Bacillus sp. And application thereof in degradation of composite organic pollution of underground water or soil

Abstract

The invention discloses a water depletion bacillus and application thereof in degradation of composite organic pollution of underground water or soil, the water depletion bacillus is named as water depletion bacillus LH-BTEX-1, the water depletion bacillus LH-BTEX-1 is preserved in China General Microbiological Culture Collection Center (CGMCC) on July 30, 2024, and the preservation number is CGMCC No.31495. The water depletion bacillus LH-BTEX-1 is named as water depletion bacillus LH-BTEX-1, and the water depletion bacillus LH-BTEX-1 is named as water depletion bacillus LH-BTEX-1 and is named as water depletion bacillus LH-BTEX-1. The bacillus can effectively degrade composite organic pollution of underground water or soil, can efficiently repair benzene series and phenol underground water and soil at the same time, is low in price, green, economical, easy to prepare and short in repair time, has good long-term stability, and can be widely applied to the field of soil remediation. The method is especially suitable for remediation of underground water and soil polluted by benzene series and phenol.

Description

Technical Field

[0001] The invention relates to a barren water bacillus and also relates to the application of the barren water bacillus in degrading the composite organic pollution of groundwater or soil. Background Art

[0002] Benzene series are important components of petroleum hydrocarbons and are common pollutants in wastewater from petroleum, chemical industry, and coking. They also have high concentrations in wastewater from industries such as synthetic rubber, paint, medicine, plastic, synthetic drugs, synthetic resin, synthetic fiber, pesticide, and dye. Benzene series are highly volatile and are easily released into the environment during the production, storage, and transportation of the petroleum industry, causing environmental pollution and posing a threat to the ecosystem and human health. Phenol is a common source of pollution in industry. It is highly toxic and widely distributed. It is present in waste output from various industrial fields such as papermaking, printing and dyeing, pharmaceuticals, plastics, and coking. Therefore, remediation of soil and groundwater contaminated by benzene, cumene, and phenol is of great significance to environmental protection.

[0003] The remediation technologies for benzene contaminated sites mainly include physical remediation, chemical remediation and biological remediation. Most physical and chemical remediation technologies have problems such as high energy consumption, damage to the physical and chemical properties of groundwater and secondary pollution to the environment. However, biological remediation has attracted more and more attention from researchers due to its advantages such as economy and environmental sustainability. Bioremediation technology achieves the absorption, transformation and degradation of benzene pollutants through the metabolic functions of microorganisms or plants. Current studies have shown that microbial remediation has a good remediation effect on a variety of benzene pollutants and has broad application prospects in the remediation of contaminated groundwater and groundwater. As the problem of groundwater and groundwater organic pollution becomes increasingly serious, it is of great significance to study low-cost, low-energy and green and sustainable bioremediation technologies.

[0004] At present, some people have used Bacillus in the degradation of benzene pollutants. For example, the patent with publication number CN 113025535 B discloses a strain of Bacillus and its application in the degradation of benzene series. However, the degradation of benzene pollutants by this Bacillus is single, and the concentration of degraded benzene is low, and the degradation rate of benzene below 25 mg / L is only 84%. 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 Bacillus paralicheniformis; the patent with publication number CN115975852A discloses the application of Bacillus siamese in the degradation of phenol; but the above strains cannot degrade benzene series and phenol at the same time, and the tolerance to benzene series and phenol is low, which is greatly limited in practical applications. Therefore, it is urgent to screen a strain that can degrade benzene series and phenol at the same time to meet people's application needs. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a barren water Bacillus, and also to provide the use of the above-mentioned barren water Bacillus in degrading complex organic pollution in groundwater or soil.

[0006] Technical solution: The barren water Bacillus described in the present invention is named Bacillus inaquosorum LH-BTEX-1, classified as Bacillus inaquosorum, and the strain number is LH-BTEX-1. It has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 33, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No. 31495, and the deposit date is July 30, 2024.

[0007] The inaquosorum Bacillus (Bacillus inaquosorum) LH-BTEX-1 of the present invention originates from groundwater of a petroleum contaminated site. After separation and purification, it grows well in a culture medium under an anaerobic environment of 30-37°C. The bacterial body of the strain "inaquosorum Bacillus" is rod-shaped, 0.4-0.6μm×1.1-3.0μm, arranged individually or in pairs, and is a Gram-positive bacterium.

