A strain of Bacillus saffron and its culture method and application

Abstract

The present invention belongs to the technical field of microbial degradation, and relates to a strain of Bacillus sabii and its cultivation method and application. The bacterium is Bacillus sabii, which is preserved in the Guangdong Provincial Microbial Culture Collection Center and its taxonomic name is Bacillus safensis subsp .safensis The deposit number is GDMCC No: 64077. The Bacillus sabdariffa DWC-2 of the present invention can grow by using petroleum hydrocarbons as a single carbon source, has a high ability to degrade petroleum, produces biosurfactants, has a high oil washing efficiency, and can be applied to microbial enhanced oil recovery, production of biosurfactants / emulsifiers, and restoration of petroleum-contaminated environments.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial degradation, and specifically relates to a strain of Bacillus saffron and a culture method and application thereof. Background Art

[0002] Oil is one of the most important energy sources in today's industrial system. Since the beginning of the 20th century, large-scale oil extraction and utilization have begun worldwide. Every year, tens of millions of tons of oil enter natural environments such as water bodies and soil. These petroleum hydrocarbon substances have the characteristics of high adhesion, high carbon content, a wide variety of organic matter and difficulty in degradation. They belong to HW08 hazardous wastes and may cause harm to animals, plants and humans. In the process of microbial degradation of petroleum hydrocarbons, due to the hydrophobicity of petroleum hydrocarbons, the contact area with microorganisms is limited to a certain extent, thereby reducing the oil treatment effect. In the process of microbial degradation of oil, microorganisms use petroleum hydrocarbons as carbon sources and energy sources, and produce decomposition enzymes through their own metabolism to crack long-chain or polycyclic hydrocarbons; in addition, microorganisms can produce substances such as biodegradation enzymes and biosurfactants that are beneficial to oil recovery during their growth and reproduction, promoting the decomposition of petroleum hydrocarbons into small molecules to achieve the purpose of degradation. Surfactants can improve the dissolution effect, reduce the oil-water interfacial tension, and improve the cleaning efficiency. It can be seen that screening microorganisms that degrade oil is the key to microbial enhanced oil recovery and bioremediation technology. Summary of the invention

[0003] Hydrocarbon degradation bacteria (HDB) are bacteria that can degrade petroleum hydrocarbons as

[0004] A general term for a class of microorganisms that biodegrade carbon sources and produce metabolites such as gases, fatty acids and biosurfactants. For a long time, finding efficient petroleum hydrocarbon degrading bacteria has been an important task for researchers in the field of oil pollution control and microorganisms. The object of the present invention is to provide a strain of Bacillus sabovici and its culture method and application. The Bacillus sabovici described in the present invention can grow using petroleum hydrocarbons as a single carbon source, has a high ability to degrade oil, and has a good emulsification effect, and has high research and application value.

[0005] The present invention provides a strain of Bacillus sabii, which is deposited in the Guangdong Provincial Microbial Culture Collection Center and has a taxonomic name of Bacillus safensis subsp . safensis , the deposit number is GDMCC No: 64077.

[0006] The present invention also provides a method for culturing the Bacillus sabdariffa described in the above technical solution, comprising the following steps: inoculating the Bacillus sabdariffa into a culture medium for fermentation and culturing to obtain a fermentation product of the Bacillus sabdariffa.

[0007] Preferably, the culture temperature is 30-50°C.

[0008] Preferably, the culture medium comprises an inorganic salt culture medium; the culture medium also comprises petroleum.

[0009] Preferably, the amount of petroleum added to the culture medium is 0.1-10 g / L.

[0010] The present invention also provides the use of the Bacillus saffron described in the above technical solution in degrading petroleum.

[0011] The present invention also provides the use of the Bacillus saffron described in the above technical solution in degrading petroleum hydrocarbons; the petroleum hydrocarbons include alkanes.

[0012] The present invention also provides the use of the Bacillus saffron described in the above technical solution in improving oil recovery and / or promoting the restoration of oil-polluted environments.

[0013] The present invention also provides the use of the Bacillus saffron described in the above technical solution in the preparation of a biosurfactant; the biosurfactant has an emulsifying effect.

[0014] The present invention also provides the use of the Bacillus saffron described in the above technical solution in improving the oil washing ability.

