WO2007125661A1 - Method for detoxification of harmful compound - Google Patents

Abstract

Disclosed is a method for detoxification of a harmful compound having at least one element selected from the group consisting of arsenic, antimony and selenium. The method comprises the steps of: allowing the harmful compound to be taken into a plant plankton capable of detoxifying the harmful compound to alkylate the harmful compound in the plant plankton, thereby detoxifying the harmful compound in the plant plankton; and allowing the detoxified compound to be excreted from the plant plankton.

Description

 Description Detoxification method for hazardous compounds Technical field

 The present invention relates to a method for detoxifying harmful compounds, and more particularly to a method for detoxifying harmful compounds using phytoplankton. Background art

 Heavy metals such as arsenic, antimony, and selenium are widely used as industrial materials such as semiconductors. However, since they are toxic to living organisms, there is concern about the impact on living organisms when they flow into the environment. Has been.

 Conventionally, as a method of removing these heavy metals, a flocculant such as polyaluminum chloride (PAC) is added to wastewater containing inorganic arsenic such as toxic arsenous acid, and arsenic is agglomerated between the flocculant and iron in raw water. A method of adsorbing, precipitating, and removing by filtration, or a method of adsorbing an arsenic compound with an active alumina or cerium-based adsorbent is generally known.

On the other hand, in the natural world, it is clear that marine organisms such as seaweed accumulate inorganic arsenic, and that part of the inorganic arsenic is converted to organic arsenic compounds such as dimethylated arsenic by physiological reactions (non- Patent Document 1: Kaise et al., 1998, Organomet. Chem., 12 137-143). As algae that take up and accumulate inorganic arsenic in this way, several kinds of microalgae such as Chlorella are known (Non-patent Document 2: Maeda et al., 1990, Appl. Organomet. Chem. , 4, 251-254, Non-Patent Document 3: Gossler et al., 1997, Appl. Organomet. Chem., 11, 57-66). These organic arsenic compounds are generally known to exhibit lower toxicity to mammals than inorganic arsenic. Dunal iella. Sal ina is known to have a large amount of arsenic accumulated in the order of As (V)> As (III)> DMA, depending on the type of arsenic added to the medium. Non-Patent Document 4: Yamaoka et al., 1999, Appl. Organomet. Chem., 13, 89-94).

 As a method for recovering and detoxifying arsenic, the PTB 1 gene involved in the arsenic resistance of the green alga Chlamydomonas rheinhardi is disrupted to increase the arsenic resistance of the algae. It is known that it accumulates in the algae and is detoxified as dimethyl arsenic (Patent Document 1: Japanese Patent Application Laid-Open No. 2000-036186). In this document, arsenic uptake follows the same pathway as phosphate uptake and is taken up competitively, so the lower the phosphate concentration, the higher the arsenic uptake and vice versa. It is also stated that the higher the, the lower the phosphate uptake.

 On the other hand, a method for decomposing harmful organic arsenic contained in chemical weapons and agricultural chemicals into inorganic arsenic using microorganisms is known (Patent Document 2: Japanese Patent Laid-Open No. 2 095-2 2 9 9 4 5). In addition, there is also known a method for removing heavy metal ions by culturing green algae acid algae in an acidic aqueous solution containing heavy metal ions such as arsenic, thereby incorporating the heavy metal ions into the living body of the acid algae. (Patent Document 3: Japanese Patent Application Laid-Open No. 8-206 684).

 As a result of investigating arsenic uptake into the phlegm repellent and the remaining amount of arsenic in the medium, most of the arsenic accumulated in the phlegm repellent was inorganic arsenic, and MMA, DMA, and TMA were slightly detected. It is also known that traces of DMA and TMA are detected (Non-Patent Document 5: Maeda et al., 1992, Appl. Organomet. Chem., 6, 399-405, Non-Patent Document 6: Maeda et al., 1992, Appl. Organomet. Chem., 6, 407-413).

 However, in the above-described method of removing heavy metals using filtration, adsorption, etc., sludge containing harmful compounds such as inorganic arsenic that still remains harmful, and adsorbents to which the harmful compounds are adsorbed are used. It is necessary to store or landfill after sealing with concrete etc. so that the harmful compound does not leak to the outside, and a large space for storage and landfill is required, making it difficult to process in large quantities There was a problem.

In addition, even if inorganic arsenic is incorporated into the above-mentioned marine organisms, only a part of the incorporated inorganic arsenic becomes an organic arsenic compound, and harmful inorganic arsenic is still accumulated in marine organisms. Further, although Patent Document 1 describes in detail the uptake of arsenic, it does not consider the point of discharging detoxified arsenic from microorganisms such as culture fluid. Patent Document 2 has a problem that dimethylarsinic acid and the like are less toxic than inorganic arsenic, and using methylated organic arsenic as inorganic arsenic increases the toxicity.

 In addition, Patent Document 3 and Non-Patent Document 4 described above do not describe that the detoxified substance is discharged out of the organism of the microorganism.

 Furthermore, in Non-Patent Documents 5 and 6, methylated arsenic in the medium is detected in a very small amount, but it is not recognized as an amount that can be industrially rendered harmless, and a little. However, there is a case where it is not detoxified, and it does not show valid data on detoxification of inorganic arsenic.

 Therefore, an object of the present invention is to provide a useful method for efficiently and systematically detoxifying harmful compounds including arsenic to solve the above problems. Disclosure of the invention

 In order to achieve the above object, the present inventors have intensively studied the relationship between chlorella culture and detoxification of harmful compounds, and as a result, have found the present invention.

 That is, the harmful compound detoxifying method of the present invention is a phytoplankton capable of detoxifying a harmful compound containing at least one element selected from the group consisting of arsenic, antimony, and selenium. And detoxifying the harmful compound by alkylating the phytoplankton in the phytoplankton, and discharging the detoxified substance outside the living body of the phytoplankton.

 In a preferred embodiment of the method for detoxifying harmful compounds of the present invention, the phytoplankton is cultured in the presence of phosphoric acid.

In a preferred embodiment of the method for detoxifying harmful compounds of the present invention, the concentration of the phosphoric acid is 0.1 to 5. O mg / L in the culture solution. Further, in a preferred embodiment of the method for detoxifying harmful compounds of the present invention, the culture of the plant plankton is selected from the group consisting of arsenic, antimony and selenium. It is characterized in that it is carried out in the presence of a reducing agent that reduces at least one metal. In a preferred embodiment of the method for detoxifying harmful compounds of the present invention, the reducing agent is a substance having an SH group.

 Further, in a preferred embodiment of the method for detoxifying harmful compounds of the present invention, the substance having an SH group is selected from the group consisting of reduced glutathione (GSH), cysteine, S-adenosylcystine, sulforafuan and thioglycolic acid. It is characterized by at least one selected.

