WO2007078089A1 - Bioelectrochemical process for denitrification using permeabilized microorganism - Google Patents
Bioelectrochemical process for denitrification using permeabilized microorganism Download PDFInfo
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- WO2007078089A1 WO2007078089A1 PCT/KR2006/005780 KR2006005780W WO2007078089A1 WO 2007078089 A1 WO2007078089 A1 WO 2007078089A1 KR 2006005780 W KR2006005780 W KR 2006005780W WO 2007078089 A1 WO2007078089 A1 WO 2007078089A1
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- microorganism
- denitrification
- microorganisms
- permeabilized
- organic solvent
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- 244000005700 microbiome Species 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 48
- 239000003960 organic solvent Substances 0.000 claims abstract description 24
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 claims description 31
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 239000006163 transport media Substances 0.000 claims description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 claims description 6
- 241000588814 Ochrobactrum anthropi Species 0.000 claims description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 4
- 241000589597 Paracoccus denitrificans Species 0.000 claims description 4
- 241000589516 Pseudomonas Species 0.000 claims description 4
- LUAZZOXZPVVGSO-UHFFFAOYSA-N Benzyl viologen Chemical compound C=1C=C(C=2C=C[N+](CC=3C=CC=CC=3)=CC=2)C=C[N+]=1CC1=CC=CC=C1 LUAZZOXZPVVGSO-UHFFFAOYSA-N 0.000 claims description 2
- PGWTYMLATMNCCZ-UHFFFAOYSA-M azure A Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 PGWTYMLATMNCCZ-UHFFFAOYSA-M 0.000 claims description 2
- FIKAKWIAUPDISJ-UHFFFAOYSA-L paraquat dichloride Chemical compound [Cl-].[Cl-].C1=C[N+](C)=CC=C1C1=CC=[N+](C)C=C1 FIKAKWIAUPDISJ-UHFFFAOYSA-L 0.000 claims description 2
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 10
- 229910052799 carbon Inorganic materials 0.000 abstract description 10
- 108090000790 Enzymes Proteins 0.000 abstract description 7
- 102000004190 Enzymes Human genes 0.000 abstract description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 45
- 229910052757 nitrogen Inorganic materials 0.000 description 20
- 230000000052 comparative effect Effects 0.000 description 12
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 10
- 229910002651 NO3 Inorganic materials 0.000 description 9
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 9
- 108090000913 Nitrate Reductases Proteins 0.000 description 7
- 239000008055 phosphate buffer solution Substances 0.000 description 7
- 230000002829 reductive effect Effects 0.000 description 7
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 description 6
- 238000011109 contamination Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000002351 wastewater Substances 0.000 description 6
- 229910001873 dinitrogen Inorganic materials 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000010865 sewage Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000010170 biological method Methods 0.000 description 3
- 230000029058 respiratory gaseous exchange Effects 0.000 description 3
- 239000010802 sludge Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108010054147 Hemoglobins Proteins 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 238000010924 continuous production Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000008823 permeabilization Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 206010011705 Cyanosis neonatal Diseases 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 108010061951 Methemoglobin Proteins 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 229910021607 Silver chloride Inorganic materials 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000012851 eutrophication Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 125000003473 lipid group Chemical group 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/005—Combined electrochemical biological processes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/46—Treatment of water, waste water, or sewage by electrochemical methods
- C02F1/461—Treatment of water, waste water, or sewage by electrochemical methods by electrolysis
- C02F1/467—Treatment of water, waste water, or sewage by electrochemical methods by electrolysis by electrochemical disinfection; by electrooxydation or by electroreduction
- C02F1/4676—Treatment of water, waste water, or sewage by electrochemical methods by electrolysis by electrochemical disinfection; by electrooxydation or by electroreduction by electroreduction
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/341—Consortia of bacteria
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/163—Nitrates
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Definitions
- the present invention relates to a method for bioelectrochemical denitrification using permeabilized microorganisms, specifically to a method for removing nitrogen in bioelectrochemical way without supply of external carbon sources, by applying electric energy from outside in the presence of a live-catalyst, microorganism which has been membrane-permeabilized by treatment with an organic solvent, and thus the enzyme activity thereof is maximized while the microorganism lacks viability, wherein the applied electric energy activates the enzymes in the permeabilized microorganism.
