Patent Application
20100279387
The present invention relates to a method for remediation of soil containing a cyanogen compound using microorganism. Specifically, the present invention relates to a method for remediation of soil containing a cyanogen compound using a microorganism capable of degrading a cyanogen compound or using a microorganism capable of degrading a cyanogen compound and a biodegradable chelating agent promoting the elution of a cyanogen compound which is difficult to extract from soil (hereinafter referred to as a “difficult-to-extract” cyanogen compound); and the microorganism used in the remediation method.
The enforcement of Soil Contamination Countermeasures Act in 2003 triggered an increase in the number of the cases of remediation of the soil polluted with a cyanogen compound. With respect to a method for remediation of the polluted soil, bio-remediation using the degrading activity of a microorganism has been studied.
Since a cyanogen compound has the property of forming a difficult-to-extract complex in the presence of metal ions, when a cyanogen compound is contained in the soil, it forms a difficult-to-extract complex due to the metal such as iron which is naturally present in the soil.
In the bio-remediation, it is difficult to efficiently decompose the difficult-to-extract complex with the form as is, and therefore not only the degrading capability of the microorganism but development of technology for solubilizing the difficult-to-extract cyanogen complex is also required to establish an efficient and reliable remediation method.
With respect to the degradation and removal of the difficult-to-extract cyanogen complex in countermeasures against soil contamination with a cyanogen compound, the microorganism belonging to the Fusarium genus having capability of degrading the cyanogen complex has been proposed (see Patent publication No. JP 3685583; Patent Document 1). However, Patent Document 1 only shows degradability of the cyanogen complex in a liquid phase using a mixed solvent and mentions nothing about the remediation performance of the microorganism in the soil.
As a method for efficiently decontaminating the cyanogen compound contained in the soil by bio-remediation, a method for controlling the pH of the contaminated soil under an weakly acid condition using a mineral acid and an organic acid and supplying the nutrient source of sugars except the nitrogen source (see JP-A-2007-75670; Patent Publication 2). However, Patent Document 2 mentions nothing about the efficiency of degrading the difficult-to-extract cyanogen complex.
In addition, as a method taking in a viewpoint of remediation by solubilizing the cyanogen complex, a method of adding water containing dissolved oxygen, NOx-N (nitrite-nitrogen and nitrate-nitrogen) and the like to the polluted soil containing a cyanogen compound and the bivalent iron ions; oxidizing the bivalent iron ions to trivalent iron ions to thereby convert them into soluble complex ions; and degrading the complex using the microorganisms in the soil is known (see JP-A-2006-255572; Patent Document 3). However, as to the remediation performance, Patent Document 3 only describes treatment of the soil in which the total cyanogen concentration is as low as 2 mg/kg (2 ppm), and the remediation capability is not known against the soil having a total cyanogen concentration of several tens of ppm which can be subject to the practical bio-remediation.
For the above reason, there has been demand for establishment of a method which enables efficient and reliable reduction of the cyanogen compound contained in the soil including the difficult-to-extract portions.
Patent Document 2: Laid-open Japanese patent publication No. 2007-75670
Patent Document 3: Laid-open Japanese patent publication No. 2006-255572
Accordingly, an objective of the present invention is to provide a method for remediation of the soil containing a cyanogen compound which enables efficient and reliable reduction of the cyanogen compound contained in the soil including the difficult-to-extract portions.
In addition, another objective of the present invention is to provide the microorganism to be used in the above-mentioned method for remediation of the soil containing a cyanogen compound.
The present inventors intensively studied to solve the above-mentioned problems. As a result, they have found that the cyanogen compound contained in the soil can be effectively and reliably cleansed by using the microorganism which belongs to the genus Arthrobacter which is capable of degrading the cyanogen compound and serves as a degradation-accelerating factor or by using the microorganism which belongs to the genus Arthrobacter and a biodegradable chelating agent which serves as a factor promoting the elution of a hardly water-soluble cyanogen compound; and accomplished the present invention.
That is, the present invention relates to a method for remediation of the soil containing a cyanogen compound described in 1 to 5 and 7 to 8 below and the microorganism used for the remediation method described in 6 below.
1. A method for remediation of a soil containing a difficult-to-extract cyanogen compound comprising adding a biodegradable chelating agent to the soil to thereby make the cyanogen compound elute and degrading the eluted cyanogen compound using the microorganism.
2. The method for remediation of a soil as described in 1 above, wherein the biodegradable chelating agent to be added is one or more of the members among citric acid, gluconic acid, tartaric acid, oxalic acid, succinic acid, carboxymethyl tartronic acid, carboxymethyloxy succinic acid, aspartate diacetic acid, L-glutamic acid diacetic acid and salts thereof.