[0008] The above-mentioned barren water bacillus can also be used to degrade complex organic pollution in groundwater or soil.

[0009] The composite organic pollution is organic pollution produced by a combination of one or more of benzene, isopropylbenzene or phenol.

[0010] Among them, the application is to inoculate Bacillus inaquosorum LH-BTEX-1 into a culture medium, anaerobically culture it at 30-37°C and pH 7.2-7.4, obtain a bacterial agent containing LH-BTEX-1 after culture, and inoculate the bacterial agent into groundwater or soil with complex organic pollution for treatment, and the treatment time is 7 to 19 days.

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

[0012] The concentration of the bacterial agent is 5-10 billion / mL; the dosage of the bacterial agent is 1-10% of the mass of groundwater or soil.

[0013] Wherein, in the groundwater or soil, the concentration of benzene is 0-3000 mg / kg or 0-3000 mg / L, the concentration of isopropylbenzene 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; more preferably, the concentration of benzene is 0-500 mg / kg or 0-500 mg / L, and the concentration of isopropylbenzene is 0-250 mg / kg or 0-250 mg / L.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The barren water Bacillus of the present invention is suitable for remediation of groundwater and soil contaminated by benzene derivatives and phenol, and the degradation rate of 40-3000 mg / L of benzene and isopropylbenzene can reach more than 90% within 20 days, and the degradation rate of 400 mg / L of phenol can reach more than 12% within 7 days; (2) The microorganisms provided by the present invention are low-cost, green and economical, easy to prepare, and have a short remediation time and good long-term stability. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0017] Figure 3 The 16S rDNA sequence of Bacillus inaquosorum LH-BTEX-1 of the present invention is compared with the standard strain;

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

[0019] Figure 5 The VITEK BCL identification card of Bacillus inaquosorum LH-BTEX-1 of the present invention;

[0020] Figure 6 The colony morphology (10 μm) of Bacillus inaquosorum LH-BTEX-1 of the present invention;

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

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

[0023] Fig. 9 The degradation effect of Bacillus ferrugineus on cumene (A) and its growth curve (B);

[0024] Fig.10 The gas chromatogram of the initial concentration of benzene and cumene in Example 3 is 1000 mg / L (reaction time 0d);

[0025] Fig.11 The gas chromatogram of the initial concentration of benzene and cumene in Example 3 is 1000 mg / L (reaction 19 days);

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

[0027] Fig.13 This is the gas chromatogram of the initial benzene concentration of 2000 mg / L in Example 3 (reaction 19d). DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments. The test materials used in the embodiments can be purchased through conventional channels.

[0029] Example 1

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

[0031] (1) Pour the sterilized LB medium into the culture dish and wait for the plate to cool before use. A total of 21 plates.

[0032] (2) Preparation of dilution solution (gradual dilution): Measure 1 mL of water samples from a contaminated site No. 1 to 7 respectively; place the seven water samples in a 250 mL Erlenmeyer flask containing 99 mL of sterile saline (with small glass beads), and then place it on a shaker for 30 min to disperse the microbial cells. Let it stand for 20-30 s to obtain a 10-1 dilution solution; then use a 1 mL sterile pipette to take 1 mL of the 10-1 dilution solution and transfer it to a test tube containing 9 mL of saline. Pipet it 3 times to mix the bacterial solution and water evenly to obtain a 10-2 dilution solution.

[0033] (3) Spreading: Mark the sterile plates with original, 10-1, and 10-2, and set three replicates for each gradient dilution; use a sterile pipette to draw 0.1 mL of the original solution into each of the three plates numbered with the original solution, and then use a spreader to spread the bacterial solution evenly on the plates, using one spreader for each dilution. Similarly, spread the 10-1 dilution and the 10-2 dilution, and remember to change the spreader.

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

[0035] (5) Purification: Single colonies were picked and isolated and purified 3 to 4 times to obtain 8 pure bacterial strains, No. 7-14.