- nitrate nitrogen can be fatal to a baby under 6 months at the concentration of more than 10 ppm, although it is relatively harmless to an adult since when it is entered into the human body, it can be rapidly removed through the kidneys. This is because the nitrite which is reduced from the entered nitrate (NO ) reacts with hemoglobin in the blood to form methemoglobin which is not capable of delivering oxygen to cell tissues, unlike hemoglobin, finally leading to a blue baby syndrome. Further, the conversion of nitrate to nitrite in the human body involves the formation of nitrosoamine which is well-known carcinogen.
- Such nitrate nitrogen is contained, especially in sewage and waste water at high concentration.
- Nitrate waste water is mainly generated and discharged from semiconductor, electric and chemical industry and fertilizer producing factory wherein nitric acid is used as a solvent or a cleanser.
- various methods including chemical, physical, biological method or the like have been researched, and some methods are practiced in industrial sites.
- most of physical/chemical methods are to substitute nitrate nitrogen to its concentrated form, which in turn, requires a secondary post- treatment process. When such post-treatment is not sufficiently conducted, it has a risk of causing further contamination.
- nitrate nitrogen is a nitrogen source or as a medium for respiration in anaerobic condition.
- microorganisms breathe, using nitrate nitrogen as the final electron recipient, instead of oxygen, which is referred as nitrate nitrogen respiration.
- nitrate nitrogen respiration a series of processes (NO " ⁇ NO " ⁇ NO ⁇ N O ⁇ N ) in which nitrate nitrogen is consequently converted, via nitrite nitrogen (nitrite), to nitrogen gas through continuous reactions, is called denitrification.
- denitrification In the first step of denitrification wherein nitrate nitrogen is converted to nitrite nitrogen, nitrate reductase is involved.
- an activated sludge process is mainly favored owing to high process stability and reliability, relatively easy process operation, low cost as compared to that of physical/chemical methods.
- the present invention has been developed to solve the problems of conventional techniques.
- the object of the present invention is to provide a highly efficient method for bioelectrochemical denitrification in which nitrate nitrogen in the course of denitrification is completely reduced to a nitrogen gas, not just reaching to the nitrite nitrogen state.
- the method according to the present invention is economic since it does not require continuous supply of external carbon sources; can prevent secondary con- tamination caused by the external carbon sources or excessive growth of microorganisms; and does not require high cost for equipment installation and wide sighting.
- the present invention relates to a method for bioelectrochemical denitrification using microorganisms with denitrification capability, characterized by applying electric energy to the reaction system comprised of nitrate nitrogen, membrane-per- meabilized microorganisms which are obtained by treatment with an organic solvent, and an electron transport medium so as to reduce the nitrate nitrogen.
- the microorganisms permeabilized by the organic solvent treatment are those obtained by treating the microorganisms with denitrification capability with an organic solvent, wherein the microorganisms with denitrification capability comprise Ochrobactrum anthropi SY509, Paracoccus denitrificans, Pseudomonas stutseri or the like.
- Ochrobactrum anthropi SY509 is preferably used.
- the microorganisms permeabilized by the organic solvent treatment are those obtained by preferably, treating the microorganisms with one or more organic solvent selected from the group consisting of chloroform, acetone, ethanol, toluene, isopropyl alcohol and dimethyl sulfoxide.
- the permeabilization of microorganisms by the organic solvent treatment can be carried out by, for example: placing microorganisms to be treated into a phosphate buffer; adding an organic solvent thereto; allowing the mixture to stand for reaction in a low temperature reaction bath at about 4?; after completing the reaction, centrifuging the resulted product; and washing the separated microorganisms with a phosphate buffer solution.
- such denitrification capability is realized by a nitrate reductase that is present in the membrane of a microorganism, wherein the enzyme is in the same type of cytochrome reductase and present at the last stage of an electron transport system, finally receiving the electron which has been delivered through various passes in the membrane of a microorganism so as to reduce the nitrate nitrogen to nitrite nitrogen. From such reductive reaction, the microorganism can form electron gradient force and thus produce the energy source, ATP.
- the electron transport medium is a material which serves to transport electrons to the nitrate reductase inside the permeabilized microorganisms obtained by the organic solvent treatment, wherein the electrons are introduced into a reaction system by the electric energy applied to the reaction system where the denitrification process is conducted.
- the electron transport medium which can be used in the present invention is one or more selected from the group consisting of neutral red, methyl viologen, benzyl viologen, azure A and safranine O, and particularly, among them, neutral red is preferred.