3. The method for remediation of a soil as described in 2 above, wherein the biodegradable chelating agent to be added is citric acid and/or citrate.
4. The method for remediation of a soil as described in 1 above, wherein the microorganism is the microorganism which belongs to genus Arthrobacter.
5. The method for remediation of a soil as described in 4 above, wherein the microorganism which belongs to genus Arthrobacter is Arthrobacter sp. No. 5 strain (FERM ABP-11019).
6. Arthrobacter sp. No. 5 strain capable of degrading a cyanogen compound (deposited in National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary under Accession No. FERM P-21400 on Oct. 18, 2007 and then internationally deposited in National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary under Accession No. FERM ABP-11019 on Sep. 16, 2008).
7. A method for remediation of the soil containing a cyanogen compound, comprising degrading the cyanogen compound in the soil using the microorganism which belongs to genus Arthrobacter.
8. The method for remediation of the soil as described in 7 above using Arthrobacter sp. No. 5 strain (FERM ABP-11019).
The method for remediation of the soil containing a cyanogen compound of the present invention enables remediation of the cyanogen compound contained in the soil efficiently and reliably.
The present invention is described in more details hereinafter.
Examples of the microorganisms which belong to the genus Arthrobacter used in the method for remediation of the soil of the present invention include Arthrobacter sp. No. 5 strain separated from the soil by using a medium where ferricyanide potassium is the only carbon source and energy source.
The observed morphology and physiological properties of Arthrobacter sp. No. 5 strain are described below:
Morphology: polymorphic bacillus;
Gram's stain: positive;
Spores: asporogenic;
Motility: nonmotile;
Requirement for oxygen: aerobic;
Oxydase: negative;
Catalase: positive;
OF (Oxidation Fermentation) test: the strain did not grow in the test medium;
Colony color: no specific colony pigment was produced.
Arthrobacter sp. No. 5 strain was identified by analyzing the base sequence of the DNA in 16SrRNA region amplified through Polymerase Chain Reaction (PCR) method using ABI PRISM 310 Genetic Analyzer (Applied Bisosystems), comparing the obtained sequence with those registered in the International Nucleotide Sequence Databases (DDBJ/EMBL/GenBank) and in the database of MicroSeq Analysis Software (Applied Biosystems) and furthermore by drawing a family tree among related species by Neighborhood Joining (NJ) method using MicroSeq Analysis Software.
As a result, Arthrobacter sp. No. 5 strain was found to be closest to Arthrobacter oxydans and Arthrobacter polychromogenes showing 0.88% of base sequence diversity among the species.
Arthrobacter oxydans
Arthrobacter polychromogenes
Arthrobacter sulfonivorans
The present strain was named Arthrobacter sp. No. 5 strain and deposited in National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (Center No. 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki 305-8566 JAPAN) under Accession No. FERM P-21400) on Oct. 18, 2007 and then internationally deposited in National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary under Accession No. FERM ABP-11019 on Sep. 16, 2008.
There is no particular limitation on the cultivation of the microorganism used in the present invention as long as it is a well-known method.
As an example of the carbon source of the medium to culture the microorganism, sugars such as glucose, sucrose, fructose and blackstrap molasses can be used singly or in combination thereof at a concentration of from 0.1 w/v % to 30 w/v % generally, preferably from about 1 w/v % to about 10 w/v %.
As an example of the nitrogen source of the culture medium, peptone, meat extract, yeast extract, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, potassium nitrate and urea can be used singly or in combination thereof at a concentration of from 0.1 w/v % to 30 w/v % generally, preferably from about 1 w/v % to about 10 w/v %.
In addition, phosphates such as potassium hydrogen phosphate and potassium dihydrogen phosphate; metal salt such as magnesium sulfate, ferrous sulfate, calcium acetate and manganese chloride; vitamins; amino acids; and nucleic acids, biotin such as thiamin and the like can be added to improve the growth of the bacterium.
The cultivation can be carried out under aerated stirred culture or under aerobic conditions at a temperature of from 20° C. to 40° C., preferably from 25° C. to 35° C. Preferable pH for the culture is in the range from 5 to 10, preferably from 7 to 8, which can be easily adjusted by adding acid or alkali.
The tank used in the cultivation step is not particularly limited as long as it is a fermentation tank capable of mixing and dispersing the bacterium during culture with culture medium components under aeration.
The soil as an object of remediation in the present invention contains a cyanogen compound: i.e. an inorganic cyanogen compound such as iron cyanide complex, copper cyanide complex, nickel cyanide complex, potassium cyanide and sodium cyanide and an organic cyanogen compound having a nitrile group.