 Moreover, in a preferred embodiment of the method for detoxifying harmful compounds according to the present invention, the phytoplankton is cultured in the presence of a carbon source.

 In a preferred embodiment of the method for detoxifying harmful compounds of the present invention, the carbon source is a saccharide or an organic acid.

 In a preferred embodiment of the method for detoxifying harmful compounds according to the present invention, the sugar is selected from the group consisting of gnolecose, galactose, funolectose, sucrose, mannose, and maltose.

 Further, in a preferred embodiment of the method for detoxifying harmful compounds according to the present invention, the organic acid is selected from the group consisting of acetic acid, citrate, malic acid, fumaric acid, succinic acid, and pyruvic acid. To do. '

 In a preferred embodiment of the method for detoxifying harmful compounds according to the present invention, the plant plankton is chlorella.

 Further, in a preferred embodiment of the method for detoxifying a harmful compound of the present invention, the harmful compound is arsenous acid, arsenic pentoxide, arsenic trichloride, arsenic pentachloride, an arsenic sulfide compound, a cyanoarsenic compound, and black arsenic. It is selected from the group consisting of compounds, and other arsenic inorganic salts.

 In a preferred embodiment of the method for detoxifying harmful compounds of the present invention, the alkylation is methylation.

In a preferred embodiment of the method for detoxifying a harmful compound of the present invention, the methylated compound is converted into a dimethyl compound or a trimethyl compound by the methylation. In a preferred embodiment of the method for detoxifying harmful compounds of the present invention, The methyl compound is characterized in that it is dimethylarsonyl ethanol (DMAE), dimethylarsonyl acetate (DMAA), dimethylarsinic acid, or arsenosugar.

 Moreover, in a preferred embodiment of the method for detoxifying harmful compounds of the present invention, the small trimethyl compound is an arsenocholine, an arsenobetaine, a trimethylarsenosuger or a trimethylarsinoxide. The invention's effect

 The harmful compound detoxification method of the present invention has an advantageous effect that detoxification can be achieved by easily and simply alkylating harmful compounds, particularly harmful compounds containing arsenic, antimony, selenium and the like. . In addition, according to the method of the present invention, harmful compounds can be rendered innocuous as much as possible, so that there is an advantageous effect that a large space such as a storage place is not required. In addition, according to the method of the present invention, harmful compounds such as inorganic arsenic are not accumulated in the cells of plant plankton, but alkylated arsenic is discharged into the culture medium, and more harmful compounds in the environment are removed. This has an advantageous effect that it can be more easily converted to a less toxic substance. Brief description of the drawings

 Figure 1 shows the results of chlorella growth with and without phosphoric acid and with or without GSH.

 Figure 2 shows the measurement results of the chemical type arsenic content in chlorella cultured with GSH added.

 FIG. 3 is a diagram showing the results of measurement of phlegm larva cultured without GSH addition.

 FIG. 4 is a diagram showing the measurement results of the medium components after culturing with the addition of GSH.

 FIG. 5 is a diagram showing the measurement results of the medium components after culturing without adding GSH.

Figure 6 shows the total amount of arsenic in chlorella and medium with and without phosphate addition and GSH. Is a diagram showing the total amount of inorganic arsenic and methylated arsenic other than inorganic arsenic.

 Fig. 7 shows the results of chemical arsenic content by alkali treatment of chlorella components. FIG. 8 is a graph showing changes over time in the growth of chlorella cultured with and without GSH addition under conditions of initial phosphate addition concentration of 1-10.

 FIG. 9 is a diagram showing the change over time of the medium components cultured with the addition of GSH. FIG. 10 is a graph showing changes over time in medium components cultured without GSH addition. FIG. 11 is a graph showing the growth results of chlorella depending on the GSH addition concentration.

 Fig. 12 shows the results of measuring the amount of arsenic of chemical type in chlorella cultured with different GSH concentrations.

 FIG. 13 is a diagram showing the measurement results of the culture medium components after culturing with different GSH concentrations.

 Fig. 14 shows the growth results of chlorella at different concentrations of Cys.

 Fig. 15 shows the measurement results of the amount of chemical arsenic in chlorella cultured with varying Cys concentrations.

FIG. 16 shows the measurement results of the culture medium components after culturing with varying _CyS concentrations. Figure 17 shows the growth results of chlorella at different concentrations of TGC.

 Fig. 18 shows the results of measuring the amount of arsenic of chemical type in chlorella cultured with different TGC concentrations.

 FIG. 19 shows the measurement results of the culture medium components after culturing with different T G C concentrations. FIG. 20 shows the growth results of chlorella depending on the glucose addition concentration.

 Fig. 21 shows the measurement results of chemical arsenic levels in chlorella cultured at different glucose concentrations.

 Figure 22 shows the measurement results of medium components after culturing with different glucose concentrations. BEST MODE FOR CARRYING OUT THE INVENTION

The method for detoxifying a harmful compound of the present invention includes incorporating a harmful compound into a detoxifying phytoplankton and alkylating the harmful compound in the phytoplankton. And detoxifying the detoxifying substance out of the phytoplankton. This is because, by continuing the cultivation of phytoplankton, harmful compounds taken into the body of the phytoplankton are alkylated, more preferably methylated, to a less toxic detoxifying substance. After being converted, the detoxifying substance can be discharged to the outside of the living body, so that the detoxifying system can be industrially used. There is no need for complicated extraction from a living body, and this is a simpler method.

 There is a method for accumulating dimethylated arsenic in the larva, but if methylation is promoted, the efficiency of discharge to the medium increases, and the amount of medium accumulated in the chlorella alga body increases. Therefore, it is considered that the method according to the present invention in which inorganic arsenic is detoxified by discharging it into the medium is efficient.

 Examples of harmful compounds include those containing at least one element selected from the group consisting of arsenic, antimony, and selenium. In addition, in this specification, a harmful compound means a compound that may flow into the environment and have some adverse effect on the organism when exposed to the organism.

 Among the harmful compounds, harmful compounds containing arsenic include arsenous acid, arsenic pentoxide, arsenic trichloride, arsenic pentachloride, arsenic sulfide compounds, cyanoarsenic compounds, black arsenic compounds, and other arsenic inorganics Examples include salts. Arsenic, for example, has an LD50 (mg / kg) (50% lethal dose in mice) of 20 or less, and is generally toxic to organisms.

 Examples of harmful compounds containing antimony include antimony trioxide, antimony pentoxide, antimony trichloride, and antimony pentachloride.

 Further, examples of harmful compounds containing selenium include selenium dioxide and selenium trioxide.

