Benzene-degrading microorganisms, benzene-degrading composition, and benzene-degrading method

Abstract

This invention provides novel benzene-degrading microorganisms exhibiting different characteristics, a benzene-degrading treatment composition, and a benzene-degrading treatment method. [Solution] Microorganisms deposited under accession number NITE BP-04196, microorganisms deposited under accession number NITE BP-04197, and compositions for benzene decomposition treatment containing them are provided. A method for benzene decomposition treatment is also provided, which includes contacting these microorganisms with benzene contained in water to be treated.

Description

[Technical Field]

[0001] This disclosure relates to benzene-degrading microorganisms, compositions for benzene degradation treatment, and methods for benzene degradation treatment. [Background technology]

[0002] Benzene is the simplest aromatic hydrocarbon and is used as a material for producing other benzene ring-containing compounds and organic compounds such as cyclohexane, as well as as an organic solvent. Benzene is used in industrial processes to manufacture a wide range of products, such as organic pigments, dyes, synthetic rubber, synthetic detergents, and pesticides. On the other hand, benzene has been reported to be toxic, and in the carcinogenicity classification criteria of the International Agency for Research on Cancer and other organizations, benzene is classified as 1A, meaning it is carcinogenic to humans. In Japan, the wastewater standard value, i.e., the upper limit of the permissible benzene concentration in wastewater, is set at 0.1 mg / L under the Water Pollution Control Law, and appropriate treatment to remove benzene is required from wastewater discharged from factories and other facilities.

[0003] Conventional methods for removing benzene have involved adsorbing it onto activated carbon and then burning it. While this method is effective, it has the problem of incurring not only the cost of purchasing activated carbon but also the cost of burning it (industrial waste disposal costs).

[0004] If benzene can be decomposed by microorganisms, it may be possible to process it safely and inexpensively. For example, Non-Patent Document 1 describes the Achromobacter xylosoxidans Y234 strain, which decomposes benzene and ethylbenzene. However, there are few reported examples of microorganisms that can decompose benzene, and their effectiveness is limited. Furthermore, industrial use of benzene-degrading microorganisms requires a large number of cells. When microorganisms are cultured using benzene as a nutrient, wastewater treatment of the culture medium itself becomes a problem. Therefore, it is desirable to culture microorganisms with organic substances other than benzene (more harmless substances) and then efficiently culture benzene with the microorganisms that have grown in this way. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Biotechnol Lett (2006) 28:1293-1298 [Overview of the project]

[0006] Different microbial strains may exhibit different substrate degradation capabilities and environmental suitability, but currently, the selection of benzene-degrading microorganisms is limited. This disclosure provides novel benzene-degrading microorganisms exhibiting different characteristics, a benzene-degrading treatment composition, and a benzene-degrading treatment method.

[0007] This disclosure includes at least the following embodiments: [1] A benzene decomposition treatment composition comprising microorganisms deposited under accession number NITE BP-04196 and / or microorganisms deposited under accession number NITE BP-04197. [2] A method for decomposing benzene, comprising contacting microorganisms deposited under accession number NITE BP-04196 and / or microorganisms deposited under accession number NITE BP-04197 with benzene contained in water to be treated. [3] The method according to [2], comprising growing the microorganisms in a BTEX-free liquid culture medium containing a protein hydrolysate before contacting the microorganisms with benzene contained in the water to be treated. [4] The method according to [3], wherein the protein hydrolysate comprises at least one selected from the group consisting of peptone, cassiteone, and tryptone. [5] The method according to [3] or [4], comprising growing the microorganism in a BTEX-free liquid medium containing the protein hydrolysate, and then contacting the microorganism with an aqueous solution having a known benzene concentration in the range of 1 to 1,000 mg / L for 14 to 48 hours before contacting the microorganism with benzene contained in the water to be treated. [6] Microorganisms deposited under accession number NITE BP-04196. [7] Microorganisms deposited under accession number NITE BP-04197. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows the time course of benzene concentration measured in a 5L strain sample (a) and an approximation to the Michaelis-Menten equation (b). [Figure 2] Figure 2 shows the time course of benzene concentration measured in a sample of strain 1U (a), and an approximation to the Michaelis-Menten equation (b). [Figure 3] Figure 3 shows a graph comparing the 5L strain and the 1U strain, with the benzene concentration of the test sample plotted on the horizontal axis and the benzene degradation rate per unit of protein on the vertical axis. [Modes for carrying out the invention]

[0009] This disclosure provides microorganisms deposited under accession number NITE BP-04196 and microorganisms deposited under accession number NITE BP-04197. In one embodiment, this disclosure provides a benzene decomposition treatment composition comprising microorganisms deposited under accession number NITE BP-04196 and / or microorganisms deposited under accession number NITE BP-04197. In another embodiment, this disclosure provides a benzene decomposition treatment method comprising contacting microorganisms deposited under accession number NITE BP-04196 and / or microorganisms deposited under accession number NITE BP-04197 with benzene contained in water to be treated.