[0036] (6) Water sample preparation: Shake the bucket containing the contaminated groundwater on site evenly, take about 1.2L and divide it into 20 100mL serum bottles, 50mL of water sample in each bottle. Then add 1.14uL of benzene and 5.75uL of isopropylbenzene to the serum bottles in turn. The water sample is obtained.

[0037] (7) Preparation of bacterial agents: Prepare 100 mL of 10% LB medium and dispense it into 12 test tubes, 5 mL of medium in each test tube. Then activate 8 indigenous strains (No. 7 to 14) and 10 self-owned strains (No. 1, No. 2-6, No. 15-18) with the medium for 1 day respectively.

[0038] (8) Determination of benzene content: Add 5 mL of activated bacterial solution directly to the serum bottle prepared previously, and then seal the bottle mouth with a rubber stopper and sealing film. Place it in a shaker at 120 rpm and 15°C for 1 week. After 1 week, take out the sample and centrifuge it at 12000 rpm. Take 1 mL of supernatant and add 9 mL of pure water into the headspace bottle.

[0039] "HJ1067-2019-Meteorological Chromatography for Determination of Benzene Series in Water Quality" uses headspace-gas chromatograph to determine the content of benzene series.

[0040] Depend on Figure 1 It can be seen that the degradation effects of the 18 strains used on cumene are all greater than 90%, but the degradation effects on benzene vary greatly (0-100%). Among them, the degradation effects of No. 2, 3, and 6 of the self-owned bacteria and No. 7, 8, 9, and 12 of the indigenous bacteria on benzene and cumene are all 100%. The No. 12 strain obtained after separation and purification was used to obtain a pure LH-BTEX-1 strain.

[0041] Example 2

[0042] The strain LH-BTEX-1 obtained in Example 1 was identified. The strain LH-BTEX-1 was identified as Bacillus inaquosorum. Figure 6 and Figure 7 As shown, the bacteria of strain LH-BTEX-1 are rod-shaped, 0.4-0.6 μm × 1.1-3.0 μm, arranged singly or in pairs, and are Gram-positive bacteria.

[0043] The complete sequence of 16S rRNA of LH-BTEX-1 strain is as follows (SEQ ID NO.1):

[0044]

[0045]

[0046] like Figure 2 As shown, a phylogenetic tree was constructed and strain LH-BTEX-1 was identified as Bacillus ferrugineus; Figure 3 Shown is the comparison analysis of the 16S rDNA sequence of LH-BTEX-1 and the standard strain.

[0047] Identification of LH-BTEX-1 strain (analysis of gyrB gene and standard strain is as follows)

[0048]

[0049]

[0050] like Figure 4 As shown, the gyrB gene of LH-BTEX-1 is compared with the standard strain; Figure 5 Shown is the VITEK BCL identification card for LH-BTEX-1.

[0051] Example 3

[0052] The benzene, cumene and phenol degradation test of the strain LH-BTEX-1 of the present invention is carried out in the following specific steps:

[0053] (1) Strain activation: Use an inoculation needle to pick up a single colony of pure bacteria and place it in LB liquid culture medium. The culture medium is shaken on a shaker at 37°C and 180 rpm for >1 day.

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

[0055] (3) Trace element mother solution: 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, CuCl2·2H2O2 mg / L, NiCl2·6H2O 24 mg / L.

[0056] (4) Mixing: Add sterilized inorganic salt culture medium and mixtures of benzene and isopropylbenzene with different concentration gradients and phenol solution into serum bottles (the initial concentrations of benzene are 250, 500, 740, 1244, and 2146 mg / L, respectively; the initial concentrations of isopropylbenzene are 40, 80, 88, 250, and 2000 mg / L, respectively; the initial concentration of phenol is 400 mg / L). Use a pipette to add 5 mL of activated bacterial solution directly into the prepared serum bottles with different pollutant concentrations, for a total of 50 mL. Seal the bottle mouth with a rubber stopper and sealing film, set up an anaerobic environment, and set the test conditions to: 160 rpm / min, 15°C, and airtight. Set up a blank group for this test. Set up a parallel group for this test.