In the present invention, phytoplankton is not particularly limited as long as it can detoxify the harmful compounds. Examples of the phytoplankton include fresh water microalgae such as chlorella, nanochlorobusis, spirulina, donariella and the like. Marine microalgae, seaweeds and seaweed. Inorganic arsenic times It is possible to use other phytoplankton as long as the effect of yielding and methylation can be achieved. Since the gutta rera used in the following examples is generally marketed and suitable for industrialization, it is a particularly preferred embodiment to use the kure relera in the present invention. In other words, chlorella is used as a feed for functional food and fry for cultivated fisheries, and has established mass culture technology, which is suitable for industrially detoxifying inorganic arsenic. However, the phytoplankton used in the present invention may be other than chlorella as long as it has an effect of absorbing inorganic arsenic contained in the solution from the solution in a short time.

 In a preferred embodiment of the present invention, phytoplankton is cultured in the presence of phosphoric acid. This is essential for the stable growth of phytoplankton. In other words, phosphoric acid is essential for nucleic acids, ATP, cell membranes, etc. By culturing while supplying this, it is possible to more efficiently incorporate harmful compounds such as arsenic and discharge harmless substances such as alkylated arsenic. Because it is.

 The concentration of phosphoric acid is not particularly limited depending on the type of phytoplankton, the culture conditions, the type of harmful compounds, etc. However, the concentration of phosphoric acid is from the viewpoint of increasing the efficiency of inorganic arsenic incorporation into chlorella. Therefore, it is preferable to adjust the concentration to 0.1 to 5. O mg / L in the culture solution.

The presence of phosphoric acid in this way is because arsenic and the like have properties similar to phosphoric acid, and arsenic is taken in by the same route as chlorella's phosphate uptake. If the concentration is lower than the concentration of arsenic, the uptake of arsenic is promoted. As a result, harmful compounds can be detoxified efficiently. On the other hand, if phosphoric acid is not added, the essential components of kurea are not supplied. This is because it may be impossible to grow. In general, phytoplankton, such as chlorella, has the ability to methylate the arsenic that has been taken in, but if the uptake of inorganic arsenic is promoted at low phosphoric acid concentrations, the toxicity of inorganic arsenic can cause growth or death. It is possible to do. After culturing in the presence of an appropriate phosphate according to the plant plankton, the arsenic that has been incorporated can be alkylated and discharged into the phytoplankton ex vivo, for example, to the medium. The amount of harmful compounds in phytoplankton is reduced, and toxicity is also manifested. There is nothing to do. Therefore, under such conditions, it is possible to stably cultivate phytoplankton that has incorporated harmful compounds, and thus it is possible to industrially achieve the detoxification of harmful compounds.

 In the present invention, the phytoplankton can be cultured in the presence of a reducing agent that reduces at least one metal selected from the group consisting of arsenic, antimony, and selenium. The presence of such a reducing agent can further promote alkylation. Furthermore, as a result of diligent research by the present inventors, it has been found that in the presence of a reducing agent, a substance that has been detoxified by alkylation in phytoplankton promotes the in vitro release of the phytoplankton. It is because it was done. If the detoxified substance is discharged outside the phytoplankton, it is easier than the extraction of the phytoplankton to obtain the detoxified substance, and even the growth management of the phytoplankton can be performed. For example, an industrial system for detoxifying harmful compounds can be provided.

 In the present invention, the reducing agent is preferably a substance having an SH group from the viewpoint that conversion to methylated arsenic can be promoted. The substance having an SH group is preferably at least one selected from the group consisting of reduced dartathione (GSH), cysteine, S-adenosylcystine, sulforafuan, and thiodaricholic acid.

 However, even in the case of oxidized dartathione (GSSG) that does not have an SH group, there are cases where it becomes reduced glutathione in a plant plankton or a medium in which the phytoplankton is cultured. Therefore, even in the presence of oxidized glutathione that does not have an SH group, conversion to methyl arsenic can be promoted sufficiently.

 Therefore, in the present invention, “in the presence of a reducing agent” refers to a substance that changes in a phytoplankton or a culture solution and acts as a reducing agent in addition to the case where the reducing agent itself is added to the culture solution. The concept including the case where it adds to a culture solution is intended.

Furthermore, in the present invention, the phytoplankton is preferably cultured in the presence of a carbon source. Phytoplankton, such as chlorella, uses inorganic arsenic Although it has the ability to chill, it captures carbon dioxide in the atmosphere as a source of methyl groups by photosynthesis and uses it for methylation. However, the use of carbon dioxide by photosynthesis alone has low methylation efficiency, and a large amount of inorganic arsenic remains in the algae. Thus, the present inventors have found that a carbon source that is insufficient for methylation is added to the culture medium and cultured, so that the carbon source can be efficiently incorporated and used for methylation. In addition, it can be cultured at a high density by adding a carbon source, increasing the total amount of algae obtained from each unit medium, and increasing the total amount of arsenic uptake and methylation. It acts synergistically to achieve the efficient detoxification of the present invention.

 That is, in order to promote methylation, a carbon source is added to the culture medium and cultured, so that it can be efficiently used for methylation as a carbon source necessary for methylation. Furthermore, it can be cultured at a high density by adding a carbon source, and the amount of dimethylated arsenic discharged into the medium increases.

 This is because force s, which is considered to be one of the factors that increase the amount of detoxifying substances released into the culture medium due to the increase in the amount of phytoplankton due to the addition of glucose, and the discharge into the culture medium (in vitro of plant plankton) UN8, an arsenosugar (see examples below), is the main, and glucose functions as a source of carbon for the methyl group to methylate to ring 8 in the phytoplankton It is guessed.

 Based on the above, in addition to continuing the cultivation of phytoplankton, phosphoric acid and GSH are also important for the release of alkylated detoxifying substances outside the body of phytoplankton.

 However, in the case of Arsenosugar, UN 8 which is a kind of Arsenosugar is rare in phytoplankton, and most of it exists outside phytoplankton (in the medium). It is presumed that the factors that make up are the factors that discharge into the culture medium.

In the present invention, the conditions under the supply of the carbon source are not particularly limited. For example, photosynthesis is not necessarily required and can be performed in a dark place or a light place. In the dark culture, cultivate in the light in advance, after culturing to the late logarithmic phase, It is also possible to add carbon source and arsenic and culture in the dark to methylate arsenic. Here, the carbon source is not particularly limited, and examples thereof include sugars or organic acids. Examples of saccharides include, but are not limited to, galecose, galactose, funolectose, sucrose, mannose, maltose and the like. Examples of the organic acid are not particularly limited, and examples include acetic acid, citrate, malic acid, fumaric acid, succinic acid, and pyruvic acid. The amount of the carbon source is not particularly limited, but from the viewpoint of achieving detoxification of harmful compounds more efficiently, in the case of Darcos, a concentration range of 1 to 100 g / L, acetic acid In the case of sodium, a concentration range of 1 to 100 g ZL is preferred.

