Bacillus Velez DHZK21 and its application

Abstract

The invention discloses a strain of Bacillus velezensis DHZK21, with a deposit number of CGMCC No. 26216 and a classification name of Bacillus velezensis. The biosurfactant produced by the strain can effectively reduce the surface tension and interfacial energy of a solvent, has strong emulsification ability, is easy to biodegrade, is non-toxic to the environment, has good thermal stability and chemical stability, is an ideal substitute for synthetic surfactants, can emulsify and reduce the viscosity of crude oil and other materials, enhance fluidity, thereby improving oil displacement efficiency and increasing oil recovery rate.

Description

Technical Field

[0001] The invention relates to Bacillus velezensis DHZK21 and application thereof, and belongs to the technical field of soil restoration using microorganisms. Background Art

[0002] Surfactants are substances that have both fixed hydrophilic and lipophilic groups. They have anti-adhesive, emulsifying, foaming, solubilizing, dispersing, and washing functions. Their applications can cover almost all fields of fine chemicals, so they are also called "industrial MSG". At present, the surfactants used in the Chinese market are mainly petroleum-based chemical surfactants. Such surfactants consume a lot of petroleum and chelating agents during production and utilization. In addition, such surfactants usually have certain biological toxicity and are difficult to be biodegraded, so they are easy to cause harm to the environment and human health. Therefore, finding new surfactants that can replace chemical surfactants is an important green environmental protection measure.

[0003] Biosurfactants are a class of surface-active substances produced by microbial fermentation metabolism, including five types: glycolipids, lipopeptides, lipoproteins, fatty acids, phospholipids and neutral derivatives. The molecular structure of biosurfactants contains non-polar hydrophobic groups composed of aliphatic hydrocarbon chains and polar hydrophilic groups. They have functional groups that are difficult to synthesize by chemical methods, have complex molecular structures, and have a low critical micelle concentration (CMC).

[0004] The process of producing surfactants by microbial fermentation is a typical green process. On the one hand, its raw materials are widely present in nature, cheap and have the ability of sustainable production; on the other hand, biosurfactants are a type of active substances obtained by microbial fermentation metabolism, which have the characteristics of low toxicity, biodegradability and environmental friendliness, so their application value is higher than that of general chemically synthesized surfactants. For example, CN107988122A provides a method for preparing a lipopeptide biosurfactant: Bacillus subtilis LPB-3 is fermented at a temperature of 35°C to obtain a lipopeptide biosurfactant. For example, CN 111690576A also provides a biosurfactant produced by Bacillus Velez, but it is used as a foliar fertilizer, but there are no effective strains and biological products in terms of bioreplacing and improving oil recovery. Summary of the invention

[0005] The present invention obtained a strain of Bacillus velezensis DHZK21 capable of producing high-performance biosurfactant by screening bacteria from farmland soil in northern Shouguang. The strain was deposited in the General Microbiological Center of China National Microbiological Culture Collection on December 15, 2022, with a deposit number of CGMCC No. 26216 and a classification name of Bacillus velezensis. The deposit address is No. 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Bacillus Velezii DHZK21 can ferment and produce biosurfactants. The biosurfactants have high surface activity, can effectively reduce the surface tension of solvents, and have the characteristics of low toxicity, biodegradability and environmental friendliness. Their application value is higher than that of general chemically synthesized surfactants.

[0007] Characteristics of Bacillus Velez DHZK21 culture medium:

[0008] Aerobic, growth temperature range 5-55 ° C, the optimum growth temperature range is 30-37 ° C. It can grow in an environment of pH 5.0-8.0, and its optimum pH is 6.5-7.0.

[0009] Beneficial effects:

[0010] Bacillus Velez DHZK21 has a wide tolerance, controllable production process, and is environmentally friendly. The biosurfactant produced is unique and superior in reducing water-oil interfacial tension, emulsifying crude oil, improving crude oil fluidity, and increasing biorecovery rate. The fermentation broth of the strain can be directly used for oil recovery without complicated purification and extraction steps, reducing unnecessary costs. Therefore, Bacillus Velez DHZK21 has good potential and prospects in biorecovery and improving oil recovery rate. BRIEF 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 of the present invention. In the accompanying drawings:

[0012] Figure 1 This is the colony morphology of the strain DHZK21;

[0013] Figure 2 This is a comparison chart of the surface tension values ​​between the fermentation broth of Bacillus Velez DHZK21 and other biosurfactants and crude oil;