- the electron transport medium can be used at the amount of 0.05-lmmol per Ig of the permeabilized microorganism obtained by the organic solvent treatment.
- the amount of the electron transport medium per Ig of the permeabilized microorganism is less than 0.05mmol, the effect of removing nitrogen is not sufficient.
- the amount is more than lmmol, improvement in the efficiency of removing nitrogen is insignificant, regarding the excessive amount of use, leading to decrease in efficiency of the whole process.
- the method for denitrification according to the present invention uses an electron transport medium for which is intentionally added to the reaction system helping the electron transportation. By the electron transport medium, the electrons can be effectively transported to the enzyme.
- At least one of the permeabilized microorganisms obtained by the organic solvent treatment and the electron transport medium can be used as being immobilized to a supporter, for reusing the permeabilized microorganisms and the electron transport medium, or for constituting the denitrification process as a continuous process.
- the method for denitrification according to one embodiment of the present invention can be carried out, for example, as follows.
- a permeabilized microorganism with denitrification capability which is permeabilized by an organic solvent treatment.
- the permeabilized microorganism can be obtained by commercially purchasing such microorganism, or alternatively by treating a microorganism with denitrification capability with an organic solvent for permeabilization, as mentioned above.
- the permeabilized microorganism and an electron transport medium are added to a reaction system comprising a phosphate buffer solution.
- contaminated materials comprising nitrate nitrogen are added, and then electric energy is applied to the reaction system for reducing nitrate nitrogen.
- either of permeabilized microorganism obtained by the organic solvent treatment or electron transport medium, or both of them can be added to the reaction system as an immobilized form to a carbon supporter or the like, for recycling or in a continuous process.
- Fig. 1 is a plot illustrating a decreasing tendency in concentration of nitrate nitrogen(NO -N) as well as the tendency of remaining nitrite nitrogen(NO -N), as the function of time, in the example 1 according to the present invention.
- Fig. 2 is a plot illustrating a decreasing tendency in concentration of nitrate nitrogen (NO -N) and the tendency of remaining nitrite nitrogen(NO -N), as the function of time, in the comparative example 1 according to the present invention.
- Ochrobactrum anthropi SY509 (Example 1); Paracoccus denitrificans (Example 2); and Pseudomonas stutseri(Examp ⁇ e 3).
- O.lg of each microorganism was added to 10ml of a phosphate buffer, then 0.05ml of chloroform as an organic solvent was added thereto, and the mixture was placed in a reaction bath at 4 0 C for 15 minutes for reaction.
- the resulted product was centrifuged to separate the microorganisms, which is then washed 3 times with a phosphate buffer solution, resulting in the permeabilized microorganism obtained by organic solvent treatment, which is to be used in bioelectrochemical denitrification.
- the permeabilized microorganisms obtained from above were diluted with a phosphate buffer solution to make the microorganism concentration of 15g/l.
- Ten ml of the diluted solution, in which the amount of microorganism was 0.15g, and 3ml of 2OmM neutral red solution, in which the amount of neutral red was O.O ⁇ mmol were placed into a 50ml volume electrochemical reactor containing 30ml of a phosphate buffer solution. Thereto, 140ppm of nitrate nitrogen was added, and electric energy was applied to carry out a nitrate nitrogen reduction.
- a carbon felt with the area of 2.0cm x 2.5cm was used as a working electrode; Pt wire was used as the counter electrode; and Ag/ AgCl electrode was used as a reference electrode.
- Ochrobactrum anthropi SY509 (Comparative example 1); Paracoccus denitrificans(Co ⁇ &m ⁇ e example 2); and Pseudomonas stutseri (Comparative example 3).
- Each microorganism was diluted with a phosphate buffer solution to make the microorganism concentration of 15g/l.
- 10ml of the diluted solution and 3ml of 2OmM neutral red solution were placed into a 50ml volume electrochemical reactor containing 30ml of a phosphate buffer solution.
- 140ppm of nitrate nitrogen was added, and the reduction of nitrate nitrogen was carried out by the same method as in the examples 1-3.
- each examples 1-3 according to the present invention which used permeabilized microorganism obtained by the organic solvent treatment to remove nitrate nitrogen showed better denitrification property, owing to the easier accessibility of substrates to the microorganism, as compared to the comparative example 1-3 which used living intact microorganisms.
