Description

Title of Invention

IRON-OXIDIZING BACTERIA WITH GOOD RESISTANCE TO ARSENIC, A METHOD FOR PREPARING SAID BACTERIA, A MEDIUM FOR SAID BACTERIA

AND A METHOD FOR PREPARING SCORODITE USING SAID BACTERIA

Technical Field

[0001]

The present invention is related to a method for preparing scorodite. More particularly, it is related to iron-oxidizing bacteria with good resistance to arsenic, a method for preparing said bacteria, a medium for said bacteria and a method for preparing scorodite using said bacteria.

Background Art

[0002]

Non-ferrous smelted raw materials such as copper ores are contaminated with various impurities, and such impurities include arsenic (As). Arsenic is a toxic element and is desired to be discarded after it has been converted to a chemically stable form, in view of the impact on the environment. In this regard, a crystal of scorodite (FeAs0 ₄ · H ₂ 0), an iron-arsenic compound, is known to be chemically stable and is suitable for a long storage.

[0003]

Patent Documents 1 and 2 disclose a method of using thermophilic iron-oxidizing bacteria, in order to produce scorodite. More specifically, they disclose that a divalent iron ion and a trivalent arsenic ion are loaded at a predetermined ratio, and Acidianus brierleyi, one species of the thermophilic iron-oxidizing bacteria, immobilizes said arsenic ion.

Citation List Patent Literature

[0004] PTL 1 : JP2013- 180034

[0005] PTL 2: JP2014-046221

Summary of Invention

Technical Problem

[0006]

As described above, the iron-oxidizing bacteria are very useful in the production of scorodite. However, arsenic that constitutes the scorodite is generally toxic. And its toxicity has an adverse effect on the activities of iron-oxidizing bacteria. Therefore, in the production of scorodite, the ability of iron-oxidizing bacteria is restricted.

[0007]

In view of the above points, an object of the present invention is to provide iron-oxidizing bacteria having resistance to arsenic and a method for preparing the bacteria. Furthermore, a further object of the present invention is to provide a method for preparing scorodite using said iron-oxidizing bacteria.

Solution to Problem

[0008]

In light of the above objects, the present inventors have studied intensively and found that the pre-culture of iron-oxidizing bacteria in an environment having a sulfur source results in an increase in resistance to the toxicity of arsenic. Based on such a finding, the present invention encompasses, in one aspect, the following inventions:

(Invention 1)

A method for preparing iron-oxidizing bacteria having resistance to arsenic, comprising the step of culturing the iron-oxidizing bacteria in an environment where a sulfur source is present. (Invention 2)

The method according to Invention 1, wherein the iron-oxidizing bacteria are thermophilic iron-oxidizing bacteria.

(Invention 3)

The method according to Invention 1, wherein an oxidation state of sulfur in said sulfur source is +5 or less.

(Invention 4)

The method according to any one of Inventions 1 to 3, wherein said step of culturing the iron-oxidizing bacteria is a step of culturing them in the presence of a sulfur source at a constant concentration.

(Invention 5)

A method for preparing scorodite, comprising the steps of:

culturing iron-oxidizing bacteria according to the method according to any one of Inventions 1 to 4; and

producing scorodite in a solution containing said iron-oxidizing bacteria, iron and arsenic. (Invention 6)

A medium for preparing iron-oxidizing bacteria having resistance to arsenic, wherein said medium contains a sulfur source.

(Invention 7)

Iron-oxidizing bacteria prepared by the method according to any one of Inventions 1 to 4. (Invention 8)

A composition for producing scorodite, wherein said composition comprises iron-oxidizing bacteria and a sulfur source.

(Invention 9)

A method for producing scorodite, comprising: culturing iron-oxidizing bacteria in an environment where a sulfur source is present; and producing scorodite in a solution containing said iron-oxidizing bacteria, iron and arsenic, wherein producing scorodite is continued until a concentration of arsenic in the solution is less than 150 mg/L.

(Invention 10)

The method according to Invention 9, wherein said iron-oxidizing bacteria are thermophilic iron-oxidizing bacteria.

(Invention 11)

The method according to invention 9 or 10, wherein an oxidation state of sulfur in said sulfur source is +5 or less.

(Invention 12)

The method according to any one of Inventions 9 to 11, wherein culturing the iron- oxidizing bacteria is a step of culturing them in the presence of a sulfur source at a constant concentration.

