganisms containing it. As supported by the experiments shown in Examples described later, the metal humic acid complex can exhibit a specific redox potential depending on its configuration. In the present invention, this characteristic is used to selectively culture a microorganism having a high affinity for a specific redox potential. As a specific operation, in the method of the present invention, a microorganism-containing sample is cultured in the presence of a metal humic acid complex and an electron donating means. Typically, a sample containing the target microorganism or a sample expected to have the target microorganism is used as the microorganism-containing sample. As long as this condition is satisfied, various samples such as various soils (for example, fields, paddy fields, forests, etc.), river water, lake water, river sediments, marine sediments, and the like can be used. A sample obtained by one or more treatments (for example, removal of a specific component, culture, etc.) can also be used.

Depending on the purpose, the metal constituting the metal humic acid complex (for example, transition metals such as manganese, iron, cobalt, etc. and magnesium, zinc, selenium) may be selected. In other words, if the metal constituting the metal humic acid complex is changed and the oxidation-reduction potential of the metal humic acid complex is changed, another type of microorganism can be selectively cultured. Thus, the method of the present invention is applicable to selective culture of various microorganisms.

One of the preferred electron donating means is an electrode (cathode), but at the same time, an electron donor such as sugar, amino acid, organic acid, organic acid salt, alcohol, aromatic compound, biodegradable plastic can be used. When an electrode is used, the metal humic acid complex may be fixed to the electrode surface (for example, a part of the electrode may be covered with the metal humic acid complex), and the method of the present invention may be carried out. According to such an aspect, it becomes possible to selectively culture a specific microorganism in a state of being accumulated on the electrode. If two or more kinds of metal humic acid complexes having different oxidation-reduction potentials are used in combination, two or more kinds of microorganisms can be selectively cultured at the same time. The culture temperature is, for example, 10 ° C to 50 ° C. The culture period is, for example, 2 days to 3 months. You may subculture during culture | cultivation. It is not necessary to unify the culture conditions throughout the entire culture process.

It is shown below that the insoluble iron humic acid complex functions as a solid phase electron carrier for anaerobic microbial dechlorination of pentachlorophenol (PCP).

1. Materials and Methods (1) Manufacturing Method of Iron Humic Acid Complex (Iron Humic Acid Complex) To synthesize iron humic acid complex (iron humic acid complex), first Aldrich Humic Acid (AHA, Aldrich Chemical Company, USA) ) Was dissolved in ultrapure water to a concentration of 1.5 g / L. Centrifuged at 15,000 g for 15 minutes to remove insoluble parts. 5 mM ferrous sulfate was added to the collected supernatant (purified humic acid) and stirred simultaneously with mixing. The pH was adjusted to 7.0 and held at 30 ° C. for 1 week. Subsequently, this solution was centrifuged at 15,000 g for 15 minutes, and the resulting precipitate, ie, iron humic acid complex, was lyophilized. A control sample (control humic acid) prepared using the same procedure using only AHA and a sample prepared using the same procedure using only ferrous sulfate (control iron) were prepared.

On the other hand, an environmental sample (environmental humic acid) extracted from Kamashima paddy soil (KM) and Yatomi paddy soil (YA) (extraction method is based on the conventional alkali method) is mixed with ferrous sulfate to form an iron humic acid complex. Synthesis was performed. The formed composites were called Fe-KMHA and Fe-YAHA.

(2) Iron fraction responsible for the electron-mediated function In order to provide information on the iron phase contained in the iron humic acid complex and to investigate the iron fraction responsible for the electron-mediated function, sequential extraction of iron was performed. . Three different types of extractants shown below were used.
(i) MgCl ₂ (1 M, pH = 7): Remove the iron fraction exchangeable with iron humic acid complex (ii) Sodium acetate (1 M, adjusted to pH 5 with acetic acid): acid soluble fraction (Iii) Alkaline solution (0.1M NaOH or Na ₄ P ₂ O ₇ ): Removes iron fraction bound to organic matter

Fractionation due to differences in elution of iron contained in iron humic acid complex According to the procedure shown in Table 1, a sequential extraction method using 20 mL of extractant and 50 mg of lyophilized iron humic acid complex ( 3 steps) were performed at room temperature. After each treatment, the extract was separated by centrifugation. The iron fraction was measured for each supernatant using an inductively coupled plasma emission spectrometer (ICP-AES). The total iron content of the iron humic acid complex was analyzed by ICP-AES after treatment with perchloric acid and nitric acid.