[0057] (5) Sampling and analysis of benzene series: After 0, 4, 10, and 19 days of reaction, samples were taken to determine the benzene series content using a headspace-gas chromatograph in accordance with the "Gas Chromatographic Method for Determination of Benzene Series in Water Quality" (HJ1067-2019), and the OD600 was determined using an enzyme marker. For gas chromatography detection, the sample was diluted 200 times; Sampling and analysis of phenol: After 0 and 7 days of reaction, samples were taken to determine the volatile phenol content using a spectrophotometer in accordance with the "4-Aminoantipyrine Spectrophotometric Method for Determination of Volatile Phenol in Water Quality" (HJ 503-2009).

[0058] Figure 8 The results show the benzene degradation effect and growth curve of strain LH-BTEX-1. Compared with the control 0 mg / L, the growth of the strain was inhibited in the early stage under different benzene concentrations, and the growth was less inhibited in the later stage. At 250 mg / L and 500 mg / L benzene concentrations, the benzene degradation rate of the strain was about 70% at 4 days, and the degradation rate increased to 85% and 95% at 10 days and 19 days, indicating that most of the benzene can be reduced within 19 days. At 740 mg / L, 1244 mg / L and 2146 mg / L benzene concentrations, the growth of the strain in the early and late stages was inhibited to a certain extent, and the corresponding degradation rate decreased slightly. The degradation rate was about 65% at 4 days, and the degradation rate remained at about 75% and 91% at 10 days and 19 days. It shows that high concentrations of benzene will inhibit the degradation ability and growth of the strain to benzene, but the degree of inhibition is not obvious.

[0059] Fig. 9The degradation effect and growth curve of strain LH-BTEX-1 on cumene are shown. Compared with the control 0 mg / L, the growth of the strain is mainly inhibited in the early stage under different cumene concentrations, and the growth is less inhibited in the later stage. At a concentration of 40 mg / L cumene, the benzene degradation rate of the strain is about 75% at 4d and 10d, and the degradation rate is above 98% at 19d, indicating that most cumene can be reduced within 19 days. At 80 mg / L and 250 mg / L cumene concentrations, the degradation rate is about 80% at 4d and 10d, and the degradation rate is about 95% at 19d. At a concentration of 2000 mg / L cumene, the degradation rate is only about 93% at 4d, and the degradation rate is as high as 99% at 19d. This shows that the strain has a strong degradation ability for high concentrations of cumene.

[0060] Table 1. Effect of Bacillus ferrugineus on phenol degradation

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

[0062] Table 1 shows the phenol degradation effect of Bacillus aquatica. It can be seen from Table 1 that the phenol degradation rate of this strain is about 12% at a phenol concentration of about 400 mg / L in 7 days.

[0063] Example 4

[0064] The strain LH-BTEX-1 of the present invention is used to perform a degradation experiment in organically contaminated groundwater, and the specific steps are as follows:

[0065] Bacillus inaquosorum LH-BTEX-1 was inoculated into a culture medium, the formula of which was 1% peptone, 0.5% yeast extract, 1% sodium chloride, 2% agar powder, and the balance was water; it was cultured anaerobically at 30-37°C and pH 7.2-7.4, and a bacterial agent containing LH-BTEX-1 was obtained after culture. The bacterial agent was inoculated into groundwater with composite organic pollution for treatment, and the degradation results are shown in Table 2:

[0066] Table 2. Results of benzene degradation by Bacillus spp. in groundwater from contaminated sites

[0067]

[0068] As shown in Table 2, after 7 days of reaction, the samples with added bacterial solution had a benzene degradation rate of more than 80% for the contaminated site water samples, among which the benzene degradation rate of 10% bacterial solution addition was as high as 99%. After 17 days of reaction, the added bacterial solution could degrade 70%-90% of the benzene in the on-site water samples.

[0069] Table 3. Degradation results of cumene by Bacillus desinergii in groundwater of contaminated sites

[0070]

[0071]

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

[0073] Table 4. Results of phenol degradation by Bacillus spp. in groundwater from contaminated sites

[0074]

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

[0076] Example 5

[0077] The strain LH-BTEX-1 of the present invention is used to perform a degradation experiment in organically contaminated soil, and the specific steps are as follows:

[0078] Bacillus inaquosorum LH-BTEX-1 is inoculated into a culture medium, the formula of which is 1% peptone, 0.5% yeast extract, 1% sodium chloride, 2% agar powder, and the balance is water; anaerobically cultured at 30-37°C and pH 7.2-7.4, and a bacterial agent containing LH-BTEX-1 is obtained after culture, and the bacterial agent is inoculated into soil with complex organic pollution for treatment.