 Further, in the present invention, the harmful compound is converted into a dimethyl compound or a trimethyl compound by anoalkylation, preferably methylation. Examples of the dimethyl compound include dimethylarsonylethanol (DMAE), dimethylenoarsoninoreacetate (DMAA), dimethylarsinic acid, and arsenosugar. Examples of the trimethyl compound include anoresenocholine, arsenobetaine, trimethyl anoleno sucrose, and trimethyl arsinoxide. This is extremely less toxic than inorganic arsenic and is relatively stable in nature.

 In addition, as an industrial method, inorganic arsenic can be detoxified using plant plankton immobilized in alginate beads or the like. Examples of the immobilization method include a carrier binding method, a crosslinking method, and a comprehensive method. A simple immobilization method may be used in which the medium is adsorbed and immobilized on a medium or the like by physical chemical adsorption.

 The use of the immobilized microorganism has the following advantages.

1) Easy separation of microorganism-immobilized beads and solution,

2) Microbe-immobilized beads can be reused,

 3) The amount of microorganism-immobilized beads can be adjusted freely according to the amount of harmful compounds such as arsenic to be treated.

Fixing agents include alginic acid, gelatin, polyacrylamide, agar, and agar. Gallose, starch, dextrin, carrageenan, chitosan, pectin and polybranol alcohol are available. Example

 Examples of the present invention will be described below, but the following examples do not limit the scope of the present invention. Example 1

Effect of presence or absence of glutathione (GSH) and phosphate concentration

 (1) Chlorella culture

 Chlorella vulgaris I AM C-629 strain, pre-cultured with Bold's Basal (BB) Medium until logarithmic growth phase, modified to 150 ml of Bold's Basal (BB) Medium at 1 X 1 05cells / ml The cells were inoculated and cultured under static light irradiation (4000 Lux, 24 hr irradiation) at a temperature of 25 ° C for 168 hours. Modified Bold's Basal (BB) Medium was added to double sodium nitrate, 10πιΜ glucose and 1 ppm arsenic acid, and 4 times dipotassium hydrogen phosphate and potassium dihydrogen phosphate. The concentration was adjusted to 1 ×, 1/4, and 1Z16, and GSH (manufactured by Sigma) 1 mM was compared with no addition of GSH.

 (2) Arsenic uptake test

 Arsenous acid as trivalent inorganic arsenic and arsenic acid as pentavalent inorganic arsenic were added to the medium so that I p pm as metal arsenic was added under fluorescent lamp irradiation (4000 Lu x, 24 hr irradiation) Then, the arsenic uptake test was carried out in chlorella by incubating at 25 ° C.

 (3) Measurement of phosphoric acid, nitric acid, and glucose concentration in the medium

 The concentration of phosphate and nitrate glucose in the medium was determined by centrifuging the medium supernatant and quantifying with MERCK RQ flex and colorimetric test paper reflect quant. The lower limit of measurement of each component is phosphoric acid 5 mg ZL PO 43-(indicated in the table is PO 4), nitric acid 5 mg / L N03- (indicated in the table is N03), and gnorlecose 1 mg / L.

(4) Arsenic content measurement Inorganic and organic arsenic in chlorella and culture medium is HP LC—I CP-MS (Inductively coupled plasma ion mass spectrometer A gi 1 ent 1 1 0 0 directly connected to high performance liquid chromatograph A gi 1 ent 7500 ce), qualitative and quantitative analysis was performed by comparing the retention time of the reaction product with the standard sample.

 (5) Arsenic analysis conditions +

 MMA, DMA, TMAO, AB, and AC are standard reagents for organic arsenic compounds, and Trichemical Laboratories reagents. As standard samples for inorganic arsenic, A s (II 1) and A s (V) Wako Pure The sodium salt of a pharmaceutical grade reagent was used. A standard solution of 10 Omg / 10 OmL of each arsenic compound was prepared by diluting with ultrapure water (Millipore).

 (Abbreviation)

 A s (III): Inorganic trivalent arsenic

 A s (V): Inorganic pentavalent arsenic

 MM A: Monomethylarsonic acid

 DMA: Dimethyaric acid

 TMAO: Trimethylanolenosoxide

 AB: Arsenobetaine (trimethylarsoyuacetic acid)

 I CP-MS equipment conditions were as follows.

R F forward power: 1.6 kW

R F reflect power: <1 W

Carrier gas flow: Ar 0.75L / min

Sampling 8.5mm

 Monitoring mass: m / z = 75 and 35 internal standard m / Z = 71

Dwell time: 0.5 sec 0. Olsec 0. Isec

Times of scan: 1 time

 HP LC conditions

Eluent: 5 mM nitric acid / 6 mM ammonium nitrate Zl.5 mM pyridinedicarboxylic acid Eluent flow rate: 0.4 mLZ min

Injection volume: 20 μL

Column: Cation exchange column Shodex RSpak 顺 -614 (150 thighs x 4.6mm i. D.) Column temperature: 40 degrees

 (6) Preparation of chlorella sample with arsenic uptake

 Centrifuge the culture medium containing chlorella, add 500 μί of methanol: pure water = 1: 1 solution to chlorella adjusted to a wet weight of about 1 O Omg, and ultrasonically disperse the pelleted chlorella. The extraction process was performed for 12 hours or more at room temperature. For the measurement of HP LC I C P—MS, the extract was diluted with ultrapure water, and the particles were removed with a 0.45 / ^ 111 Membran filter to obtain a measurement sample.

 (7) Preparation of medium sample with arsenic added

 The culture solution containing chlorella was centrifuged and the culture supernatant was separated. For the measurement of HP LC—ICP—MS, the culture supernatant was diluted with ultrapure water, and the particles were removed with a 0.45 m Membrane filter to obtain a measurement sample.

 (8) Chlorella growth results

 Table 1 and Fig. 1 show the growth results of chlorella with and without phosphate addition and GSH addition.

1

In the presence of 1 ppm of inorganic arsenic, growth was inhibited and growth was reduced when the concentration of phosphoric acid added to the medium was low. In contrast, the addition of GSH did not inhibit growth until the initial addition of phosphoric acid was 1/4. (9) Measurement results of chemical type arsenic content in chlorella

 Table 2 and Fig. 2 show the results of measurement of the amount of arsenic by chemical species in phlegm cultivated with GSH added.

リン酸の添加濃度が 1/4以下では培養終了時に培地中のリン酸が枯渴したこ とでクロレラのヒ素取り込み量が増加し、 UN 1、 A s (V)、 MMA、 UN 2、 A s (111)、 UN 4、 DMA, UN I 3、 UN 8、 U N 14の増加が顕著であった。 一方、 培地にリン酸が残存している条件ではヒ素の取り込み量は同等であった。 表 3および図 3に G SHを無添加で培養したクロレラの測定結果を示す。 2]

When the addition concentration of phosphoric acid was 1/4 or less, the amount of arsenic uptake by chlorella increased due to the depletion of phosphoric acid in the medium at the end of the culture. s (111), UN 4, DMA, UN I 3, UN 8, and UN 14 increased significantly. On the other hand, the amount of arsenic uptake was the same under conditions where phosphoric acid remained in the medium. Table 3 and Fig. 3 show the measurement results of chlorella cultured without GSH.