(3) Preparation of iron humic acid complex subjected to partial iron extraction treatment The above extractant was used for partial iron extraction. After adding 0.5 g of iron humic acid complex to 100 mL of the extractant, the mixture was shaken at 150 rpm for 24 hours, and then collected by centrifugation (15,000 g, 10 minutes). This extraction process was repeated twice, and the collected precipitate was washed with distilled water until neutralized and then lyophilized. The extracted iron humic acid complex is divided into MgCl ₂ -treated iron humic acid complex, NaOAc-treated iron humic acid complex, NaOH-treated iron humic acid complex and Na ₄ P ₂ O _7- treated iron, respectively. It was named humic acid complex.

(4) PCP-phenol dechlorination experiment An anaerobic PCP-phenol dechlorination humin culture was used as an inoculum. The culture is originally a culture that consists of anaerobic microorganisms that are soil-dependent and dechlorinates PCP to phenol, and later maintained a dechlorination activity using a humin suspension instead of soil. Subcultured 5% at a time. Anaerobic Humin Suspension Medium is a 20 ml mineral salt medium supplemented with 0.3 g lyophilized humin, 10 mM formic acid filtered through a 0.2 μm pore size filter, vitamin solution and 20 μM PCP. Before inoculation with nitrogen gas. PCP-phenol dechlorinated humin culture by transferring the culture at 5% inoculum to a container containing 20 mL inorganic medium and 5 g L ^-1 humin suspension plus 10 mM formic acid and 20 μM PCP As an anaerobic microorganism group that dechlorinates PCP to phenol (Reference 15). The culture time was 10 to 25 days, and static culture was performed at 30 ° C. The composition of the inorganic medium (L ^-1 ) is as follows: 1.0 g NH ₄ Cl; 0.05 g CaCl ₂ × 2H ₂ O; 0.1 g MgCl ₂ × 6H ₂ O; 0.4 g K ₂ HPO ₄ ; Elemental SL-10 solution (reference 20); 1 mL Se / W solution (reference 20); 15 mM MOPS (3- (N-morpholino) propanesulfonic acid) buffer (pH 7.2). PCP and its metabolites were analyzed using a gas chromatograph mass spectrometer QP2010 (Shimadzu, Kyoto) (DB-5MS capillary column, J & W Science, Folsom, CA) (Reference 21).

The effects of iron humic acid complex and control samples on microbial PCP-phenol dechlorination activity were investigated by culture experiments. A sample of iron humic acid complex and control iron was added to the medium as a lyophilized powder at a concentration of 2.5 g L ⁻¹ and autoclaved. For control HA, 10 mL of sample was added to a bottle containing 10 mL of medium and autoclaved. The effect on dechlorination was determined after at least two generations of culture. All experiments were performed in duplicate or triplicate and were repeated at least three times to confirm the results of each experiment.

(5) Adsorption experiment PCP and phenol were added at a concentration of 5 μM, 10 μM or 20 μM to a 60 mL bottle containing 20 mL inorganic medium with or without iron humic acid complex added (2.5 g / L). The cells were cultured for 5 days under the same conditions as in the culture experiment. The iron humic acid complex was removed by centrifugation, and the concentrations of PCP and phenol in the supernatant were analyzed.