(Invention 13)

A method according to any one of Inventions 9 to 12, wherein said arsenic is trivalent arsenic.

Advantageous Effects of Invention

[0009]

According to the present invention, iron-oxidizing bacteria are cultured in the presence of a sulfur source. This can result in iron-oxidizing bacteria having resistance to a toxicity of arsenic. Moreover, when such iron-oxidizing bacteria are used for the production of scorodite in an environment where arsenic is present, the iron-oxidizing bacteria grow well in such an environment. Therefore, the production of scorodite satisfactorily proceeds due to the action of iron-oxidizing bacteria. Furthermore, since such iron-oxidizing bacteria have excellent resistance to arsenic, it is possible to increase an amount of arsenic introduced when scorodite is produced. Therefore, in this sense, the production of scorodite satisfactorily proceeds.

[0010]

Incidentally, as an alternative method of reducing the negative effects due to arsenic as mentioned above, one consider an acclimated culturing of iron-oxidizing bacteria before carrying out the production of scorodite. More specifically, it is considered that during acclimated culturing, a concentration of arsenic in the culture medium is increased in a stepwise manner. For example, the acclimated culture is carried out starting with an initial arsenic concentration of 50 mg/L, and subsequently increasing the concentration by 50 mg/L, and ultimately increasing to a final concentration of 1 g/L. Then, the number of bacteria is confirmed during culturing, and at the stage after the logarithmic growth phase, the arsenic concentration is further increased. Such a method leads to a preferential growth of the iron-oxidizing bacteria having resistance to arsenic. Thus, by using such iron-oxidizing bacteria, the adverse effects of arsenic during the production of scorodite can be reduced. However, such a method spends a lot of time and efforts due to a gradual increase in the arsenic concentration. For example, it takes time to acclimate them to a targeted arsenic concentration (for example, it may take a few ten days to a few hundred days). Moreover, since any specific method for setting the degree of arsenic concentration when performing cultivation to increase the arsenic concentration after the 2nd time is not known in the art, there is a need for trial and error.

[0011]

In this regard, in one aspect of the present invention, the iron-oxidizing bacteria are cultured in the presence of a sulfur source at a constant concentration. That is, it is possible to obtain iron-oxidizing bacteria having resistance to arsenic in a simpler way as compared with the above method such as by gradually increasing the arsenic concentration.

Brief Description of Drawings

[0012] FIG. 1 is a graph showing the growth of iron-oxidizing bacteria in an environment for producing scorodite.

[0013] FIG. 2 is a graph showing a change in divalent iron in an environment for producing scorodite.

[0014] FIG. 3 is a graph showing the growth of iron-oxidizing bacteria in an environment for producing scorodite.

[0015] FIG. 4 is a photograph as observed for produced scorodite by a scanning electron microscope.

[0016] FIG. 5 is a graph showing the growth of iron-oxidizing bacteria in an environment for producing scorodite.

[0017] FIG. 6 is a graph showing a change in arsenic concentration in a solution in an environment for producing scorodite.

[0018] FIG. 7 is a graph showing a change in arsenic concentration in a solution in an environment for producing scorodite.

[0019] FIG. 8 is a graph showing a change in arsenic concentration in a solution in an environment for producing scorodite.

[0020] FIG. 9 is a graph showing a change in arsenic concentration in a solution in an environment for producing scorodite.

[0021] FIG. 10 is a graph showing a change in arsenic concentration in a solution in an environment for producing scorodite.

Description of Embodiments

[0022]

Hereinafter, specific embodiments for carrying out the present invention will be described. [0023]

1. Iron-oxidizing bacteria

1-1. Genus and species of iron-oxidizing bacteria

In one embodiment, the present invention uses iron-oxidizing bacteria. Genus and species of the iron-oxidizing bacteria include the following ones, but not particularly limited to: Acidithiobacillus genus such as Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans;

Leptospirillum genus such as Leptospirillum ferrooxidans; and

Ferroplasma genus such as Ferroplasma acidiphilum.