- Table 2 it can be found that all of the nitrate nitrogen which is primarily reduced in a deni- trification process was completely converted, via nitrite nitrogen, to a nitrogen gas in example 1.
- comparative example 1 it was shown that a significant amount of nitrate nitrogen which is primarily reduced in a denitrification process was reduced to only nitrite nitrogen.
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Hydrology & Water Resources (AREA)
- Organic Chemistry (AREA)
- Water Supply & Treatment (AREA)
- Environmental & Geological Engineering (AREA)
- Engineering & Computer Science (AREA)
- Biodiversity & Conservation Biology (AREA)
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Abstract
The present invention provides a method for bioelectrochemical denitrification using permeabilized microorganisms. Particularly, the present invention provides a method for bioelectrochemical denitrification, without supply of external carbon sources by applying electric energy from outside, in the presence of microorganisms which have been membrane-permeabilized by treating with an organic solvent, wherein the applied electric energy activates the enzymes in the permeabilized microorganisms.
Description
Description
BIOELECTROCHEMICAL PROCESS FOR DENITRIFICATION USING PERMEABILIZED MICROORGANISM
Technical Field
[1] The present invention relates to a method for bioelectrochemical denitrification using permeabilized microorganisms, specifically to a method for removing nitrogen in bioelectrochemical way without supply of external carbon sources, by applying electric energy from outside in the presence of a live-catalyst, microorganism which has been membrane-permeabilized by treatment with an organic solvent, and thus the enzyme activity thereof is maximized while the microorganism lacks viability, wherein the applied electric energy activates the enzymes in the permeabilized microorganism. Background Art
[2] The effect of civilizational development accompanied with industrialization and mass-consumption on the environment gets bigger and serious, resulting in increasing attention to the protection of water quality. Among various causes of environmental contamination, nitrogen contained in sewage or waste water causes various problems such as contamination in water supply sources and acceleration of eutrophication in a lake or water reservoir, which further leads to damages in fish farms. Therefore, the nitrogen concentration in sewage or waste water has been regulated by government, and such regulation is getting stricter.
[3] Among such environmentally hazardous nitrogen, nitrate nitrogen can be fatal to a baby under 6 months at the concentration of more than 10 ppm, although it is relatively harmless to an adult since when it is entered into the human body, it can be rapidly removed through the kidneys. This is because the nitrite which is reduced from the entered nitrate (NO ) reacts with hemoglobin in the blood to form methemoglobin which is not capable of delivering oxygen to cell tissues, unlike hemoglobin, finally leading to a blue baby syndrome. Further, the conversion of nitrate to nitrite in the human body involves the formation of nitrosoamine which is well-known carcinogen.
[4] Such nitrate nitrogen is contained, especially in sewage and waste water at high concentration. Nitrate waste water is mainly generated and discharged from semiconductor, electric and chemical industry and fertilizer producing factory wherein nitric acid is used as a solvent or a cleanser. In order to remove such nitrate nitrogen from the waste water, various methods including chemical, physical, biological method or the like have been researched, and some methods are practiced in industrial sites. However, most of physical/chemical methods are to substitute nitrate nitrogen to its concentrated form, which in turn, requires a secondary post- treatment process. When
such post-treatment is not sufficiently conducted, it has a risk of causing further contamination.
[5] Many microorganisms use nitrate nitrogen as a nitrogen source or as a medium for respiration in anaerobic condition. In anaerobic condition, microorganisms breathe, using nitrate nitrogen as the final electron recipient, instead of oxygen, which is referred as nitrate nitrogen respiration. After the nitrate nitrogen respiration, a series of processes (NO " → NO " → NO → N O → N ) in which nitrate nitrogen is consequently converted, via nitrite nitrogen (nitrite), to nitrogen gas through continuous reactions, is called denitrification. In the first step of denitrification wherein nitrate nitrogen is converted to nitrite nitrogen, nitrate reductase is involved. Therefore, biological methods for converting nitrate nitrogen to harmless nitrogen gas by using microorganisms recently come into the spotlight. As for an example of such biological methods, an activated sludge process is mainly favored owing to high process stability and reliability, relatively easy process operation, low cost as compared to that of physical/chemical methods.