(6) Microbial reduction of iron (III) oxide The effect of iron humic acid complex on the microbial reduction of iron (III) oxide was performed using Shewanella putrefaciens CN-32 It was. The S. putrefaciens CN-32 strain was cultured using Tryptic soy broth (Becton Dickinson, Sparks, USA) liquid medium at 30 ° C. under aerobic conditions, and washed and collected using a centrifuge. The iron (III) oxide reduction experiment was carried out using 30 mL of NaHCO ₃ buffer (30 mM, pH 6.8), iron humic acid complex (2.5 g L ⁻¹ ), lactic acid (20 mM) and synthesized iron (III ) Oxide (10 mM) was added, washed S. putrefaciens CN-32 strain (10 ⁹ cells mL ⁻¹ ) was inoculated, and the reduced iron (II) concentration was compared. As a control experiment, two control samples were added: a control sample inoculated with S. putrefaciens strain CN-32 without addition of iron humic acid complex with addition of iron (III) oxide, and iron (III) A control sample was prepared by inoculating the S. putrefaciens CN-32 strain with the addition of the iron humic acid complex with no oxide added. Iron (III) oxide was synthesized by a method reported in the past (Reference 22). The medium was made oxygen-free by ventilating a mixed gas of N ₂ / CO ₂ (80% / 20%).

(7) Electron-accepting ability experiment To calculate the electron-accepting ability (EAC) of the iron humic acid complex, microbial and chemical reduction assays were performed in an anaerobic chamber according to a previously reported protocol (Reference 2). 23). In the microbial reduction assay, 30 mL of NaHCO ₃ buffer (30 mM, pH 6.8) supplemented with lactate (20 mM) and synthesized iron (III) oxide (10 mM) was added with iron humic acid complex (2.5 g L ^-1) or a control sample (i.e. control iron (2.5 g L ^-1) or control HA (1.5 g L ^-1)), and the mixture was washed thereto S. putrefaciens CN-32 strain (10 ⁹ cells mL ^{- 1} ) was inoculated. For chemical reduction assays, 5 palladium-coated aluminum oxide pellets were placed in 10 mM PIPES buffer (pH 6.8) and a 2.5 g L ^-1 sample (1.5 g for control HA). L ^-1) was added, under 100% H ₂ atmosphere while shaking at 0.99 rpm, and incubated for 5 days.

To quantify the amount of electrons required to microbially or chemically reduce the iron humic acid complex, 1 ml of the sample suspension was mixed with 2 ml of 10 mM iron (III) -nitrilotriacetic acid. Reacted for more than a minute. This reaction solution is filtered through a 0.22 μm membrane filter, and 0.5 ml of the filtrate is added to 5 ml ferrozine solution (dissolved in 1 mM L ^-1 in 50 mM HEPES buffer and adjusted to pH = 7 with NaOH). The color reaction was performed, and detection and quantification were performed at a wavelength of 562 nm. The difference between the final concentration of iron (II) ions (after reduction) and the initial concentration (before reduction) was defined as EAC (e ^- equivalent (Eq) g ^-1 dry iron humic acid complex) of the sample .

(8) Electrochemical analysis For electrochemical experiments, a potentiostat (HSV-110, Hokuto Denko, Osaka, Japan) equipped with a bioelectrochemical cell was used, and the three-electrode method (graphite working electrode (5 mm x 15 cm) , Tokai Carbon, Tokyo, Japan); measured with platinum counter electrode (0.8 mm x 1 m, Niraco, Tokyo, Japan) and Ag / AgCl reference electrode (saturated potassium chloride HX-R8, Hokuto Denko, Osaka)) . The bioelectrochemical cell consisted of two chambers separated by an effective volume of 200 mL for each chamber and a proton exchange membrane (Nafion 117, DuPont, USA). The measurement conditions for cyclic voltammetry are: potential sweep rate 100 mV s ^-1 , iron humic acid complex concentration 2.5 g L ^-1 , potential range -0.8 V to 0.6 V (vs. Ag / AgCl), inorganic medium as electrolyte Using. One sample of the iron humic acid complex was measured after electrochemical reduction by applying for 18 hours at a potential of -500 mV (vs. standard hydrogen electrode) (electrolytic solution was inorganic medium).