[0024]

1-2. Thermophilic iron-oxidizing bacteria

In one preferred embodiment, thermophilic iron-oxidizing bacteria are used. The thermophilic iron-oxidizing bacteria can grow even in a high temperature environment. Therefore, the production reaction of scorodite as described below can be carried out at a high temperature. Genus and species of the thermophilic iron-oxidizing bacteria include the following ones, but not particularly limited to:

Acidianus genus such as Acidianus brierleyi, Acidianus infernus;

Sulfobacillus genus such as Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus; Acidimicrobium genus such as Acidimicrobium ferrooxidans;

Sulfolobus genus such as Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus mirabilis;

Acidiplasma genus such as Acidiplasma cupricumulans; and

Metallosphaera genus such as Metallosphaera sedula.

[0025]

2. Regarding pre-culture 2-1. Environment for pre-culture

The iron-oxidizing bacteria can be grown as long as they are in an environment containing an iron source that will be capable of being oxidized and a sulfur source, and components essential for growth, such as potassium, phosphorus and nitrogen components. In one embodiment, the environment for the pre-culture may be a culture medium. Said culture medium may be a solid culture medium or a liquid culture medium, but it is preferably a liquid culture medium. Moreover, any culture medium can be used as long as it can promote the growth of iron-oxidizing bacteria. Typical examples include 9K medium known by a person skilled in the art, as well as culture media described in known literatures (e.g., Microbiology (1996), 142, pp. 775-783), and culture media for culturing the above- mentioned iron-oxidizing bacteria, for which information has been published by depository authorities, and the like.

[0026]

In one embodiment, the pre-culture environment can be supplemented with the sulfur source. This can provide iron-oxidizing bacteria having resistance to toxicity of arsenic (e.g., trivalent arsenic or pentavalent arsenic). Moreover, in one embodiment, the environment to be used for the pre-culture (e.g., a solution containing a culture medium or minimal nutrient) may not contain arsenic, or arsenic may not be actively added to the environment to be used for the pre-culture (e.g., in the arsenic atomic weight equivalent, 0.1 g/L or less, 0.01 g/L or less, 0.001 g/L or less, or 0 g L).

[0027]

2-2. Sulfur source

In one embodiment, the sulfur source may be any simple substance or compound containing a sulfur element(s). The sulfur atom can change to a state having an oxidation number such as 6, 5, 4, 3, 2, 1, 0, -1, and -2. Sulfur in the substance served as the sulfur source may be in the state having an oxidation number of any one, or less, or more of the oxidation numbers described above (e.g., 6 or less, 5 or less, 0 or less, 0 or more, etc.). In one embodiment, it is preferably in the state where the oxidation number of sulfur is 5 or less, in other words, it is preferably in the state which is capable of allowing further oxidation. Although the following descriptions are not intended to limit the scope of the present invention, the oxidation of sulfur would have a particular effect on the bacteria, thereby altering properties of the bacteria (e.g., resistance to toxicity of arsenic). Moreover, in one embodiment, the substance served as the sulfur source may be a material which is a solid at an ordinary temperature.

[0028]

Examples of particular substances as the sulfur source include, but not limited to: sulfurous acid (H ₂ S0 ₃ ) or salts thereof; thiosulfuric acid (H ₂ S ₂ O ₃ ) or salts thereof; tetrathionic acid (H ₂ S ₄ 0 ₆ ) or salts thereof; sulfur trioxide (S0 ₃ ); sulfur dioxide (S0 ₂ ); elemental sulfur (S); hydrogen sulfide (H ₂ S); organic sulfur compounds; and the like.

[0029]

The more the sulfur source is, the better the bacteria grow, but it is not preferred in terms of economics. Moreover, an excess amount of solid sulfur source in the scorodite producing process causes a problem of making it difficult to separate it from the produced scorodite. Therefore, the amount of sulfur source contained in the solution for the pre-culture (e.g., a culture medium) is, but not limited to, in one embodiment, in terms of the weight of the sulfur atom, from 0.1 to 50 g L, and more preferably from 0.5 to 10 g/L (for example, when 98.08 g/L of H ₂ S0 ₄ is loaded and it is converted to the weight of the sulfur atom, it calculated to be 32.06 g/L; unless otherwise indicated, the same is true for other atomic weight conversions described in the present specification and the drawings). Moreover, in one embodiment, the concentration of the sulfur source may be varied during the pre- culture. For example, during pre-culture, the sulfur source is periodically added to gradually increase the concentration of the sulfur source.

