Aerobic bacteria of the genus aquifex and their use in the degradation of complex organic pollutants in groundwater or soil

Abstract

The application discloses a kind of barren water Bacillus and its application in the degradation of composite organic pollution of underground water or soil, the bacterium is named barren water Bacillus (Bacillus inaquosorum) LH-BTEX-1, on July 30, 2024, is preserved in China Microorganism Strain Preservation Management Committee General Microorganism Center, and the preservation number is CGMCC No.31495;The above-mentioned Bacillus can effectively degrade composite organic pollution of underground water or soil, can simultaneously efficiently repair benzene series and phenol underground water and soil capacity, the above-mentioned Bacillus is low in price, green economy, easy to prepare, and repair time is short, with good long-term stability, especially suitable for the repair of benzene series and phenol contaminated underground water and soil.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of barren water bacillus, also relates to the application of the above-mentioned barren water bacillus in the degradation of complex organic pollution of groundwater or soil. BACKGROUND

[0002] Benzene series is an important part of petroleum hydrocarbon, and is a common pollutant in petroleum, chemical, coking wastewater. It also has a high content in the wastewater of synthetic rubber, paint, medicine, plastic, synthetic drug, synthetic resin, synthetic fiber, pesticide, dye and other industries. Benzene series has great volatility, and is easily released into the environment during the production, storage and transportation of the petroleum industry, causing environmental pollution and harm to the ecological system and human health. Phenol is a common pollution source in industry, which has the characteristics of high toxicity and wide distribution. It exists in the output waste of papermaking, printing and dyeing, pharmaceutical, plastic, coking and other industries. Therefore, it is of great significance to repair the soil and groundwater contaminated by benzene, cumene and phenol for environmental protection.

[0003] The repair technology of benzene contaminated site mainly includes physical repair, chemical repair and biological repair. Most of the physical and chemical repair technologies have high energy consumption, destroy the physicochemical properties of groundwater and cause secondary pollution to the environment. Biological repair has attracted more and more researchers' attention due to its economy, environmental sustainability and other advantages. Biological repair technology realizes the absorption, transformation and degradation of benzene pollutants through the metabolic function of microorganisms or plants. Current studies have shown that microbial repair has good repair effect on various benzene pollutants, and has broad application prospect in contaminated groundwater and groundwater repair. With the increasing seriousness of groundwater and groundwater organic pollution, it is of great significance to study low-cost, low-energy and green sustainable biological repair technology.

[0004] At present, some people have used bacillus to degrade benzene pollutants. For example, the patent with publication number CN 113025535 B discloses a strain of bacillus and its application in degrading benzene series. However, the bacillus degrades only one kind of benzene pollutant, and the degradation concentration of benzene is low, with a degradation rate of only 84% for benzene below 25mg / L. In addition, the patent with publication number CN116445322A discloses a salt-tolerant bacillus and its application in papermaking wastewater treatment; the patent with publication number CN118272271A provides a strain of paralysin bacillus; and the patent with publication number CN115975852A discloses the application of a strain of bacillus siamensis in phenol degradation. However, the above-mentioned strains cannot simultaneously degrade benzene series and phenol, and have low tolerance to benzene series and phenol, which is greatly limited in practical application. Therefore, it is urgent to screen a strain that can simultaneously degrade benzene series and phenol to meet people's application needs. SUMMARY

[0005] The application aims to provide a Bacillus inaquosorum and application of the Bacillus inaquosorum in degradation of composite organic pollution in groundwater or soil.

[0006] The Bacillus inaquosorum is named as Bacillus inaquosorum LH-BTEX-1, classified as Bacillus inaquosorum, and has a strain number of LH-BTEX-1, and has been preserved in the China General Microbiological Culture Collection Center, located at No. 33, Institute of Microbiology, Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing, with a preservation number of CGMCC No. 31495 and a preservation date of July 30, 2024.