[0015] The present invention provides a strain of Bacillus sabogensis. The Bacillus sabogensis strain of the present invention, as a single bacterial species, can directly degrade crude oil with complex components and difficult to degrade, and can also produce biosurfactants in the process of degrading the oil, and has a high oil washing efficiency. The Bacillus sabogensis of the present invention can survive and grow in an environment containing oil as the only carbon source by only providing an inorganic salt culture medium, has a fast growth rate, and has a high ability to degrade oil. In addition, the Bacillus sabogensis provided by the present invention can also degrade C8~C9 and C 14 ~C 22 The alkanes have a good utilization effect, so it can effectively degrade the petroleum and / or petroleum products in the environment. The test results show that the Bacillus sabogensis of the present invention grows rapidly when cultured in an inorganic salt liquid medium containing crude oil, indicating that the bacteria reproduce very quickly in crude oil, which is beneficial to practical application; and the degradation rate of crude oil by the Bacillus sabogensis of the present invention reaches 56.5% in 5 days; it has the function of emulsifying and dispersing crude oil, and has a great effect on C8~C9 and C 14 ~C 22 The degradation effect of petroleum hydrocarbons is good; the fermentation liquid has a high oil washing efficiency of 68.87~87.43%; Bacillus can separate oil from the surface of oil sands. After washing, the oil sands are clean and loose, which can prevent crude oil from adhering again; it has a good effect on the repair of offshore oil spills. The oil degradation rate measured by the experimental group after 7 days is 87.82%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The colony morphology diagram provided by the present invention;

[0018] Figure 2 The phylogenetic tree result diagram provided by the present invention;

[0019] Figure 3 A growth curve diagram provided by the present invention;

[0020] Figure 4 This is a comparison chart of the blank control after 5 days of culture provided by the present invention and the effect of adding Bacillus sabdariffa DWC-2.

[0021] Biological deposit information

[0022] Bacillus safensis subsp . safensis , deposited in Guangdong Microbiological Culture Collection Center, referred to as GDMCC, on November 27, 2023. The address is 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the deposit number is GDMCC No: 64077. DETAILED DESCRIPTION

[0023] The present invention provides a strain of Bacillus sabii, which is deposited in the Guangdong Provincial Microbial Culture Collection Center and has a taxonomic name of Bacillus safensis subsp . safensis14 ~C 22 Good degradation effect of petroleum hydrocarbons.

[0024] The present invention also provides a method for culturing Bacillus shaforniensis described in the above technical solution, comprising the following steps: inoculating Bacillus shaforniensis into a culture medium for fermentation culture to obtain a fermentation product of Bacillus shaforniensis. The present invention does not specifically limit the type of the culture medium, and any culture medium that can grow Bacillus shaforniensis can be used. In the present invention, the culture medium preferably includes an inorganic salt culture medium; the culture medium preferably also includes petroleum. In the present invention, the amount of petroleum added to the culture medium is preferably 0.1~10g / L. In one embodiment of the present invention, the amount of petroleum added is preferably 1g / L; in another embodiment of the present invention, the amount of petroleum added is preferably 1.5g / L; in another embodiment of the present invention, the amount of petroleum added is preferably 5g / L. In the present invention, the inorganic salt culture medium preferably includes an inorganic salt liquid culture medium or an inorganic salt solid culture medium. In the present invention, the inorganic salt liquid culture medium preferably includes: K2HPO4 (1g / L), KH2PO4 (1g / L), MgSO4 (0.5g / L), NH4Cl (1.5g / L), CaCl2 (0.02g / L), FeCl3 (0.01g / L), pH=7.0~7.2, more preferably pH=7.0. In the present invention, the inorganic salt solid culture medium preferably includes: K2HPO4 (1g / L), KH2PO4 (1g / L), MgSO4 (0.5g / L), NH4Cl (1.5g / L), CaCl2 (0.02g / L), FeCl3 (0.01g / L), agar (15g / L), pH=7.0. In the present invention, the culture temperature is 30~50°C, more preferably 45°C. The present invention does not specifically limit the type of fermentation product, which can be a fermentation broth, a supernatant or a bacterial cell.

[0025] The present invention also provides the use of the Bacillus sabdariffa described in the above technical solution in the degradation of petroleum. The present invention can be directly applied to the degradation of crude oil. The test results show that the degradation rate of crude oil in 5 days reaches 56.5%.

[0026] The present invention also provides the use of the Bacillus sabogensis described in the above technical solution in the degradation of petroleum hydrocarbons; the petroleum hydrocarbons include alkanes. In the present invention, the alkanes include C8~C9 and C 14 ~C 22 .

[0027] The present invention also provides the use of the Bacillus saffron described in the above technical solution in improving oil recovery and / or promoting the restoration of oil-polluted environments.