(9) Characterization of iron humic acid complex For structural analysis and analysis of electron-mediated properties, elemental analysis (C, H, N, ash) and functional group analysis (Fourier transform infrared absorption spectrum (FTIR spectrum), electron Spin resonance spectrum and cyclic voltammogram) were used.

(10) Microbial reduction of nitrate The effect of iron humic acid complex on microbial reduction of nitrate (NO ³⁻ (nitrate) → NO ²⁻ (nitrite) → NH ⁴⁺ (ammonium)) was examined. The experimental method was the same as the above (4) (the same experimental method was used except that 5 mM nitrate was used instead of 20 μM PCP).

2. Results (1) Physicochemical properties of iron humic acid complex Purified AHA contains about 52.8% carbon, about 3.9% hydrogen, about 1.0% nitrogen, and about 0.8% ash. On the other hand, the iron humic acid complex contained about 23.2% carbon, about 2.8% hydrogen, about 0.4% nitrogen and about 33.6% ash. ICP-AES analysis showed that the iron humic acid complex had an iron content of 149.6 ± 13.8 g kg ⁻¹ . The results from sequential extraction show that the iron humic acid complex has 4.6% exchangeable iron fraction, 9.6% acid soluble fraction, 52.7% iron fraction combined with organic matter and poorly available (poorly available) It was revealed that the iron fraction contained 33.1%. 0.50-0.65 g insoluble iron humic acid complex (as freeze-dried powder) was obtained by complexing 1.39 g iron sulfate with 1.5 g purified AHA in aqueous solution. The recovery rate was 17.3% -22.5%.

Based on an average recovery of 20.0%, it is expected that 0.58 g iron humic acid complex (86.8 mg iron content) will be obtained from 1.39 g iron sulfate (280 mg iron content). This means that 31.0% of the iron ions were utilized to form the iron humic acid complex.

(2) Effect of iron humic acid complex on microbial dechlorination of PCP Humin extracted from soil as a solid phase fraction is a very stable electron with respect to microbial dehalogenation of PCP and tetrabromobisphenol A (TBBPA) Previously reported to show transmission ability (Refs. 15, 30). The ability to mediate soluble forms of humic acid extracted from the same soil was unstable and dehalogenation activity was lost after subculture. The insoluble iron humic acid complex was synthesized by a complex formation reaction between ferrous sulfate and humic acid, and showed stable electron transfer activity related to PCP-phenol dechlorination activity of microorganisms (Fig. 1a). On the other hand, PCP dechlorination activity was not observed in AHA (Fig. 1c). It is considered that AHA does not have the electron transfer capacity necessary for PCP dechlorination reaction of PCP-phenol dechlorinated humin culture.

The stable electron transfer properties of the iron humic acid complex were confirmed by the stable passage of PCP dechlorination activity of PCP-phenol dechlorinated humin cultures in experiments using different types of iron humic acid complexes. It was done. In the absence of PCP-phenol dechlorination humin culture or in the absence of iron humic acid complex, no dechlorination reaction was observed, PCP dechlorination activity was due to microorganisms, It can be said that a humic acid complex is necessary (Fig. 1b). PCP dechlorination was not observed when control iron or control HA samples were used (Figure 1c). Iron humic acid complexes prepared using naturally occurring humic acids, namely Fe-KMHA and Fe-YAHA, were also shown to mediate PCP dechlorination by microorganisms. These results indicate that the solid iron humic acid complex plays an important role in maintaining PCP dechlorination by microorganisms.

In the adsorption experiment, the PCP concentration decreased by 15% due to adsorption to the iron humic acid complex, but phenol was not adsorbed to the iron humic acid complex at all. No metabolites were observed in the adsorption experiments, indicating that the adsorption of PCP to the iron humic acid complex was negligible and no chemical reaction occurred between the two compounds (FIG. 1d).