[0014] Figure 3 This is a comparison chart of the water separation power of the fermentation broth of Bacillus Velez DHZK21 and other biosurfactants;

[0015] Figure 4 The E of the fermentation broth of Bacillus velez DHZK21 and other biosurfactants 24 Value comparison chart;

[0016] Figure 5 This is a comparison chart of the oil recovery efficiency between the fermentation broth of Bacillus Velez DHZK21 and other biosurfactants. DETAILED DESCRIPTION

[0017] The preferred embodiments of the present invention are described below in conjunction with 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 Screening and identification of bacterial strains

[0019] 1.1 Bacterial strain screening

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

[0021] 1.2 Molecular identification

[0022] The pure culture of the strain was taken, and the genomic DNA was used as a template. The universal primers 27F / 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGAC TT-3') were used for 16S rRNA amplification and sequencing. The gene sequence obtained by sequencing was compared by Blast search. The bacteria had a high homology with Bacillus velezensis and was identified as Bacillus velezensis. Combined with morphological and 16S rRNA sequence analysis, it was identified as Bacillus velezensis and named Bacillus velezensis DHZK21. It was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee on December 15, 2022, and the deposit number was CGMCC No.26216.

[0023] Example 2 Production of biosurfactant

[0024] The specific steps are as follows:

[0025] Step 1: Activate the strains: Streak the dormant strains on the LB plate and culture them at 37°C for 24 hours. Then pick the single colonies grown on the LB solid plate into the LB medium and activate them at 37°C for 24 hours.

[0026] Step 2, fermentation culture: the activated strain was inoculated into 50 mL of fermentation medium at a rate of 10% and cultured for 16-72 hours, and samples were taken every 2 hours to determine the surfactant production.

[0027] The fermentation broth was centrifuged at 4°C and 12000rpm for 10 minutes, the supernatant was collected, the precipitated bacteria were discarded, and then freeze-dried with a vacuum freeze dryer until constant weight was reached, and the weight of the surfactant product was weighed. The experimental results showed that the production of surfactants produced by the bacteria reached a peak at 50 hours of fermentation, and the production of surfactants gradually increased during 16-50 hours of fermentation. After 50 hours to 72 hours of fermentation, the production of surfactants remained the same and then slightly decreased. Therefore, the fermentation broth cultured for 24-72 hours can be used to prepare surfactants, and 50 hours of fermentation is the fermentation time with the highest production of surfactants produced by the bacteria.

[0028] The above culture medium is configured as follows:

[0029] LB medium was prepared according to the formula of 1 L of distilled water, 1% NaCl, 1% peptone, and 0.5% yeast extract and sterilized at 121° C. for 30 min.

[0030] LB plates were prepared according to the formula of 1 L distilled water, 1% NaCl, 1% peptone, 0.5% yeast extract, and 1.3% agar powder, and sterilized at 121° C. for 30 min.

[0031] Example 3 Performance Experimental Test of Bacillus Velezii DHZK21 Fermentation Broth

[0032] Experiment 1: Determination of surface tension of biosurfactants

[0033] The fermentation broth of Bacillus Velez DHZK21 was poured into a centrifuge tube, centrifuged at 4°C and 12000rpm for 10min, the supernatant was collected, the precipitated bacteria were discarded, and then freeze-dried with a vacuum freeze dryer to obtain the Bacillus Velez DHZK21 biosurfactant product. The surface tension between the 10mg / L Bacillus Velez DHZK21 fermentation broth product, rhamnolipid, sophorolipid, and lipopeptide and crude oil was tested using a spinning drop interfacial tension meter, and compared with the biosurfactant product produced by Bacillus Velez MMB-51 (CGMCC No. 19099). The test temperature was 50°C and the test speed was 6000r / min.

[0034] from Figure 2According to the experimental results, the fermentation broth of Bacillus Velez DHZK21 can effectively degrade the surface tension between it and crude oil, and the surface tension value (ST) can be reduced to 9mN / m, which is much better than biosurfactant products such as sophorolipids and lipopeptides. The effect is also better than the biosurfactant products produced by Bacillus Velez MMB-51 (CGMCC No.19099), which also proves that it has a good emulsification effect.