[0007] The Bacillus inaquosorum LH-BTEX-1 is obtained from groundwater in a certain oil-polluted site, and grows well in a culture medium at 30-37 DEG C in an anaerobic environment after being isolated and purified, the bacterial body of the Bacillus inaquosorum is rod-shaped, 0.4-0.6 mu m x 1.1-3.0 mu m, and arranged singly or in pairs, and is a gram-positive bacterium.

[0008] The Bacillus inaquosorum can also be applied in degradation of composite organic pollution in groundwater or soil.

[0009] The composite organic pollution is organic pollution generated by one or more combinations of benzene, isopropyl benzene or phenol.

[0010] The Bacillus inaquosorum LH-BTEX-1 is inoculated into a culture medium, anaerobically cultured at 30-37 DEG C and in an environment with a pH of 7.2-7.4, and a bacterial agent containing the LH-BTEX-1 is obtained after culture, the bacterial agent is inoculated into groundwater or soil with composite organic pollution for treatment, and the treatment time is 7-19 days.

[0011] The culture medium is LB liquid medium, and the formula is 1-2% of tryptone, 0.5-1% of yeast extract, 1-2% of sodium chloride, and 2-4% of agar powder, and further, the formula of the culture medium is preferably 1% of tryptone, 0.5% of yeast extract, 1% of sodium chloride, and 2% of agar powder, and the balance is water.

[0012] The concentration of the bacterial agent is 50-100 billion / mL, and the dosage of the bacterial agent is 1-10% of the mass of the groundwater or soil.

[0013] The concentration of benzene in the groundwater or soil is 0-3000 mg / kg or 0-3000 mg / L, the concentration of cumene is 0-2000 mg / kg or 0-2000 mg / L, and the concentration of phenol is 0-400 mg / kg or 0-400 mg / L; further preferably, the concentration of benzene is 0-500 mg / kg or 0-500 mg / L, the concentration of cumene is 0-250 mg / kg or 0-250 mg / L.

[0014] Advantages: Compared with the prior art, the application has the following remarkable advantages: (1) the Bacillus inaquosorum of the application is suitable for repairing the groundwater and soil contaminated by benzene series and phenol, and the degradation rate of 40-3000 mg / L of benzene and cumene can reach more than 90% within 20d, and the degradation rate of 400 mg / L of phenol can reach more than 12% within 7d; (2) the microorganism provided by the application is low in price, green and economical, easy to prepare, short in repair time, and has good long-term stability. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a strain degradation effect schematic diagram in the screening method of the Bacillus inaquosorum LH-BTEX-1 of the application;

[0016] Figure 2 It is a phylogenetic tree of 16S rDNA sequence of the Bacillus inaquosorum LH-BTEX-1 of the application;

[0017] Figure 3 It is a 16S rDNA sequence and standard strain comparison analysis of the Bacillus inaquosorum LH-BTEX-1 of the application;

[0018] Figure 4 It is a gyrB gene and standard strain comparison analysis of the Bacillus inaquosorum LH-BTEX-1 of the application;

[0019] Figure 5 It is a VITEK BCL identification card of the Bacillus inaquosorum LH-BTEX-1 of the application;

[0020] Figure 6 It is a colony morphology (10 μm) of the Bacillus inaquosorum LH-BTEX-1 of the application;

[0021] Figure 7The colony morphology (5 μm) of the Bacillus inaquosorum LH-BTEX-1 of the present application;

[0022] Figure 8 The degradation effect (A) and growth curve (B) of the Bacillus inaquosorum on benzene;

[0023] Figure 9 The degradation effect (A) and growth curve (B) of the Bacillus inaquosorum on cumene;

[0024] Figure 10 The gas chromatogram (reaction 0 d) of benzene and cumene with an initial concentration of 1000 mg / L in Example 3;

[0025] Figure 11 The gas chromatogram (reaction 19 d) of benzene and cumene with an initial concentration of 1000 mg / L in Example 3;

[0026] Figure 12 The gas chromatogram (reaction 0 d) of benzene with an initial concentration of 2000 mg / L in Example 3;

[0027] Figure 13 The gas chromatogram (reaction 19 d) of benzene with an initial concentration of 2000 mg / L in Example 3. DETAILED DESCRIPTION

[0028] The technical solutions of the present application are further described below in combination with the drawings and specific examples. The test materials used in the examples can be purchased through a conventional route.