[0030]

However, for the purpose of simplifying the steps, in one embodiment, the iron-oxidizing bacteria can be cultured in the presence of a sulfur source at a constant concentration. As used herein, "cultured in the presence of a sulfur source at a constant concentration" refers to continuing the above pre-culture after initiating the pre-culture in the presence of the sulfur source, without actively adding and/or deleting the sulfur source. According to this method, the steps become simpler as compared with the above methods which gradually increase the arsenic concentration, and the present method is advantageous in terms of time and effort.

[0031]

2-3. Culture conditions for pre-culture

In addition to the components of culture medium or iron-oxidizing bacteria as described above, other culture conditions can be appropriately selected by a person skilled in the art depending on situations. Typical conditions are as follows:

Temperature: from 20 to 45 °C (preferably from 30 to 45 °C);

A period of time: from 24 to 720 hours (for example, from 24 to 168 hours (preferably from 48 to 96 hours), preferably from 48 to 300 hours, more preferably from 150 hours to 250 hours); and

pH: from 0.5 to 4.0 (preferably from 1.5 to 2.5).

In a preferred embodiment using the thermophilic iron-oxidizing bacteria, other culture conditions can be appropriately selected by a person skilled in the art depending on situations. Typical conditions are as follows:

Temperature: from 45 to 85 °C (preferably from 60 to 80 °C); A period of time: from 12 to 720 hours (for example, from 12 to 168 hours (preferably from 48 to 96 hours), preferably from 48 to 300 hours, more preferably from 150 hours to 250 hours); and

pH: from 0.5 to 4.0 (preferably from 1.0 to 2.5).

[0032]

2- 4. Resistance of iron-oxidizing bacteria to arsenic

In one embodiment, the present invention includes iron-oxidizing bacteria (for example, thermophilic iron-oxidizing bacteria) having good resistance to arsenic or a composition comprising said bacteria. The iron-oxidizing bacteria include, but not limited to, the iron- oxidizing bacteria of the above-mentioned genus names (or species names). The iron- oxidizing bacteria with good resistance to arsenic or the composition comprising said bacteria can be obtained by performing the above pre-culture. The iron-oxidizing bacteria (or a composition comprising said bacteria) thus obtained are useful in the production of scorodite. In one alternative embodiment, the present invention includes a composition comprising the iron-oxidizing bacteria (e.g., the thermophilic iron-oxidizing bacteria) and the sulfur source. In such an embodiment, the iron-oxidizing bacteria can be in the state where resistance to arsenic has been enhanced by the methods such as pre-culture and the like, alternatively they may not be in the state where resistance to arsenic has been enhanced. In the latter case, the composition is firstly stored, and it may be then used by performing the pre-culture to enhance the resistance to arsenic when it is intended to use it.

[0033]

3. Scorodite

3- 1. Reaction of scorodite

As an example, scorodite is formed by the following chemical reaction:

Fe ^{3 +} + H ₃ As0 ₄ + 2H ₂ O→ FeAs0 ₄ · 2H ₂ O + 3rT It is understood that the iron-oxidizing bacteria (for example, the thermophilic iron- oxidizing bacteria) attract iron and arsenic, localize these substances and provide a place where the above chemical reaction is caused.

[0034]

Here, when iron is divalent, it is converted to trivalent iron by action of the iron-oxidizing bacteria. Also, when arsenic is trivalent, said trivalent iron would oxidize arsenic to pentavalent one. Then, the reaction as described above would occur by getting trivalent iron and pentavalent arsenic together. Moreover, in another embodiment where the iron- oxidizing bacteria are present, since the iron-oxidizing bacteria can oxidize Fe ²⁺ , the chemical reaction as described below would also occur, thereby producing scorodite:

4FeS0 ₄ + 4H ₃ As0 ₄ + 0 ₂ + 6H ₂ O→ 4FeAs0 ₄ ·2Η ₂ O + 4H ₂ S0 ₄

[0035]

3-2. Conditions for scorodite production ( ^" for the iron-oxidizing bacterial

In one embodiment, the production of scorodite is carried out in a solution. Said solution contains the iron-oxidizing bacteria, thereby facilitating the scorodite production. As the iron-oxidizing bacteria, those which have been pre-cultured in an environment containing the sulfur source are used. An amount of iron-oxidizing bacteria is not particularly limited, but a typical amount is from 1 10 ⁵ to 1 x 10 ⁸ cells/mL, preferably from 1 x 10 ⁶ to 1 x 10 ⁷ cells/mL.