(3) Iron fraction responsible for electron-mediated function of iron humic acid complex Complex with ferrous sulfate to activate and stabilize humic acid electron transfer characteristics in microbial reductive dechlorination of PCP An insoluble iron humic acid complex was prepared by formation. The effects of iron fraction removal on iron humic acid complex and changes in the ratio of iron fraction were suggested, suggesting that iron humic acid complex plays an important role in microbial reductive PCP dechlorination. .

PCP-phenol dechlorination even after removal of exchangeable iron fraction (MgCl ₂ treated iron humic acid complex) or acid soluble iron fraction (NaOAc treated iron humic acid complex) (Figures 2a and 2b) It did not affect PCP-dechlorination activity of humin cultures. Even in the NaOH-treated iron humic acid complex, which is supposed to remove the iron fraction bound to organic matter, the electron-mediated activity in the PCP dechlorination reaction of the above culture (Fig. 2c) was not lost. The activity became unstable in the iron humic acid complex treated with Na ₄ P ₂ O ₇ which was supposed to remove the bound iron fraction. Dechlorination activity was shown only in the first generation, or no dechlorination was observed (Figure 2d).

The ash content of the iron humic acid complex (37.3%) was reduced to 19.1% for the NaOH-treated iron humic acid complex and 10.6% for the Na ₄ P ₂ O _7- treated iron humic acid complex. The carbon content (21.3%) of the iron humic acid complex increased to 55.6% for the NaOH-treated iron humic acid complex and 59.1% for the Na ₄ P ₂ O _7- treated iron humic acid complex.

(4) Effect of iron humic acid complex on microbial reduction of iron (III) oxide Insoluble iron (III) oxide by S. putrefaciens CN-32 strain with and without iron humic acid complex The time course of microbial reduction is shown in FIG. Although the concentration of water-soluble iron (II) ions resulting from the reduction increased with time, small amounts of iron (II) ions were also present in the bottles without cell inoculation, with or without the addition of iron humic acid complex. Was detected. When the iron humic acid complex (2.5 g / L) was added, the microbial reduction of iron (III) oxide was promoted compared to the case where the culture was not added. A control sample without addition of iron (III) oxide, whereas iron (II) ions as high as 5.1 mM were detected when iron humic acid complex and S. putrefaciens CN-32 were added. Then, only 1.7 mM iron (II) ions were reduced from the iron humic acid complex to iron. The difference 3.4 mM in the reduced dissolved iron (II) ion concentration was 1.9 times higher than the concentration difference (1.8 mM) at the end of the culture in the condition where the iron humic acid complex was not added. It was shown that the microbial reduction of iron (III) oxide was promoted by the iron humic acid complex.

(5) Electroaccepting ability of iron humic acid complex Electron accepting ability (EAC) reduces iron humic acid complex to microbial (S. putrefaciens CN-32 strain) and chemical (H ₂ / Pd) ( 5 days), the electrical equivalent was measured and evaluated (Fig. 4). In the microbial reduction experiment, the EAC value reached 0.98 mEq / g-dry weight. In the microbial reduction experiment, the EAC value of the control iron was 0.14 mEq / g-dry weight, and the EAC value of the control humic acid was 0.10 mEq / g-dry weight (FIG. 4a). On the other hand, when the iron humic acid complex was chemically reduced, the EAC value was 0.36 mEq / g dry (FIG. 4b). In the chemical reduction experiment, the EAC value of control iron was 0.13 mEq / g-dry weight, and the control humic acid was 0.07 mEq / g-dry weight.