[0029] Example 1

[0030] The Bacillus inaquosorum LH-BTEX-1 strain of the present application is derived from the groundwater of a certain oil-contaminated site, and the strain screening method is specifically as follows:

[0031] (1) Pouring plates: pour sterilized LB medium into culture dishes, and use after the plates are cooled. A total of 21 plates are prepared.

[0032] (2) Preparation of dilutions (gradual dilution): 1-7 certain contaminated site water samples are respectively taken 1 mL; the 7 water samples are respectively placed into 250 mL triangular bottles containing 99 mL of sterile normal saline (with small glass beads), and then placed on a shaker for oscillation for 30 min to disperse the microbial cells, and then stand for 20-30 s to obtain 10-1 dilution; 1 mL of the 10-1 dilution is taken by a sterile pipette, and then transferred into a test tube containing 9 mL of normal saline, and then blown and sucked for 3 times to mix the bacterial solution and water evenly to obtain 10-2 dilution.

[0033] (3) Spreading: Label sterile plates as original, 10⁻¹, and 10⁻², with three replicates for each dilution. Use a sterile pipette to draw 0.1 mL of the original solution into each of the three numbered plates. Then, use a spreader to spread the bacterial solution evenly on the plates. Use one spreader for each dilution. Spread the 10⁻¹ and 10⁻² dilutions in the same way, remembering to change the spreader.

[0034] (4) Cultivation: Place the coated plate flat on a clean bench and let it stand for 10 minutes to allow the bacterial solution to penetrate into the culture medium. Then place the plate in a constant temperature incubator and invert it for 2-3 days.

[0035] (5) Purification: Single colonies were picked and purified 3 to 4 times to obtain 8 pure bacterial strains, numbered 7 to 14.

[0036] (6) Water sample preparation: Shake the bucket containing the contaminated groundwater evenly, and divide approximately 1.2L into 20 100mL serum bottles, 50mL per bottle. Then, add 1.14uL of benzene and 5.75uL of cumene to each serum bottle in sequence. The water sample is then obtained.

[0037] (7) Preparation of microbial agents: Prepare 100 mL of 10% LB medium and dispense it into 12 test tubes, with 5 mL of medium in each test tube. Then activate 8 native strains (7-14) and 10 native strains (1, 2-6, 15-18) with the medium for 1 day.

[0038] (8) Determination of benzene series compounds: Add 5 mL of activated bacterial solution directly to the prepared serum bottle, then seal the bottle with a rubber stopper and sealing film. Place in a shaker at 120 rpm and 15℃ for one week. After one week, remove the sample and centrifuge at 12000 rpm. Take 1 mL of the supernatant, add 9 mL of pure water, and place in a headspace vial.

[0039] The standard HJ1067-2019, "Determination of Benzene Series Compounds in Water by Gas Chromatography," specifies the determination of benzene series compound content using a headspace gas chromatograph.

[0040] Depend on Figure 1 It can be seen that the degradation efficiency of cumene by the 18 strains used was greater than 90%, but the degradation efficiency for benzene varied greatly (0-100%). Among the native strains, strains 2, 3, and 6, and strains 7, 8, 9, and 12, all showed a 100% degradation efficiency for both benzene and cumene. Strain 12, which was isolated and purified, was used to obtain the pure LH-BTEX-1 strain.

[0041] Example 2

[0042] The strain L H-BTEX-1 screened in Example 1 is identified, and the strain L H-BTEX-1 is Bacillus inaquosorum through identification; as shown in Figure 6 and Figure 7 The strain L H-BTEX-1 is rod-shaped, 0.4-0.6 μm x 1.1-3.0 μm, arranged singly or in pairs, and is a gram-positive bacterium.

[0043] The 16S rRNA full sequence of the strain L H-BTEX-1 is as follows (SEQ ID NO. 1):

[0044]

[0045]

[0046] The phylogenetic tree is constructed as shown in Figure 2 , the strain L H-BTEX-1 is identified as Bacillus inaquosorum; as shown in Figure 3 , the 16S rDNA sequence of L H-BTEX-1 and the standard strain comparison analysis.