When a cyano complex is contained in the soil, it forms a difficult-to-extract precipitate due to the metal such as iron which is naturally present in the soil, resulting in that soluble and difficult-to-extract cyanogen compounds coexist in the soil.
In order to decontaminate cyanogen compounds including difficult-to-extract portions in the present invention, cyanogen determination is conducted not only on the eluting concentration of total cyanide eluted from the soil (i.e. total eluting CN; according to “Measurement method regarding soil eluting test” in Announcement No. 18, 2003 of Ministry of Environment; unit: mg/L; the eluting standard provided by Soil Contamination Countermeasures Act <0.1) and free cyanide content in the soil (i.e. contained free CN; according to “Measurement method regarding soil eluting survey” in Announcement No. 19, 2003 of Ministry of Environment; unit: mg/L; the contained free cyanide standard provided by Soil Contamination Countermeasures Act 50) in the official method provided by Soil Contamination Countermeasures Act in Japan, but also on the concentration of total cyanide content in the soil as a Bottom Sediment Survey method (i.e. total contained CN; according to Bottom Sediment Survey of KANSUIKAN No. 127 bureau notification in 1988; unit: mg/kg). Furthermore, the total difficult-to-extract cyanide concentration in the soil (i.e. difficult-to-extract CN; unit: mg/kg) was also determined by the calculation formula of total contained CN−total eluting CN×9 to use it in evaluation. The calculation formula for the total concentration of difficult-to-extract cyanide is to be determined as the mass per the dry soil (mg/kg). The total eluting CN indicates the concentration in the filtrate of the 10% soil suspension in water. Therefore, the CN in the 90% water is assumed to be contained in the 10% soil suspended in the water, the total eluting CN is multiplied by 9 in the calculation formula to calculate the value per soil for unit conversion.
The goal level of the soil remediation of the present invention is the level which enables removal of the risk that the total eluting CN is detected due to the elution from the total contained CN: i.e. total contained CN<1 mg/kg in numerical terms. The level is based on the fact that the measuring object is the filtrate of the elute at a concentration of 10% soil in water, in which the detection limit is 0.1 mg/l, and therefore it can be concluded that at a condition of “the total contained CN<1 mg/kg, the total eluting CN falls below the detection limit.
The cyanogen concentration contained in the soil as an object of the remediation in the present invention is not particularly limited, and the remediation can be conducted at the total contained CN of 1 mg/kg to 100 mg/kg, which is a concentration in a general bioremediation.
The soil treatment method of the present invention is conducted by adding the microorganism, nutrient source, biodegradable chelating agent and the like to the soil. The addition method to the soil and the implementation scale are not particularly limited and the soil treatment can be carried out by the known method in the field of soil remediation.
The microorganism to be added to the soil may be in the form of the culture solution or diluent with water.
Though the nutrient source and biodegradable chelating agent to be added may be in either form of an aqueous solution or powder, it is desirable to be added in the form of an aqueous solution in consideration of the penetration and diffusion of the components into the soil.
When the nutrient source or biodegradable chelating agent is added in liquid form, it is allowed to leave the soil as it is or to extract the increased water from the soil.
The treatment method is carried out under ambient temperature conditions. However, in the case where the remediation performance is quantitatively measured on a laboratory scale, the treatment can be conducted by placing a soil sample in a constant-temperature bath and the like and thereby controlling the temperature at from 5° C. to 50° C., preferably from 15° C. to 35° C.
Decomposition of a cyanogen compound in the soil can be carried out by adding to the soil the microorganisms having the cyanide degradation bacterium. The microorganism to be used is not particularly limited as long as it is the microorganism having the cyanide degradation activity, but it is preferable to use the microorganism which belongs to Arthrobacter genius, and more preferable to use Arthrobacter sp. No. 5 strain.
There are no particular limitations on the concentration of the microorganism to be added to the soil as long as the concentration allows the expression of the cyanide degradation activity. However, in consideration of taking balance between the diffusion/penetration into the soil and the degradation activity, the concentration is preferably at 106 cells/g to 109 cells/g, more preferably at 107 cells/g to 108 cells/g.
A nutrient source may be added so as to maintain the cell numbers and the degradation activity of the added microorganism. The nutrient source to be added is not particularly limited as long as it can maintain the cell numbers and the degradation activity of the microorganism and, as an example of the carbon source of the medium to culture the microorganism, sugar such as glucose, sucrose, fructose and blackstrap molasses can be used singly or in combination thereof at a concentration of from 0.001 w/w % to 2.0 w/w % generally, preferably from about 0.01 w/w % to about 0.05 w/w %.