3]

 As a result of the inhibition of growth due to the toxicity of arsenic, phosphoric acid remained after culture at an initial concentration of 1/4 phosphoric acid. The chemical type arsenic content in the guttella is similar to that of the GSH addition condition, and when the phosphoric acid addition concentration is 1/4 or less, the arsenic uptake in the phlegmrel increases, and UN 1, A s ( V), MMA, UN 2, As (111), UN 4 DMA, UN 13 and UN 8 increased significantly. On the other hand, the amount of arsenic uptake was similar under the condition where phosphoric acid remained in the medium.

 (1 0) Measurement result of the amount of chemical arsenic in the medium

 Table 4 and Fig. 4 show the measurement results of medium components after culturing with GSH added.

リン酸の添加濃度が低いほど、 UN 8の排出量が顕著に増加し、 これに対応し て培地中の無機ヒ素量が減少した。 これはリン酸が培地中で枯渴する事で、 クロ レラへのヒ素の取込が増え、 取込んだヒ素を UN 8に変換し、 培地中に排出した と考えられる。 4

The lower the concentration of phosphoric acid added, the more UN 8 emissions increased, correspondingly decreasing the amount of inorganic arsenic in the medium. This is thought to be due to the fact that phosphoric acid withered in the medium increased the uptake of arsenic into chlorella, which was converted to UN 8 and discharged into the medium.

 Table 5 and Fig. 5 show the measurement results of medium components after culturing without adding GSH.

 Five]

培地中のリン酸が培地中で枯渴した試料では UN 8の排出量が顕著に増加し、 培地中の無機ヒ素量が減少した。 しかし、 GSHを添加した場合に比べ、 UN 8 の培地への排出量は lZ 3程度であり、 G SHが UN 8への変換と排出を促進す る結果となった。

In samples where phosphoric acid in the medium was withered in the medium, the amount of UN 8 excretion increased significantly and the amount of inorganic arsenic in the medium decreased. However, compared to the case where GSH was added, the amount of UN 8 released into the medium was about 1Z3, and GSH promoted the conversion to UN 8 and its release.

 (1 1) Confirmation of unidentified components in the medium

 Arsenic dimethylated arsenic to which sugar chains synthesized by algae are bound by adding 4N-NaOH equivalent to the culture medium after incubation (sample 4 in Example 1) and thermally decomposing at 100 ° C for 3 hours The sugar chain was cleaved. Dimethylated arsenic whose sugar chain is cleaved can be detected as DMA. As a result of the alkaline heat treatment, the most discharged UN 8 in the medium decreased and DMA increased. Based on this, UN 8 was presumed to be a dimethylated arsenosugar with a sugar chain attached.

 (1 2) Confirmation of unidentified components in Chlorella

Chlorella (Sample 23 from Example 3) 4N—NaOH was added to about 10 Omg, and 1 By cleaving at 00 ° C for 3 hours, the chain of dimethylated arsenic to which sugar chains synthesized by algae were bound was cleaved and compared with the components extracted with methanol. As a result of alkali treatment, UN 1, UN 2 and UN 8 decreased, MMA and DMA increased, and it was estimated that arsenic in chlorella was mostly methylated or dimethylated.

 (1 3) Total amount of arsenic in chlorella and medium

 Table 6 and Fig. 6 show the total amount of arsenic in chlorella and medium with and without phosphoric acid added and with or without GSH as the total amount of methylated arsenic except inorganic arsenic and non-inorganic arsenic.

[¾6]

Methylated arsenic in Kukurera increased under the condition that phosphoric acid in the medium was withered. Table 7 shows the confirmation of chlorella components by alkali treatment.

さらに、 表 8にリン酸添カロ量と GSH添加有無によるクロレラ及び培地中の砒素 総量の結果を示す。 [¾7]

In addition, Table 8 shows the results of the total amount of arsenic in chlorella and the medium with and without phosphate-added calories and GSH.

E 8]

表 7及び図 7から明らかなように、 NaOH 処理で糖鎖切断した結果、 MMA、 DMA が増加していることから、 UN 8は、 何らかのメチル化砒素であり、 UN 8が生成し ていることによって、 有害化合物が無害化されていることが確認できる。

As can be seen from Table 7 and Fig. 7, as a result of the increase in MMA and DMA as a result of glycan cleavage by NaOH treatment, UN 8 is some form of methylated arsenic, and UN 8 is produced. This confirms that harmful compounds have been rendered harmless.

 The methylated arsenic in the medium also increased under the condition that the phosphoric acid in the medium was withered. At that time, the amount of methylated arsenic was discharged into the medium more than the amount accumulated in chlorella, most of which was UN8. Furthermore, the addition of GSH increased the discharge of arsenic methylated into the medium. In order to obtain methylated arsenic (UN8), it was shown that it was more efficient to excrete into the medium than to accumulate in chlorella.

Example 2

Changes in medium discharge over time

 (1) Chlorella culture

 Chlorella vulgaris I AM C-629 strain pre-cultured with Bold's Basal (BB) Medium until logarithmic growth phase is modified to 150 ml of Bold's Basal (BB) Medium with 1 X

1 The cells were inoculated to a concentration of 05cells / ml, statically cultured at a temperature of 25 ° C under fluorescent light irradiation (4000 Lux, 24 hr irradiation), and sampled over time. Modified Bold's Basal (BB) Medium was added with sodium nitrate 2 times, glucose 10πιΜ, and arsenic acid 1 p pm. The concentration was compared with 1 mM GSH and no GSH added. The analysis samples were adjusted and analyzed in the same manner as described above.

 (2) Chlorella growth results

 Table 9 and Fig. 8 show the changes over time in the growth of phlegm larvae cultivated with and without GSH under the initial phosphate addition concentration of 1/10.