[0010] The microorganisms deposited under accession numbers NITE BP-04196 and NITE BP-04197 are novel strains isolated from soil by the inventors. Based on 16S rDNA sequencing analysis, they were presumed to be Achromobacter bacteria, but based on their nucleotide sequence, benzene degradation characteristics, and / or growth characteristics, they were found to be non-identical to known benzene-degrading Achromobacter xylosoxidans strains. In this disclosure, the microorganism of NITE BP-04196 is also referred to as strain 1U. In this disclosure, the microorganism of NITE BP-04197 is also referred to as strain 5L. Strains 1U and 5L were internationally deposited on November 11, 2024, at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE) (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture).

[0011] In various embodiments of the composition, the composition may contain, in addition to the microorganism, a carrier or excipient suitable for maintaining the viable microorganism, such as water. The composition may also contain cryoprotective agents such as glycerin, dimethyl sulfoxide, skim milk, or sodium glutamate. The composition may also contain benzene.

[0012] In embodiments of the benzene decomposition treatment method, the microorganisms to be contacted with benzene may be provided in the form of the above-described composition. A preferred embodiment of the method may include growing the microorganisms in a BTEX-free liquid culture medium containing protein hydrolysates before contacting the microorganisms with benzene contained in the water to be treated. This allows the microorganisms of this disclosure to grow remarkably efficiently, and a large amount of microbial cells to be obtained in a short time.

[0013] The benzene-degrading microorganisms of the present disclosure can be cultured using an aqueous solution containing benzene as a nutrient source as a medium. However, in that case, the growth rate is slow, and there are risks associated with handling a solution containing benzene for culturing. Furthermore, it may be necessary to treat the waste liquid containing benzene, which may limit its practicality. As a result of intensive research, the inventor has discovered that the benzene-degrading microorganisms of the present disclosure can grow remarkably efficiently in a BTEX-free liquid medium containing a protein hydrolyzate. This has shortened the culturing period for growth to at least half or less, and has also solved the above problems regarding safety and waste liquid treatment. However, when these microorganisms are grown in a BTEX-free liquid medium that does not contain benzene, it has been discovered that they temporarily lose their benzene-degrading ability significantly. As a solution to this problem, a re-induction method, which will be detailed in the next paragraph, has been found. As will be understood by those skilled in the art, BTEX-free means that it does not contain any of benzene ( B benzene), toluene ( T oluene), ethylbenzene ( E ethylbenzene), and xylene ( X xylene). Protein hydrolyzates are known to those skilled in the art and can be, for example, enzymatic digests of proteins. Examples of suitable protein hydrolyzates include peptone, casitone, and tryptone. That is, the protein hydrolyzate contained in the BTEX-free liquid medium may contain at least one selected from the group consisting of peptone, casitone, and tryptone. The BTEX-free liquid medium may contain, for example, 1 to 30 g / L of a protein hydrolyzate. The BTEX-free liquid medium may contain glycerin and / or glucose, which can be utilized as a carbon source. The BTEX-free liquid medium may further contain yeast extract and / or meat extract as additional nutrient sources. The BTEX-free liquid medium preferably contains, for example, casitone, glycerin, and yeast extract. More specifically, the CGY medium, which is an aqueous solution containing 5 g / L of casitone, 5 g / L of glycerin, and 1 g / L of yeast extract, is suitable.

[0014] After culturing the microorganisms of the present disclosure in a BTEX-free liquid medium and before using the microorganisms for benzene decomposition treatment of the treated water, it is preferable to contact the microorganisms with an aqueous solution having a known benzene concentration within the range of 1 to 1,000 mg / L (for example, it may be the benzene medium described later in the Examples section) for 14 to 48 hours. This benzene concentration may be, for example, within the range of 5 to 500 mg / L, or 10 to 100 mg / L. For example, the microorganisms of the present disclosure after culturing and growing in a BTEX-free liquid medium can be contacted with the above benzene aqueous solution for about 14 to 48 hours before, or after, entrapping and immobilizing them in the polymer gel described later, or before, or after, attaching and immobilizing them on the surface of the polymer carrier described later. It has been found that this preparation step significantly induces or enhances the BTEX degradation ability of the microorganisms of the present disclosure grown in a BTEX-free liquid medium. This preparation step can therefore be described as a step of re-inducing benzene degradation activity. This re-induction step may be accompanied by stirring, shaking, etc., similar to normal culturing. Those skilled in the art understand that the suspension medium of the microbial suspension can be exchanged by appropriately combining centrifugation, washing of the pellet, etc.