[0035] Experiment 2: Comparative determination of water separation power of different types of biosurfactants

[0036] Add 5mL of fermentation liquid (or other biosurfactant products) and 5mL of crude oil into a 10mL stoppered graduated test tube, and then place it in a 90℃ constant temperature box for 30 minutes. After taking out the test tube, shake it evenly for 200 times, and then place it in a 90℃ constant temperature box again. Start timing at the same time, record the volume of water separated in the test tube every 5 minutes, and calculate the water separation rate. Figure 3 As shown, compared with other biosurfactants on the market, the fermentation broth product of Bacillus Velez DHZK21 has stronger emulsifying properties, and the water separation rate can still be maintained at 10% after 400 minutes, which has greater superiority.

[0037] The formula for water separation rate is E d =V w / V.E d V is the water separation rate; w is the amount of water separated, mL; V is the volume of water phase when preparing the emulsion, mL.

[0038] Experiment 3: Comparative determination of E24 values ​​of biosurfactants

[0039] 2 mL of different hydrophobic organic substances (liquid paraffin, soybean oil, and n-hexane) were mixed with 2 mL of different surfactants (SDS, rhamnolipid, Tween 80, CTAC, and Bacillus Velezii DHZK21 fermentation broth), and the mixture was homogenized at 8000 r / min for 2 min. Then, the mixture was allowed to stand for 24 h and the height of the emulsion layer was measured. The emulsification index (E) was calculated according to the formula 24 ).

[0040] The emulsification ability of biosurfactants can be measured by the emulsification index (E 24 ) to indicate. Figure 4 As shown, whether it is liquid paraffin, soybean oil or hydrophobic organic matter such as n-hexane, the emulsification ability of the fermentation broth product of Bacillus Velez DHZK21 far exceeds that of other types of biosurfactants, and it can completely emulsify them, and the emulsification index can reach 100%.

[0041] E 24= emulsion layer height / total mixture height × 100%

[0042] Example 4 Application of Bacillus Velezii DHZK21 fermentation broth product in bioremediation

[0043] Compare the oil displacement efficiency of different biosurfactant products and their application in improving oil recovery. The experimental temperature is the reservoir temperature (80℃), the porosity is 20%, the permeability is 40×10 -3 μm 2 The core was pressurized and vacuum-saturated with formation water for 24 hours, and its porosity and permeability were accurately measured. -1 The injection rate is to saturate the crude oil until no water is produced at the production end. After saturation, the crude oil is aged in a constant temperature box at 80°C for 24 hours. A 100× biosurfactant system prepared by ionized water is injected at a certain injection rate to drive oil until the water content at the outlet is stably above 98.0%. The liquid output, oil output and injection pressure during the displacement process are recorded, and the oil recovery efficiency is calculated.

[0044] Oil recovery efficiency = oil output / liquid output × 100%

[0045] according to Figure 5 As shown in the oil recovery efficiency comparison chart, compared with different biosurfactant products, the oil recovery efficiency of Bacillus Velez DHZK21 fermentation liquid product is much higher than that of other surfactant products. When the injection volume reaches 1pv, the oil recovery efficiency can reach 80%. The results further prove that the fermentation liquid product is not only an excellent biosurfactant product, but also has outstanding superiority in biological oil recovery.

[0046] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions recorded in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Bacillus Velezii DHZK21, whose deposit number is CGMCC No.26216, and whose classification name is: Bacillus velezensis ; Deposit date: December 15, 2022, deposit address: No. 1 Beichen West Road, Chaoyang District, Beijing; Deposit unit: General Microbiology Center of China Culture Collection Administration.

2. The Bacillus Velezii DHZK21 according to claim 1, characterized in that The culture method is aerobic, 30-37°C, pH 5 6.5-7.

0.

3. Use of the Bacillus Velezii DHZK21 described in claim 1 in producing biosurfactants.

4. The use according to claim 3, characterized in that The Bacillus Velezii DHZK21 strain is activated, inoculated into a fermentation medium, and fermented for 24 hours to 70 hours. The fermentation liquid is then centrifuged, and the supernatant is collected to obtain a liquid rich in biosurfactants.

5. Use of the Bacillus Velezii DHZK21 described in claim 1 in petroleum biorecovery.

6. A biosurfactant, characterized in that The product is prepared by fermentation with the Bacillus Velezii DHZK21 described in claim 1.

7. A method for producing a biosurfactant, characterized in that: The Bacillus Velezii DHZK21 strain described in claim 1 is activated, inoculated into a fermentation medium, and fermented for 24 h to 72 h, then the fermentation liquid is centrifuged, and the supernatant is collected to obtain a liquid rich in biosurfactants.

8. The production method according to claim 7, characterized in that The liquid rich in biosurfactant is freeze-dried to obtain the Bacillus Velezii DHZK21 biosurfactant product.

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