As an example of the nitrogen source, peptone, meat extract, yeast extract, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, potassium nitrate and urea can be used singly or in combination thereof at a concentration of from 0.001 w/w % to 2.0 w/w % generally, preferably from about 0.01 w/w % to about 0.05 w/w %.
In addition, phosphates such as potassium hydrogen phosphate and potassium dihydrogen phosphate; metal salt such as magnesium sulfate, ferrous sulfate, calcium acetate and manganese chloride; vitamins; amino acids; and nucleic acids, biotin such as thiamin and the like can be added as needed.
The timing of adding a nutrient source is not particularly limited and a nutrient source may be added separately before adding the microorganism, added concurrently with the microorganism, or added separately after adding the microorganism. The number of times the nutrient source is added is not limited either.
Solubilization of a difficult-to-extract cyanogen compound can be carried out by adding a biodegradable chelating agent. The chelating agent to be used is not particularly limited as long as it is biodegradable. As an example of the chelating agent, citric acid, gluconic acid, tartaric acid, oxalic acid, succinic acid, carboxymethyl tartronic acid, carboxymethyloxy succinic acid, aspartate diacetic acid, L-glutamic acid diacetic acid and salts thereof can be used singly or in combination thereof at a concentration in soil of from 0.001 w/w % to 2.0 w/w % generally, preferably from about 0.01 w/w % to about 0.05 w/w %. The particularly preferable components of the chelating agent are citric acid and salts thereof. The timing of adding a biodegradable chelating agent is not particularly limited and the chelating agent may be added separately before adding the microorganism, added concurrently with the microorganism, or added separately after adding the microorganism. The number of times the chelating agent is added is not limited either. When the cyanide degradation bacteria exists in the soil, the chelating agent may be used without being combined with the addition of the bacteria. However, the most preferable condition is using Arthrobacter sp. No. 5 strain and potassium citrate in combination.
The chelating agent to be used is not particularly limited and may be a free-acid type agent or a metal-salt type agent. In order to keep the pH in the soil neutral, it is also possible to employ a method of using the free-acid type and the metal-salt type chelating agents in combination.
The reason why to restrict the chelating agent to a biodegradable one is to prevent the residue accumulation in the soil and to minimize the environmental impact.
In the soil remediation method of the present invention, the mechanism to realize solubilization of the difficult-to-extract cyanogen compound is attributed to the result that the iron ion, which is a cause for insolubilization of the iron ferrocyanide, for example, which was altered from the soluble potassium ferricyanide due to the bivalent iron ion in the soil, is taken up into the chelating agent.
Hereinafter, the present invention will be explained in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.
After cultivating Arthrobacter sp. No. 5 strain (Accession No. in National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary: FERM ABP-11019) as the microorganism in the agar slant medium of Nutrient Broth (NB) in a test tube of 18 mm diameter at 35° C. for 24 hours, 1 ml of the suspension in which the cultured bacterium was suspended in 10 ml of sterile water was inoculated to 100 ml of the medium shown in Table 3 placed in a 500 ml-volume flask, and subjected to shake culture at 35° C. and 150 rpm for 12 hours. Furthermore, 20 ml of the shake culture solution was divided and inoculated into 2 l of the medium shown in Table 3 placed in a 5 l-volume fermentation tank to thereby carry out the cultivation for 30 hours under conditions at 35° C., 1000 rpm and airflow rate of 1 l/min. (0.5 vvm), while controlling the pH not less than 7 using 15% ammonia water and controlling the glucose concentration within the range from 5 g/l to 50 g/l by intermittently adding 50% glucose fluid.
As a result, 3 l of the culture solution having the number of cells of 1×1011 and turbidity of 105 was obtained. The turbidity was determined by measuring the absorbance at a wavelength of 660 nm using a spectrophotometer (produced by Hitachi, Ltd.; Model U-1800 Ratio Beam Spectrophotometer).
30 ml each of the liquid additives shown in the entries of Conditions 1 to 4 in Table 4 was added to each of soil samples and mixed at Day 0 and Day 20 in the treatment of allowing 1 kg of soil having total contained CN of 40.0 mg/kg, total eluting CN of 2.2 mg/l and total difficult-to-extract CN of 20 mg/kg (specific gravity of 1.7 kg/1; water content of 25 mass %) placed in a 1 l-volume polyethylene container with a lid to stand in a constant-temperature bath at 35° C. for 40 days, and the remediation was observed by measuring total eluting CN, total contained CN and total difficult-to-extract CN of the soil samples.