(6) Fourier transform infrared absorption spectrum (FTIR spectrum)
The FTIR spectrum of the iron humic acid complex (Figure 5a) showed a clear difference compared to AHA and control iron (Figure 5b). AHA showed a spectral band typical of humic substances. When AHA is complexed with ferrous sulfate, a clear change is observed at wavelengths between 1750 and 1200 cm ^-1 , with several sharp new peaks at 1735 cm ^-1 , 1700 cm ^-1 , 1235 cm ^-1 And small peaks appeared at 1500-1377 cm ⁻¹ , 1605-1500 cm ⁻¹ and 1735-1605 cm ⁻¹ . On the other hand, in the Na ₄ P ₂ O _7- treated iron humic acid complex that showed unstable mediated activity in the microbial PCP dechlorination reaction, the peak in the range of 1750 to 1200 cm ⁻¹ was reduced or eliminated.

(7) Cyclic voltammetric properties of the iron humic acid complex The electrochemical reduction characterized the redox properties of the iron humic acid complex. The analysis results are shown in FIG. The cyclic voltammograms (CV) of both the iron humic acid complex (FIG. 6a) and the control humic acid (control-HA, FIG. 6b) showed no obvious peaks. In general, humic substances and metal complexes thereof lack electrode activity (Reference Document 24), and thus no clear peak is often observed in CV. However, a clear peak was obtained in the CV of the iron humic acid complex after electrical reduction at -500 mV (vs standard hydrogen electrode) for 18 hours. From the peak potential, the redox potential of the iron humic acid complex was estimated to be 0.10 V (versus the standard hydrogen electrode), whereas the redox potential of control iron was estimated to be 0.08 V. On the other hand, even when electrical reduction was performed, irregularly shaped CV was observed in the Na ₄ P ₂ O _7- treated iron humic acid complex (a redox _couple could not be observed) (FIG. 6C).

(8) Effect of iron humic acid complex on microbial reduction of nitrate When iron humic acid complex was present, nitrate began to be rapidly metabolized to nitrous acid, and nitrous acid was metabolized to ammonium (Fig. 8). ). On the other hand, in the absence of the iron humic acid complex, the metabolism of nitrate to nitrous acid was slow, and the metabolism of nitrous acid to ammonium was very small (Fig. 8).

3. Discussion As a result of the above experiment, it was discovered that a stable microbial PCP dechlorination reaction can be realized by using an insoluble iron humic acid complex (by complex formation of ferrous sulfate and commercial humic acid) ( Figure 1a). Moreover, it was shown that the complex prepared using humic acid which exists naturally, ie, Fe-KMHA and Fe-YAHA, also has the microbial dechlorination activity of PCP (a result is not shown). In other words, solid-phase iron humic acid complex was effective in maintaining PCP dechlorination in various environments. Furthermore, it was proved that the iron humic acid complex also promotes the microbial reduction of iron (III) oxide (Fig. 3), suggesting the existence of various redox transmission functions in the iron humic acid complex. Further studies showed that the iron humic acid complex promotes not only microbial PCB dechlorination, microbial reduction of iron, but also microbial reduction of nitrate (Figure 8).

The electron-accepting ability test revealed that the iron humic acid complex has an electric capacity that can be reduced microbially or chemically, that is, an electron-accepting capacity (FIG. 4). The electron accepting capacity of the microbial redox active moiety was more than 2.5 times the electron accepting capacity of the chemical redox active moiety. Moreover, it was shown that the iron humic acid complex has an electron accepting capacity of 0.98 mEq / g-dry weight higher than the electron accepting capacity reported for dissolved humic substances (Ref. 25) (FIG. 4a).