[0015] When the microorganisms are contacted with benzene contained in the treated water, the treated water can be wastewater, for example, industrial wastewater discharged from factories, etc. The 5L strain was found to have a faster decomposition rate and be suitable for the decomposition of benzene in a relatively high concentration range. The 1U strain was found to have the characteristic that it can maintain a relatively fast decomposition rate even when the benzene concentration is low, for example, 20 mg / L or less, or 10 mg / L or less. Since it is beneficial to efficiently treat benzene-containing wastewater close to the drainage standard value of 0.1 mg / L and clear the standard value, this characteristic of the 1U strain is highly useful.

[0016] In certain embodiments, the microorganisms are immobilized by being encapsulated in a polymer gel acting as a water-insoluble solid carrier, and then brought into contact with the treated water containing benzene (e.g., industrial wastewater). This embodiment is particularly preferred because it prevents the leakage of the microorganisms themselves and ensures that even small amounts of microorganisms can be reliably immobilized and utilized. The technique of encapsulating and immobilizing live microorganisms in a polymer gel is known as inclusive immobilization and is publicly known.

[0017] Examples of polymer gels that can be used for encapsulation and immobilization include, but are not limited to, gels based on natural polymers such as agar, alginic acid, and carrageenan, as well as gels based on synthetic polymers such as polyalkylene glycol (particularly polyethylene glycol or polypropylene glycol), polyacrylamide, and polyvinyl alcohol. Polymer gels with encapsulated and immobilized microorganisms can be used, for example, in the form of beads with a diameter of 2 to 10 mm, more preferably 3 to 5 mm. Instead of encapsulation and immobilization in a polymer gel, microorganisms may be attached and immobilized on the surface of a porous polymer carrier, which is typically sponge-like. Crosslinking agents known to those skilled in the art, such as glutaraldehyde, may be used for attachment and immobilization.

[0018] The 1U and 5L strains may similarly exhibit the ability to decompose not only benzene but also other BTEX compounds, namely toluene, ethylbenzene, and / or xylene. Therefore, similar to the benzene decomposition methods described above, compositions and decomposition methods for BTEX containing toluene, ethylbenzene, and / or xylene, utilizing the 1U and / or 5L strains, are also conceivable. [Examples]

[0019] Soil samples were collected from areas thought to have a history of contact with benzene. Microorganisms capable of degrading benzene were searched for in these soil samples, and several candidate microorganisms were isolated. From these, two benzene-degrading microorganisms, strain 1U and strain 5L, capable of degrading benzene and proliferating, were identified. Although strains 1U and 5L had different 16S rDNA sequences, they were presumed to be Achromobacter bacteria based on sequence similarity.

[0020] While strains 1U and 5L can utilize benzene, their growth was very slow when cultured in benzene medium, requiring approximately one month to obtain a sufficient yield from the colonies. However, strains 1U and 5L could be rapidly grown when cultured in BTEX-free liquid medium containing at least protein hydrolysates (e.g., peptone, cassitone, and / or tryptone) (e.g., BTEX-free CGY liquid medium containing cassitone, glycerin, and yeast extract). The bacterial pellets grown in this manner for four days were washed three times with inorganic medium and resuspended in benzene medium. The benzene medium in this example was composed of benzene 50 mg / L, K2HPO4 62.5 mg / L, KH2PO4 9.76 mg / L, (NH4)2SO4 500 mg / L, MgSO4·7H2O 200 mg / L, CaCl2·2H2O 25 mg / L, NaCl 50 mg / L, FeCl3·6H2O 20 mg / L, NaHCO3 6.35 mg / L, with the remainder being water. It was found that the benzene-degrading activity of the 1U and 5L strains had an inducible aspect. That is, it was observed that more complete benzene-degrading activity was exhibited after further incubation or culture in benzene medium for approximately 14-48 hours than immediately after growth in BTEX-free liquid medium. In the following experiments, benzene-degrading activity was tested after growth of microorganisms in BTEX-free liquid medium (CGY medium), followed by resuspending in benzene medium and post-culturing for 12 hours.

[0021] In typical experiments, 10 mL of the above post-culture medium was added to 40 mL of fresh benzene medium to prepare the test sample. A control sample (Abiotic Control) was also prepared using the same post-culture medium that had been autoclaved and sterilized instead of the post-culture medium. These samples were kept in sealed vials and shaken at 100 rpm at 28°C for several hours, and samples were taken over time to measure the benzene concentration in the solution by gas chromatography (GC-FID).