Results are shown below according to the ranking by soil remediation performance after 40 days.
The most favorable condition was Condition 4 in which all of the cyanogen-degrading bacterium, nutrient source and elution promoter were added. The sample was found to have total contained CN of 0.9 mg/kg, total eluting CN<0.1 mg/l, and total difficult-to-extract CN of 0.9 mg/kg, which passed the standards provided by Soil Contamination Countermeasures Act, and reached a cleanup level such that the risk of the total eluting CN being detected due to the elution from the total contained CN was eliminated. When analyzing the purification tendency, the degradation by the added bacterium and the elution effects by the elution promoter appeared prominently as in
The second-best condition was Condition 2 in which the cyanogen-degrading bacterium and the nutrient source were added. The sample was found to have total contained CN of 15.0 mg/kg, total eluting CN<0.1 mg/l, and total difficult-to-extract CN of 15.0 mg/kg, which passed the standards provided by Soil Contamination Countermeasures Act, but did not reach a cleanup level such that the risk of the total eluting CN being detected due to the elution from the total contained CN was eliminated. When analyzing the purification tendency, the remediation at the initial stage proceeds at a fast pace owing to the degradation effect by the added bacterium as in
The third-best condition was Condition 3 in which the nutrient source and the elution promoter were added. The sample was found to have total contained CN of 22.0 mg/kg, total eluting CN of 2.1 mg/l, and total difficult-to-extract CN of 3.0 mg/kg. Not only the risk of the total eluting CN being detected due to the elution from the total contained CN remained but the total eluting CN was detected and the soil sample did not pass the standards provided by Soil Contamination Countermeasures Act either. When analyzing the purification tendency, since the cyanogen-degrading bacterium is not added in Condition 3, the degradation by the indigenous bacterium in the soil activated by the added nutrient source continues at a slow pace as in
The worst condition was Condition 1 in which only water was added. The sample was found to have total contained CN of 38.0 mg/kg, total eluting CN of 2.1 mg/l, and total difficult-to-extract CN of 19.6 mg/kg and the soil was found hardly cleaned up from the initial state (see
30 ml of the liquid additive shown in the entry of Condition 4 in Table 4 was added to the soil sample and mixed at Day 0 and Day 20 in the treatment of allowing 1 kg of soil having total contained CN of 10.0 mg/kg, total eluting CN of 0.2 mg/l and total difficult-to-extract CN of 8.1 mg/kg (specific gravity of 1.7 kg/1; water content of 25 mass %) placed in a 1 l-volume polyethylene container with a lid to stand in a constant-temperature bath at 15° C. for 40 days, and the remediation was observed by measuring total eluting CN, total contained CN and total difficult-to-extract CN of the soil sample.
As a result, the sample after 40 days was found to have total contained CN of 0.9 mg/kg, total eluting CN<0.1 mg/l and total difficult-to-extract CN of 0.9 mg/kg, which passed the standards provided by Soil Contamination Countermeasures Act, and reached a cleanup level such that the risk of the total eluting CN being detected due to the elution from the total contained CN was eliminated (see
In the treatment of allowing 1 ton (T) of soil having total contained CN of 80.0 mg/kg, total eluting CN of 0.2 mg/l and total difficult-to-extract CN of 39.5 mg/kg (specific gravity of 4.5 kg/1; water content of 30 mass %) placed in a 1 ton-volume container to stand, the operation of pouring 300 l of the liquid additive shown in the entry of Condition 5 in Table 4 to the soil from the top surface of the container and making 300 l of the liquid exhausted and recovered from a bottom valve was carried out in two batches at Day 0 and Day 20; and the remediation was observed by measuring total eluting CN, total contained CN and total difficult-to-extract CN of the soil sample. As a result, the soil sample after 50 days of the treatment was found to have total contained CN of 0.9 mg/kg, total eluting CN<0.1 mg/l, and total difficult-to-extract CN of 0.9 mg/kg, which passed the standards provided by Soil Contamination Countermeasures Act, and reached a cleanup level such that the risk of the total eluting CN being detected due to the elution from the total contained CN was eliminated. Also, CN of the drainage recovered from the bottom valve at passing the liquid was 20 mg/l at the first batch of Day 0, and fell below the detection limit (<0.1 mg/l) at the second batch of Day 20.
The experiment was carried out at an ambient temperature, and the average temperature was 28° C. from Day 0 to Day 20 and 25° C. from Day 20 to Day 50.
Arthrobacter sp.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-273699 | Oct 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2008/069019 | 10/21/2008 | WO | 00 | 4/21/2010 |