According to the results of sequential extraction experiments, the iron humic acid complex has an exchangeable iron fraction of 4.6%, an acid soluble fraction of 9.6%, an iron fraction bound to organic matter of 52.7% and an iron fraction of hardly extracted 33.1%. It became clear that it contained. Probably humic acid and hydrolysable iron fractions (highly hydrated iron ions, eg Fe _n (OH) _m (H ₂ O) _x ^{(3n-m) +} or Fe _m O _n (OH) _x ^{(3m The formation} of crystalline iron oxide is suppressed by the interaction of ^{-2n-x) +} ), and it is considered that low crystalline iron oxide such as ferrihydrite was formed (Reference Document 26). Activity was observed in the iron humic acid complex even after treatment with 1 M MgCl ₂ , 1 M sodium acetate and 0.1 M NaOH (Figure 2a, Figure 2b, Figure 2c). It was suggested that the iron humic acid complex has a stable active ingredient. On the other hand, in the Na ₄ P ₂ O _7- treated iron humic acid complex, a mediating effect on PCP dechlorination activity was observed only in the first generation, or no dechlorination was observed (FIG. 2d), It was shown that the electron-mediated ability is unstable. Na ₄ P ₂ O ₇ is much more capable of extracting organic-bound iron fractions from samples containing amorphous or imperfect crystalline iron oxyhydroxides such as ferrihydrite than NaOH and aqueous acetylacetone solutions. (References 27 and 28). The results of the iron humic acid complex treated with Na ₄ P ₂ O ₇ showed much lower ash (10.6%) than the sample treated with NaOH (19.1%) are supported by this report (Ref. 27, 28). Therefore, it was suggested that the redox-mediated function of the iron humic acid complex is due to the organic matter-bound iron fraction eluted by Na ₄ P ₂ O ₇ or the iron fraction with low crystallinity.

In the FTIR spectrum of the iron humic acid complex (Fig. 5a), a clear change is observed at a wavelength of 1735 to 1377 cm ^-1 , and peaks due to carboxylic acid groups and phenolic hydroxyl groups involved in complex formation with iron are observed. Has been obtained (FIG. 5a). On the other hand, some sharp new peak 1735 cm ^{^-1,} 1700 cm ^-1, found at 1235 cm ^-1, C = O stretching vibration of the carboxyl group, COO ^- of symmetric stretching and CO stretching vibration of phenolic hydroxyl group Was confirmed (reference document 29). In the Na ₄ P ₂ O ₇ treated iron humic acid complex, no peak was observed in the wave number range of 1750 to 1200 cm ⁻¹ .

From the measurement result by cyclic voltammetry, which is an electrochemical analysis method, it was revealed that a redox active site exists in the iron humic acid complex (FIG. 6a). The redox potential of the iron humic acid complex was estimated to be 0.10 V (vs. standard hydrogen electrode). On the other hand, since no redox _couple could be observed in the cyclic voltammogram of the Na ₄ P ₂ O _7- treated iron humic acid complex, the iron fraction responsible for the electron-mediated function was treated by Na ₄ P ₂ O ₇ treatment. It is considered as an organic matter-bound iron fraction to be extracted or an iron fraction with low crystallinity.

As described above, it has been clarified that the insoluble iron humic acid complex has a stable electron-mediated function in the microbial dechlorination reaction of PCP, the microbial reduction reaction of iron, and the microbial reduction of nitrate. The reaction center responsible for this electron-mediated function is considered to be an organic matter-bound iron fraction extractable with Na ₄ P ₂ O ₇ or an iron fraction with low crystallinity. Further research is necessary to clarify the chemical reality of the site responsible for the electron-mediated function of the iron humic acid complex.

The metal humic acid complex acts as an electron transfer medium (electron acceptor and deliverer) that mediates surplus electrons generated in oxidation-reduction reactions such as decomposition of organic substances by microorganisms. The metal humic acid complex can maintain an effect as an electron transfer medium for respiratory growth of microorganisms for a long period of time at the site where it is added. A system using a metal humic acid complex as an electron transfer medium (electron transfer system) can be applied to bioremediation.