[0036]

3-3. Conditions for scorodite production (culture medium)

It is also important to satisfactorily grow the iron-oxidizing bacteria during the scorodite production, because it is considered that if the iron-oxidizing bacteria grow, then the speed of producing scorodite will be increased accordingly. Therefore, it is preferable that it includes the iron ion source and arsenic ion source required for scorodite, as well as a culture medium suitable for the growth of the iron-oxidizing bacteria. As with the pre- culture, the culture medium is not particularly limited, and may be a solid or liquid culture medium, but is typically a liquid culture medium. Also, as with the pre-culture, the liquid culture media to be used include typically 9K culture medium, culture media described in known literatures (e.g., Microbiology (1996), 142, pp. 775-783), and culture media for culturing the above-mentioned iron-oxidizing bacteria, for which information has been published by depository authorities, and the like.

[0037]

In one embodiment, the culture medium during the scorodite production may further comprise Yeast extract. This can further improve the production of scorodite. An amount of yeast extract added is not particularly limited, and is typically from 0.01 to 0.1% by weight (more preferably from 0.02 to 0.1% by weight).

[0038]

3-4. Conditions for scorodite production (for the iron ion)

The iron ion sources making up scorodite may be divalent or trivalent. Typically, they include, but not particularly limited to, divalent iron compounds such as ferrous sulfate (FeS0 ₄ ), ferrous hydroxide (II, III), and iron disulfide (FeS ₂ ); iron sulfide minerals comprising divalent irons such as pyrite and magnetic pyrite; and the like. Further, they can be used in combination with one or two or more of these. An amount of the iron ion source is not particularly limited, but it is preferable that a molar ratio to an arsenic ion as described below, i.e., a molar ratio of Fe/As is finally 1 to 2, preferably 1.2 to 1.5.

[0039]

3-5. Conditions for scorodite production (for the arsenic ion)

The arsenic ion sources making up scorodite may be trivalent or pentavalent. Also, the arsenic ion sources include typically arsenous acid (H3ASO3) and arsenic acid (H ₃ As0 ₄ ), metaarsenous acid, and the like. An amount of the arsenic ion source is not particularly limited, but it is from 0.5 to 3 g/L (an amount converted to the weight of arsenic atoms). Further, in the sense that the production efficiency of scorodite is improved, the amount of arsenic ion source is preferably 1 g/L or more, and more preferably 2 g/L or more. In one embodiment, the iron-oxidizing bacteria for use in the present invention can be grown even in an environment where the arsenic ion is present at the concentration described above. Further, in the sense that the production efficiency of scorodite is improved, the amount of arsenic ion source may also be 2 g/L or more, or 5 g/L or more. Furthermore, the upper limit value is not particularly defined, but it can be optionally determined from the economic point of view, such as a point of view for which no effect is produced by even further increase in the concentration. For example, it is lOg/L or less, 9 g/L or less, 8 g/L or less, 7 g/L or less, 6 g/L or less, 5 g/L or less, 4 g/L or less, or 3 g/L or less.

[0040]

Further, the ratio of the iron ion source and the arsenic ion source as described above is not particularly limited, but typically, the molar ratio of Fe/As is finally from 1 to 2, and preferably from 1.2 to 1.5.

[0041]

3-6. Conditions for scorodite production (for other conditions)

In addition to the components of the culture medium or the iron-oxidizing bacteria as described above, other culture conditions can be appropriately selected by a person skilled in the art depending on situations. Typical conditions are as follows:

Temperature: from 20 to 45 °C (preferably from 30 to 45 °C);

A period of term: one week or more (preferably two weeks or more); and

pH: from 0.5 to 3.0 (preferably from 1.2 to 2.5).

In a preferred embodiment using the thermophilic iron-oxidizing bacteria, other culture conditions can be also appropriately selected by a person skilled in the art depending on situations. Typical conditions are as follows:

Temperature: from 45 to 90 °C (preferably from 60 to 85 °C);

A period of time: from 1 to 28 days (preferably 2 days or more, 15 days or less, or 20 days or less); and

pH: from 0.5 to 3.0 (preferably from 1.0 to 2.0).

[0042]

Also, if the pre-culture of the iron-oxidizing bacteria has been carried out prior to the scorodite production, it may be carried out such that the components required for the production of scorodite are added to the environment of the pre-culture. Alternatively, only the iron-oxidizing bacteria subjected to the pre-culture may be recovered to add said bacteria to a fresh scorodite producing environment. The former case leads to the continuing presence of the sulfur source, and is thus preferred in terms of efficiency of the production process or resistance to arsenic.