[0028] The present invention also provides the use of the Bacillus saffron described in the above technical solution in the preparation of a biosurfactant; the biosurfactant has an emulsifying effect.

[0029] The present invention also provides the use of the Bacillus sabogensis described in the above technical solution in improving the oil washing ability. The Bacillus sabogensis described in the present invention has the ability to produce surfactants, can reduce interfacial tension, has good oil washing efficiency (68.87-87.43%), and has high oil washing ability.

[0030] In order to further illustrate the present invention, a strain of Bacillus sabogen provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0031] The culture medium involved in the following examples is:

[0032] ① Inorganic salt liquid culture medium: K2HPO4 (1g / L), KH2PO4 (1g / L), MgSO4 (0.5g / L), NH4Cl (1.5g / L), CaCl2 (0.02g / L), FeCl3 (0.01g / L), pH=7.0.

[0033] ②Inorganic salt solid culture medium: K2HPO4 (1g / L), KH2PO4 (1g / L), MgSO4 (0.5g / L), NH4Cl (1.5g / L), CaCl2 (0.02g / L), FeCl3 (0.01g / L), agar (15g / L), pH=7.0.

[0034] ③Petroleum liquid culture medium: Add 5g crude oil to 1L of inorganic salt liquid culture medium.

[0035] ④LB solid medium: peptone (20 g / L), NaCl (10 g / L), beef extract (20 g / L), agar (15 g / L), pH = 7.0.

[0036] Example 1

[0037] Morphological characteristics of strains.

[0038] Inoculate a single colony into LB solid medium, place the plate upside down in a constant temperature incubator, and culture at 45℃ for 48h. The colony is round, slightly yellow and opaque, with a smooth and moist surface and a diameter of 2~4mm. Figure 1 shown.

[0039] Example 2

[0040] Isolation, screening and purification of Bacillus saffron.

[0041] The sample of the produced liquid was added to 100 mL of petroleum liquid culture medium at an inoculum volume of 5% (v / v) for enrichment culture, and cultured in a shaking incubator at 45°C and 150 r / min for 7 days. When the culture medium became turbid, the culture medium was aspirated and transferred back to fresh petroleum liquid culture medium at an inoculum volume of 5%, and the enrichment culture was continuously transferred 3 times under the same culture conditions as above.

[0042] The dilution plate method is used for separation. After the culture solution is diluted in series, 100 μL of the dilution solution is applied to the LB solid medium and cultured for 48 hours. After the colonies grow, observe the colony morphology, use a sterile inoculation loop to select colonies with large morphological differences from the above LB solid medium, and use the plate streak separation method to inoculate them on the LB solid medium. Place them in a constant temperature incubator at 45°C and invert for 48 hours. After the colonies grow, pick a single colony and inoculate it into the LB solid medium in the same way. Repeat the above streak separation process until a purified colony with a single morphology is formed. The single colony is back-inoculated in the LB solid medium and the inorganic salt solid medium containing 1g / L crude oil. The one that can grow in both culture media is the oil-degrading bacteria, and finally a strain of oil-degrading bacteria is obtained.

[0043] Example 3

[0044] Molecular identification of Bacillus saffron.

[0045] Bacterial species were identified by 16S rRNA gene sequence. Gene fragments were amplified using universal primers 27F-AGTTTGATCMTGGCTCAG (SEQ ID NO.2) and 1492R-GGTTACCTTGTTACGACTT (SEQ ID NO.3). PCR conditions were as follows: pre-denaturation at 94°C for 4 min; 94°C for 45 s, 55°C for 45 s, 72°C for 1 min, for a total of 30 cycles; standing at 72°C for 10 min, and cooling at 4°C. The PCR amplification product was analyzed using 1.7% agarose gel. The 16S rRNA gene was sequenced and analyzed using the BLAST algorithm. The nucleotide sequence of the 16S rRNA gene obtained by sequencing is shown in SEQ ID NO.1. The analysis results were compared with the common sequences in the NCBI database, and then a phylogenetic tree was constructed using MEGA11 software. The phylogenetic tree is shown in Figure 2 As shown, it can be seen that the strain of Bacillus shafusus DWC-2 and Bacillus safensis subsp . safensis strain FO-36b had a very high similarity, and the strain DWC-2 of Bacillus sabogensis was identified as Bacillus sabogensis, i.e. Bacillus safensis subsp . safensisDWC-2 was then sent to Guangdong Microbiological Culture Collection Center for preservation with the collection number GDMCC No: 64077.