[6]
Disclosure of Invention Technical Problem
[7] In recent years, various methods for improving the efficiency of a denitrification process as above described method using microorganisms have been investigated. As a result of such investigation, various processes such as A2O, UTS, Bardenpho or the like have been suggested to improve the efficiency of the above-mentioned activated sludge process. However, most of these suggested processes still mainly utilize activated sludge, hence the whole denitrification efficiency is generally low, and they require continuous supply of external carbon sources for maintaining the activity of microorganisms during the denitrification process. Such continuous supply of external carbon sources may cause secondary contamination owing to the carbon sources themselves and excessive growth of microorganisms, let alone economic disadvantage. Further, it also has a limit that it requires high cost for equipment installation and wide sighting. Technical Solution
[8] The present invention has been developed to solve the problems of conventional techniques. The object of the present invention is to provide a highly efficient method for bioelectrochemical denitrification in which nitrate nitrogen in the course of denitrification is completely reduced to a nitrogen gas, not just reaching to the nitrite nitrogen state. The method according to the present invention is economic since it does not require continuous supply of external carbon sources; can prevent secondary con-
tamination caused by the external carbon sources or excessive growth of microorganisms; and does not require high cost for equipment installation and wide sighting.
[9] The present invention relates to a method for bioelectrochemical denitrification using microorganisms with denitrification capability, characterized by applying electric energy to the reaction system comprised of nitrate nitrogen, membrane-per- meabilized microorganisms which are obtained by treatment with an organic solvent, and an electron transport medium so as to reduce the nitrate nitrogen.
[10] According to the method for denitrification of the present invention, the microorganisms permeabilized by the organic solvent treatment are those obtained by treating the microorganisms with denitrification capability with an organic solvent, wherein the microorganisms with denitrification capability comprise Ochrobactrum anthropi SY509, Paracoccus denitrificans, Pseudomonas stutseri or the like. Among the said microorganisms, Ochrobactrum anthropi SY509 is preferably used.
[11] According to the method for denitrification of the present invention, the microorganisms permeabilized by the organic solvent treatment are those obtained by preferably, treating the microorganisms with one or more organic solvent selected from the group consisting of chloroform, acetone, ethanol, toluene, isopropyl alcohol and dimethyl sulfoxide. The permeabilization of microorganisms by the organic solvent treatment can be carried out by, for example: placing microorganisms to be treated into a phosphate buffer; adding an organic solvent thereto; allowing the mixture to stand for reaction in a low temperature reaction bath at about 4?; after completing the reaction, centrifuging the resulted product; and washing the separated microorganisms with a phosphate buffer solution.
[12] In the microorganisms with denitrification capability, such denitrification capability is realized by a nitrate reductase that is present in the membrane of a microorganism, wherein the enzyme is in the same type of cytochrome reductase and present at the last stage of an electron transport system, finally receiving the electron which has been delivered through various passes in the membrane of a microorganism so as to reduce the nitrate nitrogen to nitrite nitrogen. From such reductive reaction, the microorganism can form electron gradient force and thus produce the energy source, ATP.
[13] When using living microorganisms wherein the denitrification process is done by passing through the foregoing complex internal procedures, in denitrification process, it is advantageous in terms of cost, processability, stability or the like, as compared to the denitrification carried out by using a purified nitrate reductase. However, there are also some problems of using living microorganisms in denitrification such that substrates cannot be sufficiently moved into the microorganisms, and external carbon
sources should be continuously supplied to maintain the microorganisms alive.
[14] However, according to the method for denitrification of the present invention, it is possible to solve such conventional problems by using membrane-permeabilized microorganisms which are obtained by an organic solvent treatment. The organic solvent treatment makes some loss in the lipid part of the microorganism membrane, making the microorganism membrane thin. In this state, the biological activity of the microorganism per se is lost, however the activity of a nitrate reductase which is present inside the thin microorganism membrane is still maintained. Then, the access of substrates to the enzyme becomes facilitated through such thin cytoplasmic membrane, and supply of external carbons sources is not necessary since the microorganisms are in biologically inactivated state. Further, as it is described later, the denitrification process becomes more efficient since electrons can be directly transferred to the enzymes through an electron transport medium.
[15] In the method for denitrification of the present invention, the electron transport medium is a material which serves to transport electrons to the nitrate reductase inside the permeabilized microorganisms obtained by the organic solvent treatment, wherein the electrons are introduced into a reaction system by the electric energy applied to the reaction system where the denitrification process is conducted. The electron transport medium which can be used in the present invention is one or more selected from the group consisting of neutral red, methyl viologen, benzyl viologen, azure A and safranine O, and particularly, among them, neutral red is preferred.