[0043]

Further, in the embodiment of the present invention, the concentration of arsenic in the solution can be considerably reduced. Conventionally, it has been considered that an arsenic concentration above a certain level has been needed to produce scorodite. Therefore, it has been considered that as the producing process of scorodite proceeds, the concentration of arsenic in the solution is reduced, and when it is reduced to a certain level, the production reaction of scorodite substantially stops. However, by using the method according to the above embodiment, the concentration of arsenic can be reduced to a concentration lower than the conventional concentration, without substantial stop of the production reaction of scorodite.

[0044] Accordingly, the solution with the reduced concentration of arsenic allows to reduce or omit a step of adding arsenic or concentrating the solution. According to the method according to the above embodiment, the production process of scorodite can be continued, even after the arsenic concentration leaded to less than 150 mg/L, less than 100 mg/L, or less than 50 mg/L.

[0045]

Further, in the embodiment of the present invention, arsenic in the solution can be reduced in a short period of time. For example, the period of time required to decrease the concentration to less than any of the concentrations described above is 20 days or less, and more preferably 15 days or less.

[0046]

4. Regarding acclimated culture

In one aspect, the present invention may carry out an acclimated culture before performing the pre-culture as described above. Its specific conditions are not particularly limited, but for example, the acclimated culture can be carried out starting with an initial arsenic concentration of 50 mg/L, subsequently increasing the concentration by 50 mg/L, and ultimately increasing to a final concentration of lg/L. Also, other conditions are as follows, but not particularly limited to:

Temperature: from 45 to 90 °C (preferably from 60 to 85 °C);

A period of time: from 10 to 100 days (preferably 20 days or more); and

pH: from 0.5 to 3.0 (preferably from 1.0 to 2.0).

The combination of the acclimated culture with the pre-culture results in better progress of the growth of the iron-oxidizing bacteria as compared with the case where only the acclimated culture is carried out, so that the production efficiency of scorodite is increased. Examples [0047]

Hereinafter, Examples of the present invention will be illustrated, but these Examples are presented in order to provide better understanding of the present invention and its advantages, and in no way intended to limit the present invention.

[0048]

Examples 0-3

200 mL of 9K inorganic salt medium was put into a culture flask, a pH of the medium was adjusted to 1.5 with dilute sulfuric acid. This was inoculated with Acidianus brierleyi, one of the thermophilic iron-oxidizing bacteria (5 x 10 ⁵ cells/mL). Further, to this medium elemental sulfur was added to a final concentration of 10 g/L. Then, a shaking culture was carried out at 70 °C for four days.

[0049]

This medium was then transferred to a 500 ml shaking flask. When transferring , a concentration of bacteria was adjusted such that it was 1 x 10 ⁶ cells/mL. Arsenic acid (H3ASO4) and ferrous sulfate (FeS0 ₄ ) were then added as described in Table 1 to adjust As ⁵⁺ and Fe ²⁺ at the predetermined ratio. Further, Yeast extract was added such that the final concentration was 0.02%, and shaken for 12 days while maintaining the temperature at 70 °C. A change in bacterial concentration during shaking was measured by observing the bacteria on Nikon LABOPHOT-2, counting the number of bacteria within the observation field, and converting it to the number of bacteria in the whole sample. Further, a change in the concentration of As and/or Fe in the medium was measured on an ICP atomic emission spectrometer (ICP-AES, available from Seiko Instruments Co., Ltd., SPS7700). [0050]

[Table 1]

[0051]

A change in the growth rate of bacteria was shown in Table 2 and FIG. 1. Referring to Examples 0-3, up to 5 days they were in the logarithmic growth phase, and after 5 days they became saturated. When comparing Example 3 with a relatively higher arsenic concentration to Example 1 with a relatively lower arsenic concentration, their growth rates were comparable to each other. It was demonstrated that in any case, the culturing in the presence of the sulfur source resulted in better growth of the bacteria even in the subsequent environment where arsenic was present.

