Bacillus velezensis DHZK22 and its applications

Abstract

The present invention relates to the technical field of petroleum sludge degradation, and discloses a Bacillus velezensis DHZK22, with the preservation number of CGMCC No. 26217, and the taxonomic name is: Bacillus velezensis; this strain can efficiently degrade hydrocarbon compounds in highly petroleum-polluted soil (petroleum hydrocarbon concentration > 3%), so that it meets the greening land standard and returns to its original use.

Description

Technical Field

[0001] The present invention relates to Bacillus velezensis DHZK22 and its application, belonging to the technical field of using microorganisms for soil remediation. Background Art

[0002] As the main energy source for industrial production and daily life, oil often leaks accidentally during its exploration, production, refining, transportation, and storage processes, which is the main cause of water and soil pollution. Since most animals and plants cannot decompose hydrocarbons, the pollutants gradually accumulating in tissues can lead to the death or mutation of animals and plants. Therefore, the pollution of soil by hydrocarbons will cause serious damage to the local ecosystem. Traditional soil remediation techniques include mechanical, burial, evaporation, dispersion, and washing. However, these techniques are not only expensive, but also cause secondary pollution to the environment and cannot completely degrade pollutants. Bioremediation technology refers to using certain life metabolic activities of microorganisms to remove and degrade certain pollutants in the environment. Bioremediation technology has low costs and does not cause secondary pollution to the environment. The biodegradation of natural microbial populations is one of the main mechanisms for removing oil and other hydrocarbon pollutants in the environment and is more economical than other remediation techniques.

[0003] The treatment of oily sludge by microorganisms is mainly through the growth and metabolism of crude oil-degrading bacterial communities. Taking crude oil as the nutrients required for their growth and reproduction, they degrade crude oil into CO2 and H2O through their own metabolic processes and discharge them out of the body, thereby reducing the oil content in oily sludge and achieving the purpose of safe treatment. The treatment of oily sludge by microorganisms is divided into biostimulation and bioaugmentation technologies. Biostimulation technology is to activate the indigenous bacteria in the soil under artificially optimized environmental conditions to allow them to carry out a better growth and metabolic process, thus being more conducive to degrading petroleum pollutants; bioaugmentation technology is a technology that first domesticates highly efficient degrading bacteria and then inoculates them into oily sludge. It can overcome the limitations of biostimulation technology itself. Therefore, we generally adopt bioaugmentation to add exogenous microorganisms to achieve better treatment effects. At present, there are still defects in soil microbial remediation technology, such as a small number of available strains, low remediation efficiency, and few application cases and experiences of microbial remediation. There is an urgent need to increase the number of engineering applications of highly efficient strains.

[0004] Bacillus is a common genus of bacteria in soil. There are many known species that can degrade oily sludge pollutants. For example, Dong Ding et al. reported Bacillus subtilis that can efficiently degrade petroleum hydrocarbons in 2017, and Sharma R et al. screened Bacillus amyloliquefaciens in 2018. However, there are few reports on Bacillus velezensis in degrading highly petroleum-polluted soil. Wang Dong et al. screened Bacillus velezensis 12-5B in 2023, which can degrade petroleum hydrocarbons in low-concentration pollution, and the degradation rate only reached 93.5%. The screened Bacillus velezensis DHZK22 can efficiently degrade hydrocarbon compounds in petroleum-polluted soil. The petroleum hydrocarbon degradation rate can reach 99% in only 5 days, enabling the polluted soil to meet the green land standard and return to its original use. Summary of the Invention

[0005] In the present invention, a strain of Bacillus velezensis DHZK22 capable of efficiently degrading petroleum hydrocarbon compounds was screened from the beach sand soil in Xiaying Town, Changyi City, Weifang City. This strain was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 25, 2022, with the deposit number CGMCC No. 26217. The taxonomic name is: Bacillus velezensis; the deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Bacillus velezensis DHZK22 can directly use petroleum hydrocarbon compounds as a carbon source to degrade petroleum hydrocarbons in petroleum-polluted soil, thereby alleviating the polluted soil area and restoring it to the national green land standard.