[Table 9] Addition condition Medium residual amount Growth amount Culture time

 Sample As (V) GSH P04 Qulcose N03 P04 Dulcose N03 Bacterial concentration Total yield hr ppm mM mM IDR / L mR / L mf? / L cells / ml mg / 150nil

9 72 I 1 1/10 10 X2 19 1480 362 5.00E + 05 38.7

10 96 ι 1 1/10 10 X2 8 1100 328 7.74E + 06 162.9

11 120 ι 1 1/10 10 X2 <5 270 188 1.03B + 07 543.7

12 144 1 1 1/10 10 X2 <5 1 88 2.85E + 07 796.7

13 168 1 1 1/10 10 X2 <5 0 49 3.88E + 07 885.6

14 240 I 1 1/10 10 X2 5 0 54 3.70E + 07 868.1

15 72 I 0 1/10 10 X2 19 1560 392 1.55E + 06 40.6

16 96 I 0 1/10 10 X2 11 1140 336 2.00E + 06 134.9

17 120 I 0 1/10 10 X2 5 750 274 5.25E + 06 281.1

18 144 I 0 1/10 10 X2 <5 335 208 9.25E + 06 482.9

19 168 1 0 1/10 10 X2 <5 340 234 1.23E + 07 517.4

The culture with GSHl mM added grew better and the effect of inorganic arsenic toxicity was less.

 (3) Measurement results of chemical type arsenic content in the medium

 Table 10 and Fig. 9 show the changes over time in the medium components cultured with GSH added.

[Table 10]

培養開始後 1 20時間で培地中のリン酸が枯渴し、 その後、 経時的に UN 8の 培地への排出量が増加し、 培養 1 68時間では培地中の無機ヒ素の 48%が UN 8に変換され、 240時間では 5 5 %が UN 8に変換された。 これに対応して培 地中の無機ヒ素量が減少した。

In 20 hours after the start of culture, phosphoric acid in the medium was depleted, and thereafter, the amount of UN 8 discharged into the medium increased over time, and in 1 hour of culture, 48% of the inorganic arsenic in the medium was In 240 hours, 55% was converted to UN8. Correspondingly, the amount of inorganic arsenic in the culture medium decreased.

 Tables 11 and 10 show the changes over time in the medium components cultured without GSH.

[Table 1 1]

Phosphoric acid in the medium withered 20 hours after the start of cultivation, but UN 8 was discharged into the medium. The amount was confirmed after 144 hours in culture. In 1680 hours of culture, 13% of the inorganic arsenic in the medium was converted to UN8. Correspondingly, the amount of inorganic arsenic in the medium decreased. Compared with GSH addition, UN 8 was discharged although it was 1 Z 4. Example 3

Effect of dartathione concentration

 (1) Chlorella culture

 Chlorella vulgaris IAM C-629 strain, which has been pre-cultured with Bold's Basal (BB) Medium until the logarithmic growth phase, is modified to 150 ml of Bold's Basal (BB) Medium at 1 X 1 05cells / ml The cells were inoculated and cultured under static light irradiation (4000 Lux, 24 hr irradiation) at a temperature of 25 ° C for 168 hours. Modified Bold 's Basal (BB) Medium is 2/100% hydrogen phosphate and 2% hydrogen dihydrogen phosphate, 2 times sodium nitrate, 10mM glucose, 1ppm arsenate The GSH concentration was adjusted to 0, 0.01, 0.1, and ImM. Preparation of analysis sample The opiate analysis was carried out in the same manner as described above.

 (2) Chlorella growth results

 Table 12 and Fig. 11 show the growth results of chlorella depending on the concentration of GSH.

[Table 1 2]

リン酸初期添加濃度 1/1 0の条件下では培養 1 6 8時間で培地中のリン酸は 全ての条件で枯渴した。 生育量は G SHの添加量が高いほど増加し、 無機ヒ素に よる生育阻害が抑制された。

Under the condition of initial addition concentration of phosphoric acid 1/10, phosphoric acid in the medium was withered under all conditions in 1 68 8 hours of culture. The amount of growth increased as the amount of GSH added increased, and inhibition of growth by inorganic arsenic was suppressed.

 (3) Measurement result of chemical type arsenic content in chlorella

Tables 1 and 3 and Fig. 12 show chemical types of arsenic in chlorella cultured at different GSH concentrations. The measurement result of quantity is shown.

13 ]

The higher the concentration of GSH added, the higher MMA UN'2 UN4 DMA and UN I 3 UN 8 UN 14 and the higher the total arsenic content.

 (4) Measurement results of chemical type arsenic content in the medium

 Table 14 and Fig. 13 show the measurement results of medium components after culturing with different GSH concentrations.

[Table 14]

The higher the GSH addition concentration, the more the UN 8 emissions increased and the amount of inorganic arsenic in the medium decreased. At a GSH concentration of 1 mM, 50% of the inorganic arsenic in the medium was converted to UN 8 in 1968 hours of culture.

 (5) Total amount of arsenic in chlorella and medium

 Table 15 shows the total amount of arsenic in chlorella and the medium as the total amount of methylated arsenic except inorganic arsenic and non-inorganic arsenic.

[Table 1 5]

培養後のリン酸は全ての GSH濃度の条件で枯渴しており、 ク口レラ中へのメチ ル化ヒ素の蓄積と培地への排出が確認された GSH無添加と 0. 0 ImM添加条件で はメチル化ヒ素のクロレラ中蓄積と培地排出に大きな差は無いが、 GSHO. ImM 以上の添加ではクロレラ中蓄積おょぴ培地への排出は GSH濃度の増加ともに増大 した。 その際、 メチル化ヒ素量はクロレラへ蓄積される量より、 培地中へ排出さ れる量の方が多く、 その大部分が UN8であった。 メチル化ヒ素 （UN8) を得るには ク口レラへの蓄積より、 培地に排出させる方が効率が高いことが示された。 実施例 4

Phosphoric acid after culture was withered under all conditions of GSH concentration. Accumulation of arsenic methylate in the larva and discharge into the medium were confirmed. No GSH addition and 0.0 ImM addition condition so There was no significant difference between the accumulation of methylated arsenic in chlorella and the excretion of the medium, but the addition of GSHO. ImM or more increased the excretion of chlorella in the acetic acid accumulation medium with an increase in GSH concentration. At that time, the amount of methylated arsenic was discharged into the medium more than the amount accumulated in chlorella, most of which was UN8. To obtain methylated arsenic (UN8), it was shown that it was more efficient to excrete it into the culture medium than to accumulate it in the larva. Example 4

Effect of cysteine concentration

 (1) Chlorella culture

 Modified chlorella (Chlorella vulgaris IAM C—629 strain) pre-cultured with Bold's Basal (BB) Medium until logarithmic growth phase.

The cells were inoculated to 1 05 cells / ml, and incubated at 240 ° C for 240 hours at a temperature of 25 ° C under fluorescent light irradiation (4000 Lux, 24 hr irradiation). Modified Bold 's Basal (BB) Medium is 1/10 of dihydrogen phosphate and dihydrogen phosphate, double sodium nitrate, 10 mM glucose, 1 ppm arsenate And the systemine (Cys) was adjusted to concentrations of 0, 1, and 4 mM. Analysis sample preparation and analysis were carried out in the same manner as described above.

 (2) Chlorella growth results

 Table 16 and Fig. 14 show the growth results of chlorella at different concentrations of Cys.