[0046] Example 4

[0047] Degradation of crude oil by Bacillus saffron DWC-2.

[0048] (1) Growth curve

[0049] DWC-2 was inoculated into petroleum liquid culture medium and cultured in a shaker at 150 r / min and 45°C. The density of the bacteria (OD 600 value), with time as the horizontal axis, OD 600 The value is used as the vertical scale to draw its growth curve, and the result is as follows Figure 3 As shown in the growth curve, the bacteria grew rapidly in the petroleum liquid medium, and the OD 600 The value reaches 2.118, which indicates that the bacteria reproduce very quickly in the petroleum liquid culture medium, which is conducive to practical applications.

[0050] (2) Crude oil degradation rate

[0051] The strain DWC-2 seed solution (OD 600 The crude oil degradation rate was determined by UV spectrophotometry after the culture medium containing oil and inorganic salt (value of 1.4) was added to the sterilized oil-containing inorganic salt liquid medium (containing crude oil 1g / L) at 45℃ and 150rpm for 5 days. The blank control was set without adding Bacillus sabogenes DWC-2. Figure 4 This is a comparison chart of the blank control and the effect of adding Bacillus sabdariffa DWC-2 after 5 days of culture. Figure 4 As shown, the culture sample with the addition of strain Bacillus sabogenes DWC-2 has no obvious oil layer, while the oil layer and water layer in the blank control are clearly separated, indicating that Bacillus sabogenes DWC-2 has a significant effect on oil degradation. The degradation rate of crude oil by Bacillus sabogenes DWC-2 in 5 days reached 56.5%.

[0052] Example 5

[0053] Determination of emulsification properties of fermentation broth of Bacillus saffron DWC-2.

[0054] The DWC-2 seed solution (OD 600 The value was 1.4) and inoculated into 100 mL of petroleum liquid culture medium, and cultured in a shaker at 150 r / min and 45°C for 5 days to obtain a fermentation broth. The petroleum liquid culture medium without inoculation of Bacillus sabdariffa DWC-2 was used as a blank control.

[0055] Take two graduated test tubes and add 3 mL of diesel and crude oil respectively. Add 7 mL of fermentation liquid to each test tube and shake vigorously for 3 minutes. Let it stand at room temperature for 24 hours and then measure. Divide the height of the emulsion layer by the total height of the organic phase and multiply it by 100%, which is EI-24. If EI-24>50%, the emulsification is considered stable.

[0056] The emulsification activity of the fermentation broth of Bacillus shaforniensis DWC-2 for diesel and crude oil was greater than 50, 54% and 65% respectively, showing the application value of emulsifying crude oil to improve oil recovery and emulsifying and solubilizing hydrocarbon pollutants to enhance their biodegradation.

[0057] Example 6

[0058] Determination of surface and interfacial tension of fermentation broth of Bacillus saffron DWC-2.

[0059] The surface tension of the fermentation broth of Bacillus fusus DWC-2 (prepared by the same method as in Example 5) was detected by a kino surface tension meter (platinum plate method), and a petroleum liquid culture medium without inoculation of Bacillus fusus DWC-2 was used as a blank control. The results are shown in Table 1. The surface tension data were analyzed. Compared with the blank control, the surface tension of the fermentation broth was reduced after the action of Bacillus fusus DWC-2, and the surface tension decreased from 62.503 before the action to 24.882, with a reduction rate of 60.19%, which indicates that Bacillus fusus DWC-2 has a strong function of secreting surfactants.

[0060] Table 1 Surface tension of DWC-2 GDMCC No: 64077 fermentation broth

[0061]

[0062] The interfacial tension of the fermentation broth of Bacillus fusus DWC-2 was tested by TX500C spinning drop interfacial tension meter, and the petroleum liquid medium without inoculation of Bacillus fusus DWC-2 was used as the blank control. The results are shown in Table 2. The interfacial tension data were analyzed. Compared with the blank control, the interfacial tension was reduced after the action of Bacillus fusus DWC-2, and the interfacial tension decreased from 22.39 before the action to 2.47, with a reduction rate of 88.97%. This shows that compared with the blank control, Bacillus fusus DWC-2 can effectively reduce the interfacial tension.