[0047] The identification of the strain L H-BTEX-1 (gyrB gene and standard strain comparison analysis is as follows)

[0048]

[0049]

[0050] The gyrB gene of L H-BTEX-1 and the standard strain comparison analysis are as shown in Figure 4 ; as shown in Figure 5 , the VITEK BCL identification card of L H-BTEX-1.

[0051] Example 3

[0052] The benzene, cumene and phenol degradation test of the strain L H-BTEX-1 of the application is carried out, and the specific steps are as follows:

[0053] (1) Strain activation: the single colony of the pure strain is picked up with an inoculation needle into LB liquid medium, and the culture solution is incubated at 37℃ in a shaking incubator at 180r / min for >1d.

[0054] (2) Inorganic salt medium: K2HPO4·3H2O 1g / L, KH2PO4 1g / L, (NH4)2SO4 0.5g / L, MgSO4·7H2O 0.36g / L, KNO3 0.5g / L, CaCl2 0.001g / L, trace element stock solution 1mL / L.

[0055] (3) Trace element mother liquor: FeCl2·4H2O 1500 mg / L, Na2MoO4·2H2O 24 mg / L, ZnCl2 70 mg / L, MnCl2·4H2O 6 mg / L, CoCl2·6H2O 190 mg / L, MnSO4·7H2O 100 mg / L, CuCl2·2H2O 2 mg / L, NiCl2·6H2O 24 mg / L.

[0056] (4) Mixing: Add the sterilized inorganic salt medium and the mixture of benzene and cumene and the phenol solution with different concentration gradients (the initial concentration of benzene is 250, 500, 740, 1244, 2146 mg / L respectively; the initial concentration of cumene is 40, 80, 88, 250, 2000 mg / L respectively; the initial concentration of phenol is 400 mg / L) into the serum bottle, and then add 5 mL of activated bacterial solution directly into the serum bottle with different pollutant concentrations using a pipette gun, a total of 50 mL. Seal the bottle opening with a rubber plug and sealing film, set an anaerobic environment, and set the test conditions as follows: 160 rpm / min, 15°C, and closed. This test sets a blank group. This test sets a parallel group.

[0057] (5) Sampling and analysis of benzene series: After 0, 4, 10, and 19 days of reaction, sampling is performed according to "Determination of Benzene Series in Water by Gas Chromatography" (HJ1067-2019) using headspace-gas chromatography to determine the content of benzene series and using an enzyme marker to determine OD600. When gas chromatography is used for detection, the sample is diluted by 200 times; sampling and analysis of phenol: after 0, 7 days of reaction, sampling is performed according to "Determination of Volatile Phenol in Water by 4-Aminoantipyrine Spectrophotometry" (HJ 503-2009) using a spectrophotometer to determine the content of volatile phenol.

[0058] Figure 8 The results show that the strain LH-BTEX-1 has good degradation effect on benzene and growth curve. Compared with the control group of 0 mg / L, the growth of the strain is inhibited in the early stage, and the inhibition is smaller in the later stage. At the benzene concentrations of 250 mg / L and 500 mg / L, the benzene degradation rate of the strain is about 70% in 4 days, and the degradation rate increases to more than 85% and 95% in 10 days and 19 days, respectively, indicating that most of the benzene can be reduced within 19 days. At the benzene concentrations of 740 mg / L, 1244 mg / L, and 2146 mg / L, the growth of the strain is inhibited to a certain extent in the early and late stages, and the corresponding degradation rate decreases slightly, with a degradation rate of about 65% in 4 days, and the degradation rate remains at about 75% and 91% in 10 days and 19 days, respectively. It is shown that high concentration of benzene can inhibit the degradation ability and growth of the strain, but the inhibition is not obvious.