 1 6]

リン酸初期添加濃度 1/1 0の条件下では培養 240時間で培地中のリン酸お よびダルコース全ての条件で枯渴した。 生育量は何れの条件でも殆ど同等であつ た。

Phosphoric acid in the medium after 240 hours of culture under conditions of initial phosphate addition concentration of 1/10 And withered under all conditions of dull course. The growth was almost the same under all conditions.

 (3) Measurement result of chemical type arsenic content in chlorella

 Table 17 and Fig. 15 show the results of measurement of the amount of arsenic of chemical type in chlorella cultured with different concentrations of Cys.

Ho 1 7 ']

The higher the concentration of Cys added, the higher UN1 and MMA, and the higher the total arsenic content. (4) Measurement results of chemical type arsenic content in the medium

 Tables 18 and 16 show the measurement results of the medium components after culturing with varying Cys concentration.

Ho 1 8]

The higher the concentration of Cys added, the higher the UN8 emissions and the lower the amount of inorganic arsenic in the medium.

(5) Total amount of arsenic in chlorella and medium Table 19 shows the total amount of arsenic in chlorella and the medium as the total amount of methylated arsenic except inorganic arsenic and inorganic arsenic.

[Table 1 9]

クロレラ中へのメチル化ヒ素の蓄積と培地への排出が確認された、 Cy s無添 加に比べ C y s添加条件では藻体中のメチル化ヒ素の蓄積が増加した。 培地ヘメ チル化ヒ素の排出も Cy s濃度の増加ともに増大し、 Cy s添加によるクロレラ のヒ素のメチル化の促進効果が確認された。 実施例 5

Accumulation of methylated arsenic in algal cells was increased under the Cys-added condition compared to the addition of Cys, in which accumulation of methylated arsenic in Chlorella and discharge into the medium was confirmed. The excretion of hematylated arsenic in the medium also increased with the increase in the concentration of Cys, confirming the effect of promoting the methylation of chlorella arsenic by adding Cys. Example 5

Effect of thioglycolic acid concentration

 (1) Chlorella culture

 A modified chlorella (Chlorella vulgaris I AM C-629 strain) pre-cultured with Bold's Basal (BB) Medium until the logarithmic growth phase is changed to 10 Oml of Bold's Basal (BB) Medium.

The cells were inoculated to 10 ⁵ cells / ml, and cultured under static light irradiation (4000 Lux, 24 hr irradiation) at a temperature of 25 ° C for 240 hours. The modified Bold 's Basal (BB) Medium has 2-10 hydrogen phosphate and 2N hydrogen phosphate, 2 times sodium nitrate, 10 mM gnolecose and 1 ρ pm arsenate. The sodium thioglycolate (TGC) was adjusted to concentrations of 0, 1, 2, 4, 8, and 16 mM. Preparation of the analysis sample and analysis were performed in the same manner as described above.

 (2) Chlorella growth results

Table 20 and Figure 17 show the growth results of chlorella at different concentrations of TGC. [Table 20]

リン酸初期添加濃度 1/10の条件下では培養 240時間で培地中のリン酸、 グルコースは全ての条件で枯渴した。 生育量は TGC添加濃度 8、 1 6 mMが若干少 なくなつたが、 何れの条件でも殆ど同等であった。

Under conditions where the initial concentration of phosphoric acid was 1/10, phosphoric acid and glucose in the medium were withered in all conditions after 240 hours of culture. Growth was slightly lower at TGC addition concentrations of 8 and 16 mM, but was almost the same under all conditions.

 (3) Measurement result of chemical type arsenic content in chlorella

 Table 21 and Fig. 18 show the results of measurement of the amount of arsenic by chemical species in larvae cultured at different TGC concentrations.

 twenty one ]

 As

 Trial TGC UNI As (V) MMA DMA 藝 UN6 'UN7 UN8 Total

 (III)

 MM ppm ppm PPm ppm ppm ppm ppm Pm PPm

 Ppm

 27 0. 3.016 1.316 0.310 0.015 0.008 0.062 0.010 0.006 0.015 4.757

28 1 2.334 0.551 0.121 0.009 0.002 0.019 0.012 1-3.049

29 2 2.636 0.470 0.120 0.008 0.003 0.014 0.010 1-3.260

30 4 2.944 0.518 0.157 0.004 0.002 0.018 0.011 ― 3.654

31 8 4.373 0.892 0.258 0.007 0.003 0.022 0.013 0.003 0.002 5.571

32 16 4.631 0.927 0.293 0.005 0.003 0.022 0.015 0.007 0.003 5.907 The higher the concentration of TGC, the more UN1 and MMA tended to increase, and the total arsenic content also increased.

 (4) Measurement results of chemical type arsenic content in the medium

 Table 22 and Fig. 19 show the measurement results of the medium components after culturing with different TGC concentrations.

[Table 22]

The higher the concentration of TGC added, the more UN 2 'and UN 8 were discharged, and the amount of inorganic arsenic in the medium decreased. In addition, as the concentration of TGC increased, the amount of U N 7 discharged into the medium decreased.

 (5) Total amount of arsenic in chlorella and medium

 Table 23 shows the total amount of arsenic in chlorella and medium as the total amount of inorganic arsenic and methylated arsenic other than inorganic arsenic.

[Table 23]

Accumulation of methylated arsenic in the larva was confirmed and discharged into the culture medium. No TGC added Compared with the addition of TGC, the accumulation of methylated arsenic in the algal cells increased. To medium Arsenic arsenic emissions also increased with increasing TGC concentration. When 16 mM TGC was added, about 80% of the inorganic arsenic in the medium was converted to methylated arsenic. Chlorella arsenic methylation by adding TGC The promotion effect was confirmed. Example 6

Effect of glucose concentration

 (1) Chlorella culture

 Chlorella vulgaris IAM C-629 strain, pre-cultured with Bold's Basal (BB) Medium until logarithmic growth phase, is modified to 1 x 100 ml of Bold's Basal (BB) Medium

The cells were inoculated at 105 cells / ml, and cultured under static light irradiation (4000 Lux, 24 hr irradiation) at a temperature of 25 ° C for 240 hours. The modified Bold's Basal (BB) Medium is 1/0 for 2 hydrogen phosphate and 2 hydrogen phosphate, 4 times for sodium nitrate, 2, 4, 8, 16 and 32 for glucose. Adjustment was made by adding 64 mM arsenic acid to lp pm. Preparation of the analysis sample and analysis were performed in the same manner as described above.

 (2) Chlorella growth results

 Table 24 and Fig. 20 show the growth results of chlorella depending on the glucose concentration.