[0063] Table 2 Interfacial tension of DWC-2 GDMCC No: 64077 fermentation broth

[0064]

[0065] In the process of microbial degradation of petroleum hydrocarbons, surfactants play a very important role as a medium. Since petroleum hydrocarbons are highly hydrophobic, especially polycyclic aromatic hydrocarbons in crude oil are extremely hydrophobic, if the microbial cells are highly hydrophilic, it is difficult for the microorganisms and petroleum hydrocarbons to contact each other and react. In the process of strengthening the treatment of oil-containing pollutants, the Bacillus sabogensis DWC-2 of the present invention can be used as a surfactant to reduce the oil-water interfacial tension and enhance the mutual contact between the Bacillus sabogensis DWC-2 cells and crude oil, thereby improving the degradation efficiency of Bacillus sabogensis DWC-2 on petroleum hydrocarbons, and then improving the bioremediation efficiency.

[0066] Example 7

[0067] Effects of Bacillus saffron DWC-2 on crude oil alkanes

[0068] In a 250mL Erlenmeyer flask, add 5g crude oil, 150mL inorganic salt liquid culture medium, and 10% (v / v) DWC-2 seed liquid, and culture at 45℃ and 150rpm for 7-15 days. After the culture, collect the fermentation liquid, centrifuge at 3500rpm for 15min, and use gas chromatography to detect and analyze the crude oil after the action of DWC-2 bacteria, and determine the degradation of different carbon alkanes by DWC-2 bacteria. The oil sample before action in Table 3 refers to the oil sample without inoculation: In a 250mL Erlenmeyer flask, add 5g oil sample and 150mL inorganic salt liquid culture medium, and culture under the same conditions.

[0069] It can be seen from Table 3 that after DWC-2 degrades petroleum hydrocarbons, C8~C9 and C 14 ~C 22 The content decreased, indicating that Bacillus shaforniensis DWC-2 had a better degradation effect on C8~C9 and medium- and long-chain alkanes.

[0070] Table 3 Carbon number distribution data of n-alkanes in crude oil before and after the action of Bacillus shafoetus DWC-2

[0071]

[0072] Example 8

[0073] Oil washing experiment

[0074] Crude oil and quartz sand were mixed evenly in a mass ratio of 1:5 to prepare oil sand, placed in a 60°C oven for aging for 24 hours, and then taken out and sealed for use. The fermentation broth of Bacillus shafusus DWC-2 was heated at 45°C for 8 hours, and then diluted in different proportions. Pour 30 mL of fermentation broth into a centrifuge tube, put in 15 g of oil sand (the initial oil content of the oil sand is 2.5 g), shake evenly 5 times, put it in a 60°C oven and let it stand, and take it out after 24 hours. After pouring out the upper liquid, rinse repeatedly with distilled water until the water phase is colorless. The dried oil sand is weighed (centrifuge tube + oil sand), the mass is m1, and the mass of the centrifuge tube is m0. The oil washing efficiency η is calculated by the following formula:

[0075]

[0076] Table 4 Fermentation broth oil washing efficiency

[0077]

[0078] This specific example shows that the fermentation liquid has a high oil washing efficiency and can separate the oil from the surface of the oil sand. After the oil washing, the oil sand is clean and loose, which has the effect of preventing crude oil from adhering again.

[0079] Example 9

[0080] Remediation effect of Bacillus saffron DWC-2 on marine oil spills

[0081] In the experiment, a 250 mL wide-mouth conical flask was used as a container, and an experimental group and a control group were set up respectively. Three parallel samples were set up for each group, and the experiment was carried out for 7 days at 45 ° C and 150 rpm. 10% DWC-2 bacterial solution and 90 mL of natural seawater were added to the experimental group, and then 0.15 g of crude oil was added. 100 mL of natural seawater was directly added to the control group, and then 0.15 g of crude oil was added. After 7 days, the oil degradation rate measured in the experimental group was 87.82%, which shows that the Bacillus shafusus DWC-2 described in the present invention has a repair effect on offshore oil spills.

[0082] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A strain of Bacillus saffron ( Bacillus safensis subsp. safensis ) DWC-2, characterized in that, The Bacillus shaforniensis DWC-2 is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 64077.

2. Use of the Bacillus sabdariffa DWC-2 described in claim 1 in degrading petroleum.

3. Use of the Bacillus sabdariffa DWC-2 of claim 1 in the degradation of petroleum hydrocarbons; the petroleum hydrocarbons include alkanes.

4. Use of the Bacillus sabdariffa DWC-2 according to claim 1 in improving oil recovery and / or promoting the remediation of oil-contaminated environments.

5. Use of the Bacillus sabdariffa DWC-2 according to claim 1 in the preparation of a biosurfactant; the biosurfactant has an emulsifying effect.

6. Use of the Bacillus sabdariffa DWC-2 described in claim 1 in improving oil washing ability.

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