[0059] Figure 9The degradation effect of the strain LH-BTEX-1 on cumene and the growth curve are shown, and the growth of the strain is inhibited in the early stage and the inhibition is small in the later stage under different cumene concentrations, relative to the control 0 mg / L. The cumene degradation rate of the strain is about 75% at 4 d and 10 d under the cumene concentration of 40 mg / L, and the degradation rate is above 98% at 19 d, indicating that most of the cumene can be reduced within 19 days. The degradation rate is about 80% at 4 d and 10 d under the cumene concentrations of 80 mg / L and 250 mg / L, and the degradation rate is about 95% at 19 d. The degradation rate is about 93% at 4 d under the cumene concentration of 2000 mg / L, and the degradation rate is as high as 99% at 19 d. It is indicated that the strain has strong degradation capacity for high-concentration cumene.

[0060] Table 1, degradation effect of Bacillus inaquosorum on phenol

[0061] Serial number Reaction 0 d phenol concentration (mg / L) Reaction 7 d phenol concentration (mg / L) Phenol degradation rate (%) 1 385 309 19.74 2 404 381 5.69

[0062] Table 1 is the degradation effect of Bacillus inaquosorum on phenol. As shown in Table 1, the phenol degradation rate of the strain is about 12% at 7 d under the phenol concentration of about 400 mg / L.

[0063] Example 4

[0064] The strain LH-BTEX-1 of the application is used for degradation experiment in organic contaminated groundwater, and the specific steps are as follows:

[0065] The Bacillus inaquosorum LH-BTEX-1 is inoculated in a culture medium, and the formula of the culture medium is as follows: 1% of proteose peptone, 0.5% of yeast extract, 1% of sodium chloride, 2% of agar powder, and the rest is water; the culture medium is cultured anaerobically at 30-37°C and pH 7.2-7.4, and then a bacterial agent containing the LH-BTEX-1 is obtained, the bacterial agent is inoculated in the composite organic contaminated groundwater for treatment, and the degradation results are shown in Table 2:

[0066] Table 2, degradation results of Bacillus inaquosorum on benzene in contaminated site groundwater

[0067]

[0068] As shown in Table 2, after 7 d of reaction, the sample added with the bacterial liquid has a benzene degradation rate of more than 80% on the contaminated site water sample, and the benzene degradation rate is as high as 99% under the addition of 10% of the bacterial liquid, and after 17 d of reaction, the bacterial liquid can degrade 70%-90% of benzene in the on-site water sample.

[0069] Table 3, degradation results of Bacillus inaquosorum on cumene in contaminated site groundwater

[0070]

[0071]

[0072] As shown in Table 3, after 7 days of reaction, the sample added with the bacterial solution has 100% degradation rate of cumene (initial concentration less than 10 mg / L) in the contaminated site water sample.

[0073] Table 4, degradation results of phenol in the contaminated site groundwater by Bacillus inaquosorum

[0074]

[0075] As shown in Table 3, after 7 days of reaction, the sample added with the bacterial solution has 47% degradation rate of phenol (initial concentration 417 mg / L) in the contaminated site water sample.

[0076] Example 5

[0077] The strain LH-BTEX-1 of the application is used for degradation experiment in the organic contaminated soil, and the specific steps are as follows:

[0078] The Bacillus inaquosorum LH-BTEX-1 is inoculated in the culture medium, the formula of the culture medium is 1% of proteose peptone, 0.5% of yeast extract, 1% of sodium chloride, 2% of agar powder, and the rest is water; anaerobic culture is carried out at 30-37°C and pH 7.2-7.4, and the bacterial agent containing LH-BTEX-1 is obtained after culture, and the bacterial agent is inoculated in the composite organic contaminated soil for treatment.

[0079] Table 5, degradation results of cumene in the contaminated site soil by Bacillus inaquosorum

[0080]

[0081] As shown in the above table, compared with the blank sample, after 7 days of reaction, the sample added with the bacterial solution has more than 90% degradation rate of cumene in the contaminated site soil sample, after 17 days of reaction, the sample added with the bacterial solution can degrade nearly 100% cumene in the site soil sample, and the sample with a rotation speed of 40 r has better effect than the sample at rest.