[0007] Characteristics of the culture medium of Bacillus velezensis DHZK22:

[0008] Aerobic, the growth temperature range is 5 - 55 °C, and the optimal growth temperature range is 30 - 37 °C. It can grow in an environment with a pH of 5.0 - 8.0, and its optimal pH is 7.0.

[0009] Beneficial effects:

[0010] Bacillus velezensis DHZK22 has a wide tolerance, a controllable production process, and is environmentally friendly. It can directly use petroleum hydrocarbons as a carbon source for growth, thereby efficiently degrading the content of petroleum hydrocarbons and being used for the remediation of petroleum hydrocarbon-polluted soil. Therefore, Bacillus velezensis DHZK22 has good potential and prospects in the remediation of highly petroleum-polluted soil. Description of the Drawings

[0011] The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. In the accompanying drawings:

[0012] Figure 1 It is the colony morphology diagram of strain DHZK22;

[0013] Figure 2 It is the growth and degradation rate of DHZK22; among which (a) is the variation and comparison diagram of the growth of DHZK22 with the degradation time; (b) is the variation and comparison diagram of the degradation rate of DHZK22 with the degradation time;

[0014] Figure 3 It is the degradation rate diagram of DHZK22 for polycyclic aromatic hydrocarbons (PAH) in crude oil;

[0015] Figure 4 It is the petroleum hydrocarbon degradation rate diagram of DHZK22 at different pH values;

[0016] Figure 5 It is the petroleum hydrocarbon degradation rate diagram of DHZK22 at different temperatures. Specific Embodiments

[0017] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0018] Example 1 Strain Screening and Identification

[0019] 1.1 Strain Screening

[0020] In the separation and screening, the sample used is petroleum-contaminated soil. The sample is stirred with sterilized deionized water, and the sample solution is enriched and cultured through LB medium and diluted and coated on LB plates.

[0021] 1.2 Molecular Identification

[0022] The pure culture of the strain was taken, and genomic DNA was used as a template. The 16S rRNA was amplified and sequenced using the universal primers 27F / 1492R (27F: 5’-AGAGTTTGATCCTGGCTCAG-3’, 1492R: 5’-GGTTACCTTGTTACG ACTT-3’). The gene sequence obtained by sequencing was searched and aligned by Blast. This bacterium had a high homology with Bacillus velezensis and was identified as Bacillus velezensis. Combining morphological and 16S rRNA sequence analysis, it was identified as Bacillus velezensis and named Bacillus velezensis DHZK22. It was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 25, 2022, with the deposit number CGMCC No. 26217.

[0023] Example 2 Determination of Petroleum Hydrocarbon Degradation Rate

[0024] The specific steps are as follows:

[0025] The isolated and purified strain was cultured in LB liquid medium for 24 h. The strain was inoculated into 100 mL of MSM medium containing 40 g / L of n-hexadecane, diesel, and crude oil at a ratio of 1%, and the initial OD was controlled at about 0.025. The pH of the medium was adjusted to 7, and it was cultured with shaking at 30 °C and 150 rpm. The growth of the bacteria was observed, and the content of petroleum hydrocarbons and OD in the bacterial liquid were detected regularly. 600 .

[0026] Detection method for petroleum hydrocarbon content: High-performance gas chromatography was used to determine the amount of compounds in n-hexadecane, diesel, and crude oil. Each bottle of residual hydrocarbon compounds was extracted with chloroform, diluted, and then injected into the gas phase. By summing up all detectable peaks in the SIM mode, the total amount of residual hydrocarbon compounds in each sample was calculated.