[0079] Table 5. Degradation results of cumene by Bacillus ferrugineus in contaminated soil

[0080]

[0081] It can be seen from the above table that compared with the blank sample, after 7 days of reaction, the samples with added bacterial solution have a degradation rate of more than 90% for the contaminated soil samples. After 17 days of reaction, the addition of bacterial solution can degrade nearly 100% of the isopropylbenzene in the soil samples on site. The effect of the samples with a rotation speed of 40r is better than that of the samples left stationary.

[0082] Table 6. Results of benzene degradation by Bacillus ferrugineus in contaminated soil

[0083]

[0084]

[0085] It can be seen from the above table that compared with the blank sample, after 7 days of reaction, the samples with added bacterial solution have a benzene degradation rate of more than 70% for the benzene in the contaminated soil samples. After 17 days of reaction, the addition of bacterial solution can degrade nearly 100% of the benzene in the soil samples on site. The effect of the samples with a rotation speed of 40r is better than that of the samples left stationary.

[0086] Example 6

[0087] The strain LH-BTEX-1 of the present invention was subjected to a benzene degradation experiment at a low dosage ratio in organically contaminated groundwater, wherein the concentration of the bacterial agent was 5-10 billion / mL (range value); the dosage 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. Results of benzene degradation by Bacillus spp. in groundwater from contaminated sites (low addition ratio)

[0089]

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

[0091] In summary, when the concentrations of benzene and cumene are high, i.e., 1000-2000 mg / L, or even when the concentration of cumene is 3000 mg / L, the growth of the strain is subject to certain restrictions. However, in terms of reduction degradation, the degradation rates of benzene and cumene by LH-BTEX-1 within 19 days are still as high as 92% and 98%, respectively. 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 within 19 days are 96% and 98%, respectively. The phenol degradation rate of this strain at a phenol concentration of about 400 mg / L in 7 days is about 12%. The dosage of bacterial solution is preferably 10% of the mass of soil or groundwater. Therefore, the Bacillus inaquosorum LH-BTEX-1 of the present invention can synchronously and efficiently repair groundwater and soil contaminated by the complex organic matter of benzene series and phenol, with a short repair time and good long-term stability.

Claims

1. A barren water Bacillus, characterized in that The bacterium was named Bacillus inaquosorum LH-BTEX-1 and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 30, 2024, with the deposit number CGMCC No.31495.

2. Use of the barren water bacillus according to claim 1 in degrading complex organic pollution in groundwater or soil.

3. The use according to claim 2, characterized in that: The composite organic pollution is organic pollution produced by a combination of one or more of benzene, cumene or phenol.

4. The use according to claim 3, characterized in that: Bacillus inaquosorum LH-BTEX-1 is inoculated into a culture medium and cultured under an anaerobic environment to obtain a bacterial agent containing LH-BTEX-1. The bacterial agent is inoculated into groundwater or soil with complex organic pollution for treatment.

5. The use according to claim 4, characterized in that: The culture medium is LB liquid culture medium, and the formula is: 1%-2% tryptone, 0.5%-1% yeast extract, 1%-2% sodium chloride, 2%-4% agar powder, and the balance is water.

6. The use according to claim 4, characterized in that: The conditions of the anaerobic environment are 30-37° C. and pH 7.2-7.

4.

7. The use according to claim 4, characterized in that: The culture duration is 2 to 3 days.

8. The use according to claim 4, characterized in that: The concentration of the bacterial agent is 5-10 billion / mL; the dosage of the bacterial agent is 1-10% of the mass of groundwater or soil.

9. The use according to claim 4, characterized in that: In the groundwater or soil, the concentration of benzene is 0-3000 mg / kg or 0-3000 mg / L, the concentration of isopropylbenzene 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.

10. The use according to claim 4, characterized in that: The treatment lasts for 7 to 19 days.

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