[0022] Figure 1a shows the time course of benzene concentration measured in a 5L strain sample. A significant decrease in benzene concentration was observed starting 6 hours after the start of the test (18 hours after the microorganism first came into contact with benzene). Within 5 hours from that point, benzene was decomposed to below the lower limit of quantification. Figure 1b shows an approximation to the Michaelis-Menten equation based on the measurements in Figure 1a. In this measurement, V max The ratio was 14.71, and the measured protein concentration derived from bacteria was 96.0 mg / L, so the maximum specific degradation rate of the 5L strain was 0.153 mg-Benzene mg -1 -Protein h -1 This was the calculation.

[0023] Figure 2a shows the time course of benzene concentration measured in a sample of strain 1U. The benzene concentration began to decrease significantly from 2 hours after the start of the test (14 hours after the bacteria first came into contact with benzene), and within 8 hours from that point, the benzene was decomposed to below the lower limit of quantifiable value. Figure 2b shows an approximation to the Michaelis-Menten equation based on the measurements in Figure 2a. In this measurement, V max The ratio was 4.97, and the protein concentration was 41.6 mg / L, so the maximum specific degradation rate of the 1U strain was 0.120 mg-Benzene mg -1 -Protein h -1 This was the calculation.

[0024] FIG. 3 related to FIGS. 1b and 2b shows a graph comparing the 5L strain and the 1U strain by plotting the benzene concentration of the test sample on the horizontal axis and the benzene degradation rate per protein mass on the vertical axis. The K m [mg L -1 of the 5L strain is 15.10, and the K m of the 1U strain is 3.79. As described above, the 5L strain has a high maximum specific degradation rate and a high K m and was considered suitable for the degradation of benzene in the high-concentration range. In contrast, the 1U strain is understood to have the characteristic of maintaining a relatively fast degradation rate even when the benzene concentration is low and is suitable for the degradation of benzene in the low-concentration range.

[0025] Non-Patent Document 1 describes that for the Achromobacter xylosoxidans Y234 strain that degrades benzene, the oxygen consumption rate is 0.041 mg-O2 / mg-CDW / h. Since the chemical formula for the oxidation of benzene is C6H6 + 7.5O2 → 6CO2 + 3H2O, based on the chemical oxygen demand, 7.5 moles of O2 (molecular weight 32) are required to oxidize 1 mole of benzene (molecular weight 78). That is, ideally, 0.325 parts by mass of benzene can be decomposed with 1 part by mass of oxygen. If the CDW concentration representing the cell dry weight is fixed at, for example, 1000 mg-CDW / L, the above oxygen consumption rate becomes 41 mg-O2 / L / h. When this amount of oxygen is converted to the amount of benzene decomposition, the benzene degradation rate becomes 13.3 mg-Benzene / L / h. On the other hand, the 5L strain has a benzene degradation rate of 0.153 mg-Benzene mg -1 -Protein h -1 . Assuming that the protein accounts for 55% of the bacterial CDW (Trends Microbiol. 2016, 24:12-25), this benzene degradation rate is 0.08415 mg-Benzene mg -1 -CDW h -1As a result, in the case of 1000 mg-CDW / L, the degradation rate is 84.15 mg-Benzene / L / h, which is more than six times higher. Similarly, the benzene degradation rate of strain 1U is calculated to be 66.00 mg-Benzene / L / h in the case of 1000 mg-CDW / L, which is also about five times higher. Strains 1U and 5L in this disclosure represent BTX-degrading microorganisms with extremely high degradation rates.

Claims

1. A benzene decomposition treatment composition comprising microorganisms deposited under accession number NITE BP-04196 and / or microorganisms deposited under accession number NITE BP-04197.

2. A method for decomposing benzene, comprising contacting microorganisms deposited under accession number NITE BP-04196 and / or microorganisms deposited under accession number NITE BP-04197 with benzene contained in water to be treated.

3. The method according to claim 2, further comprising growing the microorganisms in a BTEX-free liquid culture medium containing a protein hydrolysate before contacting the microorganisms with benzene contained in the water to be treated.

4. The method according to claim 3, wherein the protein hydrolysate comprises at least one selected from the group consisting of peptone, cassiteone, and tryptone.

5. The method according to claim 3 or 4, comprising growing the microorganism in a BTEX-free liquid culture medium containing the protein hydrolysate, and then contacting the microorganism with an aqueous solution having a known benzene concentration in the range of 1 to 1,000 mg / L for 14 to 48 hours, before contacting the microorganism with benzene contained in the water to be treated.

6. Microorganisms deposited under accession number NITE BP-04196.

7. Microorganisms deposited under accession number NITE BP-04197.

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