[0082] Table 6, degradation results of benzene in the contaminated site soil by Bacillus inaquosorum

[0083]

[0084]

[0085] From the above table, compared with the blank sample, the sample added with the bacterial solution has a benzene degradation rate of more than 70% for the contaminated site soil sample after 7 days of reaction, and the sample added with the bacterial solution can degrade nearly 100% of benzene in the site soil sample after 17 days of reaction, and the sample with a rotation speed of 40 r is better than the sample at rest.

[0086] Example 6

[0087] The strain LH-BTEX-1 of the present application was used for a benzene degradation experiment with a low addition ratio in an organic contaminated groundwater, wherein the concentration of the bacterial agent was 50-100 billion / mL (range value), and the addition amount of the bacterial agent was 0.017-0.084% of the mass of the groundwater or soil, and the results were as follows:

[0088] Table 7: Benzene degradation results of Bacillus inaquosorum in contaminated site groundwater (low addition ratio)

[0089]

[0090] From the above table, when the addition ratio of the bacterial solution is reduced to below 0.1%, the degradation effect on benzene (with an initial concentration of about 300 mg / L) in the site groundwater is still good, and the degradation effect increases with the increase of the addition ratio, from 60% (when the addition ratio of the bacterial solution is 0.017%) to 100% (when the addition ratio of the bacterial solution is 0.084%).

[0091] In summary, when the concentrations of benzene and cumene are relatively high, i.e. 1000-2000 mg / L, or even when the concentration of cumene is 3000 mg / L, the growth of the strain is limited to a certain extent, but the degradation rate of benzene and cumene by LH-BTEX-1 is still as high as 92% and 98% respectively within 19 days. When the concentrations of benzene and cumene are relatively low, i.e. 40-750 mg / L, the growth of the strain is less inhibited in the later stage. In terms of degradation effect, the degradation rates of benzene and cumene by LH-BTEX-1 are 96% and 98% respectively within 19 days. The strain has a phenol degradation rate of about 12% for a phenol concentration of about 400 mg / L within 7 days. The addition amount of the bacterial solution is preferably 10% of the mass of the soil or groundwater. Therefore, the Bacillus inaquosorum LH-BTEX-1 of the present application can simultaneously and efficiently repair the groundwater and soil contaminated by benzene series and phenol, has a short repair time, and has good long-term stability.

Claims

1. A paucisporus Bacillus sp. characterized in that, The strain is named Bacillus inaquosorum LH-BTEX-1, and was preserved in China General Microbiological Culture Collection Center on July 30, 2024, with a preservation number of CGMCC No. 31495.

2. Use of the oligotrophic A. hydrophila according to claim 1 for the degradation of complex organic pollution of ground water or soil, characterized in that, The complex organic pollution is organic pollution caused by one or more of benzene, cumene or phenol.

3. Use according to claim 2, characterized in that, Bacillus inaquosorum LH-BTEX-1 is inoculated into a culture medium and cultured in an anaerobic environment, and a bacterial agent containing LH-BTEX-1 is obtained after culture, which is inoculated into the complex organic pollution groundwater or soil for treatment.

4. Use according to claim 3, characterized in that, The culture medium is LB liquid medium, and the formula is as follows: 1%-2% of tryptone, 0.5%-1% of yeast extract, 1%-2% of sodium chloride, 2%-4% of agar powder, and the rest is water.

5. Use according to claim 3, characterized in that, The anaerobic environment is at a temperature of 30-37 DEG C and a pH of 7.2-7.

4.

6. Use according to claim 3, characterized in that, The culture is for 2-3 days.

7. Use according to claim 3, characterized in that, The concentration of the bacterial agent is 50-100 billion / mL, and the dosage of the bacterial agent is 1-10% of the mass of the groundwater or soil.

8. Use according to claim 3, characterized in that, The concentration of benzene in the groundwater or soil is 0-3000 mg / kg or 0-3000 mg / L, the concentration of cumene is 0-2000 mg / kg or 0-2000 mg / L, and the concentration of phenol is 0-400 mg / kg or 0-400 mg / L.

9. Use according to claim 3, characterized in that, The treatment is for 7-19 days.

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