[0052]

[Table 2]

[0053]

Also, a change in the concentration of the divalent iron ion is shown in Table 3 and FIG. 2. They demonstrated that in all of Examples 0-3, oxidation of divalent to trivalent form of the iron ion occurred. [0054]

[Table 3]

[0055]

Comparative Example 1

Pre-culture was carried out by the same manner as in Example 3, and the production of scorodite were further carried out in the presence of As ⁵⁺ (0.5 g/L) and Fe ²⁺ (2.0 g/L). However, in the pre-culture, ferrous sulfate as an iron source was added such that it was 2 g/L as an iron atom, in place of elemental sulfur. A change in the growth rate of the bacteria is shown in Table 4 and FIG. 1. No bacteria were grown, which is believed to be due to the toxicity of arsenic. In addition, a change in the concentration of divalent iron ion is shown in Table 5 and FIG. 2. Comparison with Examples 0-3 showed that the oxidation rate of iron was decreased. Thus, it demonstrated that the action of oxidizing iron by the bacteria was inhibited, thereby inhibiting the growth.

[0056]

[Table 4] [0057]

[Table 5]

[0058]

Example 4

The pre-culture and the production process of scorodite were carried out in the same manner as in Example 1, with the exception that the amount of As ⁵⁺ was lg/L, and the amount of Fe ²⁺ was 1 g/L. A change in the growth rate of the bacteria is shown in Table 6 and FIG. 3. They showed a growth rate comparable to that of Examples 1-3, though As ⁵⁺ was higher than that of Examples 1-3. Also, it was confirmed that scorodite was produced. The recovered scorodite particles were observed by means of SEM (FIG. 4).

[0059]

Example 5

The pre-culture and the production process of scorodite were carried out in the same manner as in Example 4, with the exception that the amount of As ⁵⁺ was 2 g/L, and the amount of Fe ²⁺ was 2 g/L. A change in the growth rate of the bacteria is shown in Table 7 and FIG. 3. They showed that although As ⁵⁺ was higher than that of Example 4, the bacteria were still grown. Further, it was confirmed that as similar to the case of Example 4, scorodite was produced. [0060]

[Table 6]

[0062]

Comparative Example 2

The pre-culture and the production process of scorodite were carried out in the same manner as in Comparative Example 1. In the pre-culture, 0.5 g/L of an iron ion was added in place of elemental sulfur. As the iron ion source, ferrous sulfate was used. Further, in the pre-culture, an arsenic ion was also added such that it was in an amount of 0.5 g/L. As the arsenic ion, arsenic acid was used. A change in the growth rate of the bacteria is shown in Table 8 and FIG. 3. No bacteria were grown. Even if they were pre-cultured using arsenic, resistance to arsenic was not improved, in contrast to the pre-culture with the sulfur source. In addition, it was confirmed that scorodite was not produced, and yellow iron precipitates were produced.

[0063]

[Table 8] [0064]

Comparative Example 3

200 mL of 9K inorganic salt medium was put into a 500 ml shaking flask, and a pH of the medium was adjusted to 1.5 with dilute sulfuric acid. This was inoculated with Acidianus Brierleyi, one of the thermophilic iron-oxidizing bacteria (2.1 x 10 ⁵ cells/mL). It should be noted that this bacterium was previously subjected to a general acclimated culture. In other words, using sodium metaarsenite as an As source, the culturing was performed by allowing the bacteria to gradually acclimate from a lower concentration of As, so that resistance to As up to 1 g/L as a concentration of trivalent As was achieved. The bacterial concentration was adjusted such that it was 2.1 x 10 ⁶ cells/mL, and As ³⁺ and Fe ²⁺ were adjusted to 2 g/L, respectively, by adding arsenic acid (H ₃ As0 ₄ ) and ferrous sulfate (FeS0 ₄ ). Further, yeast extract was added such that its final concentration was 0.02 %, and shaken for 11 days while maintaining the temperature at 70 °C. A change in the bacterial concentration during shaking was measured by observing the bacteria on Nikon LABOPHOT-2, counting the number of bacteria within the observation field, and converting it to the number of bacteria in the whole sample. Further, a change in the concentration of As and/or Fe in the medium was measured on an ICP atomic emission spectrometer (ICP-AES, available from Seiko Instruments Co., Ltd., SPS7700).