[0027] The results are as Figure 2 shown. As can be seen from (a), when the initial OD was controlled to be the same (both 0.025), Bacillus velezensis DHZK22 had a fast growth rate, and the OD could reach more than 2. Therefore, it can be inferred that when DHZK22 grew in the inorganic medium containing n-hexadecane, diesel, and crude oil, the availability of n-hexadecane and diesel was relatively faster than that of crude oil.

[0028] According to the results in (b), Bacillus velezensis DHZK22 has obvious effects on the degradation of highly contaminated soil with n-hexadecane, diesel oil and crude oil. On the fifth day, the degradation rates can all reach 99%. Compared with the degradation of crude oil, the degradation rates of n-hexadecane and diesel oil are faster.

[0029] Example 3: Degradation analysis of each component of crude oil by Bacillus velezensis DHZK22

[0030] The strain was inoculated into 100 mL of MSM medium containing 40 g / L of crude oil at a ratio of 1% and cultured for 4 days with shaking at 30 °C and 150 rpm. Gas-phase detection and analysis were performed on polycyclic aromatic hydrocarbons (PAHs) in crude oil before and after degradation.

[0031] According to Figure 3 it can be seen that from the perspective of the degradation rate of polycyclic aromatic hydrocarbons (PAHs), the overall degradation rate is 95%, and the degradation rates of naphthalene, fluorene, phenanthrene, 1-methylnaphthalene and 2-methylnaphthalene all reach more than 98%. Thus, Bacillus velezensis DHZK22 shows good degradation ability for PAHs and is a highly potential indigenous strain that can be used for the treatment of petroleum pollution.

[0032] Example 4: Effect of pH on the degradation rate of petroleum hydrocarbons by Bacillus velezensis DHZK22

[0033] Take five 250 mL conical flasks and add 100 mL of MSM medium respectively. Adjust the pH to 5, 6, 7, 8, and 9 respectively, add 40 g / L of n-hexadecane, diesel oil, and crude oil, inoculate Bacillus velezensis DHZK22 at 1%, and culture at 30 °C and 150 r / min for 5 days. Then measure the content of petroleum hydrocarbons (n-hexadecane, diesel oil, crude oil) by gas-phase detection and calculate the degradation rate of petroleum hydrocarbons (1 - C / C0). The results are shown in Figure 4 , and it is found that the degradation rate of more than 95% of petroleum hydrocarbons can be maintained between pH 7 - 9. The degradation rate is the highest at pH 7, reaching 99%. Moreover, the acidic environment has a greater impact on the degradation rate of petroleum hydrocarbons (n-hexadecane, diesel oil, crude oil) than the alkaline environment.

[0034] Example 5: Effect of temperature on the degradation rate of petroleum hydrocarbons by Bacillus velezensis DHZK22

[0035] Add 40 g / L of n-hexadecane, diesel oil, and crude oil to 100 mL of MSM medium, inoculate Bacillus velezensis DHZK22 at 1%, and culture at 20, 25, 30, 35, and 40 °C respectively. After 5 days, measure the content of petroleum hydrocarbons (n-hexadecane, diesel oil, crude oil) by gas-phase detection and calculate the degradation rate of petroleum hydrocarbons (1 - C / C0). The results are shown in Figure 5, The results show that the optimum degradation temperature is around 30°C, and the degradation rate of petroleum hydrocarbons can reach 99%. When the temperature is lower than 30°C, the degradation rate gradually increases with the increase of temperature. When the temperature is higher than 30°C, the higher the temperature, the lower the degradation rate.

[0036] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or equivalently replace some of the technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Bacillus velezensis DHZK22, with the preservation number of CGMCC No. 26217, and the taxonomic name is: Bacillus velezensis ; Preservation date: December 25, 2022, Preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Preservation unit: China General Microbiological Culture Collection Center.

2. The Bacillus velezensis DHZK22 according to claim 1, characterized in that, The culturing method thereof is aerobic, at 30-37 °C, and pH 5.0-8.

0.

3. Application of the Bacillus velezensis DHZK22 as claimed in claim 1 in the remediation of petroleum-polluted soil.

Images