Recently, research has been actively conducted on “microbial fuel cells” that collect electric electrons generated in redox reactions in organic substance decomposition by microorganisms to obtain electrical energy, and “microbe electroculture” that grows microorganisms using electrical energy. ing. The system of the present invention can also be applied to microbial fuel cells and microbial electroculture devices. By selecting the metal at the active center of the metal humic acid complex, it is possible to produce an electron transfer medium having a specific redox potential. If such an electron transfer medium is used, it can be expected that specific types of microorganisms can be selectively cultured.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

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Claims

An electron transfer system comprising a metal humic acid complex as an electron transfer medium, an electron donating means, and a microorganism as an electron acceptor.
The electron transfer system according to claim 1, wherein the metal constituting the metal humic acid complex is a transition metal, magnesium, zinc or selenium.
The electron transfer system according to claim 2, wherein the transition metal is manganese, iron, or cobalt.
The electron transfer system according to claim 1, wherein the metal constituting the metal humic acid complex is a metal having a plurality of redox potentials.
The electron donating means comprises a combination of a reducing organic compound and a reducing microorganism that receives electrons from the reducing organic compound and reduces the metal humic acid complex. The electronic transmission system according to Item.
6. The reducing organic compound is one or more organic compounds selected from the group consisting of sugars, amino acids, organic acids, organic acid salts, alcohols, aromatic compounds, and biodegradable plastics. Electronic transmission system.
The electron transmission system according to any one of claims 1 to 4, wherein the electron donating means is a cathode electrode.
The electron transfer system according to claim 7, wherein electrons are supplied from a reducing organic compound to the anode electrode electrically connected to the cathode electrode through a microorganism.
Electron donation comprising a combination of a reducing organic compound and a reducing microorganism that reduces the metal humic acid complex by receiving electrons from the reducing organic compound to the anode electrode electrically connected to the cathode electrode 8. The electron transmission system according to claim 7, wherein electrons are supplied by the means.
The electron transfer system according to any one of claims 1 to 9, wherein the microorganism as an electron acceptor is a dehalogenated microorganism, a bacterium having an iron reducing ability, or a bacterium having a nitrate reducing ability.
The electron transfer system according to any one of claims 1 to 9, wherein the microorganism as an electron acceptor is a microorganism that can exchange electrons with an extracellular solid phase.
The microorganism as an electron acceptor is a Geobacter bacterium, a Schwanella bacterium, a Dehalobacter bacterium, a Desulfitobacterium genus, a Desulfromonas bacterium, a Clostridium bacterium, a Bacteroides bacterium, a Desulfobrybacteria bacterium, a Sedientibacter genus The electron transfer system according to any one of claims 1 to 9, which is one or more bacteria selected from the group consisting of bacteria and sulfurospirillum bacteria.
An environmental purification method comprising decomposing an organic halogen compound in a polluted environment by the electron transmission system according to claim 10.
An environmental purification method comprising the step of adding a metal humic acid complex to a contaminated environment.
The environmental purification method according to claim 14, further comprising a step of adding a reducing organic compound as an electron donor to the contaminated environment.
Furthermore, the electron donating means includes a step of adding a reducing organic compound and a reducing microorganism that receives the electron donation from the reducing organic compound and reduces the metal humic acid complex to a contaminated environment. The environmental purification method as described.
Furthermore, the environmental purification method of Claim 14 including the step which installs the cathode electrode as an electron donor means in a contaminated environment.
The environmental purification method according to any one of claims 14 to 17, further comprising a step of adding a dehalogenated microorganism as an electron acceptor to the contaminated environment.
An environmental purification kit comprising a metal humic acid complex as an electron transfer medium and a reducing organic compound as an electron donor.
A combination of a metal humic acid complex as an electron transfer medium, a reducing organic compound as an electron donating means, and a reducing microorganism that receives the electron donation from the reducing organic compound and reduces the metal humic acid complex. A kit for environmental purification.
An environmental purification kit including a metal humic acid complex as an electron transfer medium and a cathode electrode.
The environmental purification kit according to any one of claims 19 to 21, further comprising a dehalogenated microorganism as an electron acceptor.
A method for selectively culturing microorganisms, comprising culturing a microorganism-containing sample in the presence of a metal humic acid complex and an electron donor.
An electron transfer medium consisting of a metal humic acid complex.