[0065]

Example 6

200 mL of 9K inorganic salt medium was put into a shaking flask, and a pH of the medium was adjusted to 1.5 with dilute sulfuric acid. This was inoculated with Acidianus Brierleyi, one of the thermophilic iron-oxidizing bacteria (5 x 10 ^s cells/mL). It should be noted that this bacterium was previously subjected to a general acclimated culture. In other words, using sodium metaarsenite as an As source, the culturing was performed by allowing the bacteria to gradually acclimate from a lower concentration of As, so that resistance to As up to 1 g/L as a concentration of trivalent As was achieved. Further, to this medium elemental sulfur was added to a final concentration of 10 g/L. Then, a shaking culture was carried out at 70 °C for 4 days. This medium was then transferred to a 500 ml shaking flask. When transferring, the bacterial concentration was adjusted such that it was 2.5 x 10 ⁶ cells/mL. As ³⁺ and Fe ²⁺ were then adjusted to 2 g/L, respectively, by adding arsenic acid (H3ASO4) and ferrous sulfate (FeS0 ₄ ). Further, yeast extract was added such that its final concentration was 0.02 %, and shaken for 9 days while maintaining the temperature at 70 °C. A change in the bacterial concentration during shaking was measured by observing the bacteria on Nikon LABOPHOT-2, counting the number of bacteria within the observation field, and converting it to the number of bacteria in the whole sample. Further, a change in the concentration of As and/or Fe in the medium was measured on an ICP atomic emission spectrometer (ICP-AES, available from Seiko Instruments Co., Ltd., SPS7700).

[0066]

Changes in the growth rates of the bacteria in Comparative Example 3 and Example 6 are shown in Table 9 and FIG. 5. When the culture with sulfur was not carried out, the bacteria were grown up until the 6 ^th day, but thereafter decreased. At the end of this study, precipitates were formed. However, when confirmed by XRD, no scorodite was detected. On the other hand, when the culture with sulfur was carried out, the bacteria continued to grow up until the end of the study. At the end of this study, precipitates were formed. When confirmed by XRD, scorodite was detected. Accordingly, it could be confirmed that even if the arsenic ion was trivalent, the culturing in the presence of the sulfur source had an effect of enhancing resistance to arsenic. [0067]

[Table 9]

[0068]

Examples 7-9 and Comparative Examples 4-5

200 mL of 9K inorganic salt medium was put into a shaking flask, and a pH of the medium was adjusted to 1.5 with dilute sulfuric acid. This was inoculated with Acidianus Brierleyi, one of the thermophilic iron-oxidizing bacteria, under the conditions shown in Table 10. Further, to this medium elemental sulfur was added to the final concentration shown in Table 10. A shaking culture was then carried out at 70 °C for a period of time shown in

Table 10.

[0069]

[Table 10]

[0070]

This medium was then transferred to a 500ml shaking flask. When transferring, the concentration of bacteria was adjusted so as to meet the conditions shown in Table 11. Then, sodium arsenate (HNa ₂ As0 ₄ ) (pentavalent arsenic) or arsenious acid (NaAs0 ₂ ) (trivalent arsenic) and ferrous sulfate (FeS0 ₄ ) were added as described in Table 11 to adjust As or As and Fe at the defined ratio. Further, Yeast extract was added such that its final concentration was 0.02 %, and shaken for up to 28 days while maintaining the temperature at 70 °C. A change in the concentration of As and/or Fe in the medium was measured on an ICP automic emission spectrometer (ICP-AES, available from Seiko Instruments Co., Ltd., SPS7700). Furthermore, the concentrations of the As solutions on the 15 ^th day from initiation of the production of scorodite for Example 7, and on the 28 ^th day from initiation of the production of scorodite for Examples 8-9 and Comparative Examples 4-5 were considered to be the final concentrations. The results are shown in Table 11 and FIGs. 6-10.

[0071]

[Table 11]

[0072]

It was confirmed that in all of the tests described above, scorodite was produced. Further, as shown in Comparative Examples 4 and 5 (FIGs. 9-10), when the pre-culture with the sulfur source was not carried out, the As concentration in the solution could be simply reduced to at lowest 160 mg/L. However, as shown in Examples 7-9 (FIGs. 6-8), when pre-cultured using the sulfur source, the As concentration in the solution could be reduced to the significant concentration. In other words, they demonstrated that even if the As concentration in the solution was decreased, the production of scorodite could be progressed. [0073]

Further, from comparison of Example 7 (FIG. 6) with Comparative Example 4 (FIG. 9), it is understood that when using trivalent arsenic as an arsenic source, the pre-culture with the sulfur source can rapidly reduce the As concentration in a short period of time. In other words, the production rate of scorodite was shown to be increased by the method of the present invention.