[Table 24]

 Conditions for addition Medium residual amount Growth amount

 As (V) Ku, 'Leucaus P04 N03 Ku,' Lucos P04 N03

 Bacteria degree Total yield

 Ppm mk mM mg / L mg / mg / L cells / ml mg / 100ml

33 1 2 1/10 X4 0 <5 720 2.93E + 07 150.9

34 1 4 1/10 X4 0 <5 570 3.93E + 07 282.9

35 1 8 1/10 X4 0 5 500 6.55E + 07 498.9

36 1 16 1/10 X4 0 <5 233 6.98E + 07 874.2

37 1 32 1/10 X4 0 + 5 <5 1.01E + 07 1395.2

38 1 64 1/10 X4 4 <5 <5 1.57E + 08 1978.0 Under the condition where the initial concentration of phosphoric acid was 110, phosphoric acid and glucose in the medium were withered under all conditions after 2400 hours of culture. The glucose amount ₃ 2 mM or more and Blight thirst also nitric acid. The amount of growth increased as the glucose concentration increased.

 (3) Result of measurement of the amount of arsenic by chemical type in Chlorella

 Table 25 and Fig. 21 show the results of measurement of the amount of arsenic of chemical type in chlorella cultured at different glucose concentrations.

[Table 2 5]

グルコースの添加濃度が高いほど、 U N 6が増加したが、 他の化学種別ヒ素は すべて減少した。

The higher the concentration of glucose added, the more UN 6 increased, but all other arsenic species decreased.

 (4) Measurement results of chemical arsenic content in the culture medium

Table 26 and Fig. 22 show the measurement results of the culture medium components after culturing with different glucose concentrations.

[Table 26] Gelcos UNI LHVC LN2 Asfln) UNI DMA U B U 7

 B UN? "UN3 Total mM PPm m ppm PPm ppm Pm ppm ppm ppm ppm ppm ppm ppm ppm ppm

33 2 Q003 0.830 Q008 Q02B Q082 Q895

34 4 Q003 Q7B0 QOOI QOOI Q07C Q856

35 8 QO0S Q222 Q017 QO0Q Q00 Qoie QCB3 Q00B Q025 oice Q289 Q743

36 16 QG03 Q017 Q006 Q012 Q002 Q004 αοοθ aoie am 008 Q060 Q51-1 Q684

37 32 Q008 Q011 QOOS QOI1-Q003 α028 aooe Q0I Q081 Q5 9 Q654

38 64 Q004 QOI2 QOCS Q011-Q003 czi QOCB Qoce Q0S8 Q639 Q749 The higher the glucose concentration, the higher the UN 8 emissions and the lower the amount of inorganic arsenic in the medium.

 (5) Total amount of arsenic in chlorella and medium

 Table 27 shows the total amount of arsenic in chlorella and medium as the total amount of inorganic arsenic and methylated arsenic other than inorganic arsenic.

[Table 2 7.]

グルコース濃度が高いほどク口レラ中の総ヒ素は減少し、 培地中の無機ヒ素は メチル化ヒ素に変換された。 ク口レラ中のヒ素がグルコースの増加により.減少す るが、 これは培地中の無機ヒ素が少なくなるため、 細胞中のヒ素の含有量も低下 すると考えられる。 培地中の無機ヒ素のメチル化はグルコース添加によりクロレ ラの生育量が増加するため、 より多量にメチル化が進んだと考えられる。 ダルコ ース添加量を増加させることで、クロレラをより多く増殖させ、無機ヒ素の 9 5 % をメチル化できることが確認された。 産業上の利用可能性

The higher the glucose concentration, the lower the total arsenic in the larva and the inorganic arsenic in the medium was converted to methylated arsenic. The amount of arsenic in phlegm is decreased due to an increase in glucose, but this is thought to decrease the content of arsenic in the cells because the amount of inorganic arsenic in the medium decreases. Methylation of inorganic arsenic in the medium is thought to have progressed to a greater extent because the growth of chlorella increased with the addition of glucose. It was confirmed that by increasing the amount of darcos added, more chlorella was grown and 95% of inorganic arsenic could be methylated. Industrial applicability

The method of the present invention makes detoxification of harmful compounds including arsenic more practical and industrial. It is possible to provide. Harmful compounds including arsenic are converted to more harmless compounds by alkylation, and these harmless compounds are extremely stable and safe, so they are widely used in the fields of industrial waste treatment, sludge and soil environmental protection. It is extremely effective in this field.

Claims

1. A harmful compound containing at least one element selected from the group consisting of arsenic, antimony, and selenium is incorporated into a phytoplankton capable of detoxifying the harmful compound, and the phytoplankton is capable of detoxifying the harmful compound. Toxic harmful compounds Detoxifying the harmful compound by detoxifying the phytoplankton, and detoxifying the phytoplankton.  2. The method according to claim 1, wherein the phytoplankton is cultured in the presence of phosphonic acid.  Surrounding  3. The method according to claim 2, wherein the concentration of the phosphoric acid is 0.1 to 5. O mg / L in the culture solution.  4. The phytoplankton is cultured in the presence of a reducing agent that reduces at least one metal selected from the group consisting of arsenic, antimony, and selenium. The method described in 1.  5. The method according to claim 4, wherein the reducing agent is a substance having an SH group. 6. The method according to claim 5, wherein the substance having an SH group is at least one selected from the group consisting of reduced dartathione (GSH), cysteine, S-dedenosylcystine, sulforafuan, and thiodaricholic acid. .  7. The method according to any one of claims 1 to 6, wherein the cultivation of the phytoplankton is performed in the presence of a carbon source.  8. The method according to claim 7, wherein the carbon source is a saccharide or an organic acid.  9. The method according to claim 8, wherein the saccharide is selected from the group consisting of glucose, galactose, fructose, sucrose, mannose and maltose. 10. The method of claim 8, wherein the organic acid is selected from the group consisting of acetic acid, succinic acid, phosphonic acid, fumaric acid, succinic acid, and pyruvic acid. 11. The method according to any one of claims 1 to 7, wherein the phytoplankton is an alley.  1 2. The harmful compound is selected from the group consisting of arsenous acid, arsenic pentoxide, arsenic trichloride, arsenic pentachloride, arsenic sulfide compound, cyanoarsenic compound, black arsenic compound, and other arsenic inorganic salts Claims 1-1 1 A method as claimed in any one of the preceding claims.  The method according to any one of claims 1 to 12, wherein the alkylation is methylation.  14. The method according to claim 13, wherein the harmful compound is converted into a dimethyl compound or a trimethyl compound by the methylation.  15. The method according to claim 14, wherein the dimethyl compound is dimethyla / lesonylethanol (DMAE), dimethylarsonyl acetate (DMAA), dimethylarsinic acid, or arsenosjuger.  16. The method according to claim 14, wherein the trimethyl compound is arsenocholine, arsenobetaine, trimethylarsenosugar or trimethylarsinoxide.