[16] According to the method for denitrification of the present invention, the electron transport medium can be used at the amount of 0.05-lmmol per Ig of the permeabilized microorganism obtained by the organic solvent treatment. When the amount of the electron transport medium per Ig of the permeabilized microorganism is less than 0.05mmol, the effect of removing nitrogen is not sufficient. When the amount is more than lmmol, improvement in the efficiency of removing nitrogen is insignificant, regarding the excessive amount of use, leading to decrease in efficiency of the whole process.
[17] For the nitrate reductase in microorganisms to function properly, electrons should be received from NADH which is an electron donor. However, when the enzymatic reaction is processed by using permeabilized microorganisms obtained by the organic solvent treatment and applying electric energy from outside, as in the present invention, there would be a problem that direct electron transportation cannot be occurred between the nitrate reductase in the permeabilized microorganism and the device for applying electric energy. Therefore, in order to solve such problem, the method for denitrification according to the present invention uses an electron transport medium for which is intentionally added to the reaction system helping the electron
transportation. By the electron transport medium, the electrons can be effectively transported to the enzyme.
[18] In the meantime, in the method for denitrification according to the present invention, at least one of the permeabilized microorganisms obtained by the organic solvent treatment and the electron transport medium can be used as being immobilized to a supporter, for reusing the permeabilized microorganisms and the electron transport medium, or for constituting the denitrification process as a continuous process.
[19] The method for denitrification according to one embodiment of the present invention can be carried out, for example, as follows.
[20] First to be done is to prepare a permeabilized microorganism with denitrification capability, which is permeabilized by an organic solvent treatment. Depending on the species, the permeabilized microorganism can be obtained by commercially purchasing such microorganism, or alternatively by treating a microorganism with denitrification capability with an organic solvent for permeabilization, as mentioned above. Then, the permeabilized microorganism and an electron transport medium are added to a reaction system comprising a phosphate buffer solution. Thereto, contaminated materials comprising nitrate nitrogen are added, and then electric energy is applied to the reaction system for reducing nitrate nitrogen.
[21] In the above description, either of permeabilized microorganism obtained by the organic solvent treatment or electron transport medium, or both of them can be added to the reaction system as an immobilized form to a carbon supporter or the like, for recycling or in a continuous process.
[22]
Brief Description of the Drawings
[23] Fig. 1 is a plot illustrating a decreasing tendency in concentration of nitrate nitrogen(NO -N) as well as the tendency of remaining nitrite nitrogen(NO -N), as the function of time, in the example 1 according to the present invention.
[24] Fig. 2 is a plot illustrating a decreasing tendency in concentration of nitrate nitrogen (NO -N) and the tendency of remaining nitrite nitrogen(NO -N), as the function of time, in the comparative example 1 according to the present invention.
[25]
Mode for the Invention
[26] Hereinafter, the present invention is further described in detail through examples and comparative examples, without any intention of limiting the scope of the present invention with such examples and comparative examples.
[27]
[28] Examples
[29] Examples 1-3
[30] Three different microorganisms having denitrification capability were used in the examples 1-3: Ochrobactrum anthropi SY509(Example 1); Paracoccus denitrificans (Example 2); and Pseudomonas stutseri(Examp\e 3). O.lg of each microorganism was added to 10ml of a phosphate buffer, then 0.05ml of chloroform as an organic solvent was added thereto, and the mixture was placed in a reaction bath at 40C for 15 minutes for reaction. After completing the reaction, the resulted product was centrifuged to separate the microorganisms, which is then washed 3 times with a phosphate buffer solution, resulting in the permeabilized microorganism obtained by organic solvent treatment, which is to be used in bioelectrochemical denitrification.
[31] Then, the permeabilized microorganisms obtained from above were diluted with a phosphate buffer solution to make the microorganism concentration of 15g/l. Ten ml of the diluted solution, in which the amount of microorganism was 0.15g, and 3ml of 2OmM neutral red solution, in which the amount of neutral red was O.Oβmmol were placed into a 50ml volume electrochemical reactor containing 30ml of a phosphate buffer solution. Thereto, 140ppm of nitrate nitrogen was added, and electric energy was applied to carry out a nitrate nitrogen reduction. In the electrochemical reduction, a carbon felt with the area of 2.0cm x 2.5cm was used as a working electrode; Pt wire was used as the counter electrode; and Ag/ AgCl electrode was used as a reference electrode.
[32] During the reduction of nitrate nitrogen, a part of the solution was taken from the reactor as a sample, at each time when 30 minutes, 60 minutes, 90 minutes and 120 minutes were elapsed from the start of the reaction. The concentration of nitrate nitrogen(NO -N) in each sample for the examples 1-3, was determined, and the results were represented in the following Table 1. Further, the concentration of nitrite nitrogen(NO -N) in the sample of the example 1 was determined, and the result was represented in the following Table 2. Further, regarding the example 1, the resulted concentrations of nitrate nitrogen(NO -N) and nitrite nitrogen(NO -N) were represented in Fig. 1.
[33]
[34] Comparative examples 1-3
[35] Three different microorganisms having denitrification capability were used in the comparative examples 1-3: Ochrobactrum anthropi SY509(Comparative example 1); Paracoccus denitrificans(Coπφ&mύγe example 2); and Pseudomonas stutseri (Comparative example 3). Each microorganism was diluted with a phosphate buffer solution to make the microorganism concentration of 15g/l. 10ml of the diluted solution and 3ml of 2OmM neutral red solution were placed into a 50ml volume electrochemical reactor containing 30ml of a phosphate buffer solution. Thereto, 140ppm
of nitrate nitrogen was added, and the reduction of nitrate nitrogen was carried out by the same method as in the examples 1-3.
[36] During the reduction of nitrate nitrogen, samples were taken from the reactor as described in the above Examples, and then the concentration of nitrate nitrogen(NO - N) in each sample for the comparative examples 1-3 was determined. The results were represented in the following Table 1. Further, the concentration of nitrite nitrogen(NO -N) in the sample of the comparative example 1 was determined, and the result was represented in the following Table 2. Further, regarding the comparative example 1, the resulted concentrations of nitrate nitrogen(NO -N) and nitrite nitrogen(NO -N) were represented in Fig. 2.
[37] Table 1 Table 1. Concentration of nitrate nitrogen(NO -N) in samples (ppm)
[38] [39] [40] Table 2 Table 2. Concentration of nitrite nitrogen(NO -N) in samples (ppm)
[41] [42] From Table 1, it can be recognized that each examples 1-3 according to the present invention which used permeabilized microorganism obtained by the organic solvent treatment to remove nitrate nitrogen, showed better denitrification property, owing to the easier accessibility of substrates to the microorganism, as compared to the
comparative example 1-3 which used living intact microorganisms. From Table 2, it can be found that all of the nitrate nitrogen which is primarily reduced in a deni- trification process was completely converted, via nitrite nitrogen, to a nitrogen gas in example 1. However, in comparative example 1, it was shown that a significant amount of nitrate nitrogen which is primarily reduced in a denitrification process was reduced to only nitrite nitrogen.
[43]
Industrial Applicability
[44] As described so far, according to the present invention, it is possible to economically remove nitrate nitrogen at high efficiency without a risk of secondary contamination, by completely and securely converting all the nitrate nitrogen which is a primarily reduced state in the course of denitrification and general contaminant contained in sewage or waste water to nitrogen gas.
[45]
Claims
[1] A method for bioelectrochemical denitrification using microorganism with deni- trification capability, characterized by the method comprises applying electric energy to the reaction system comprising nitrate nitrogen, a membrane -per- meabilized microorganism which is obtained by treatment with an organic solvent, and an electron transport medium so as to reduce the nitrate nitrogen.
[2] The method for bioelectrochemical denitrification according to claim 1, wherein the membrane-permeabilized microorganism is those obtained by treating Ochrobactrum anthropi SY509, Paracoccus denitrificans or Pseudomonas stutseri with an organic solvent.
[3] The method for bioelectrochemical denitrification according to claim 1, wherein the organic solvent is one or more selected from the group consisting of chloroform, acetone, ethanol, toluene, isopropyl alcohol and dimethyl sulfoxide.
[4] The method for bioelectrochemical denitrification according to claim 1, wherein the electron transport medium is one or more selected from the group consisting of neutral red, methyl viologen, benzyl viologen, azure A and safranine O.
[5] The method for bioelectrochemical denitrification according to claim 1, wherein the amount of the electron transport medium is 0.05-lmmol per Ig of the membrane-permeabilized microorganism.
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