Bacillus complex microbial inoculant and application thereof in promoting anaerobic degradation of petroleum hydrocarbon by microbial flora

Abstract

The invention discloses a bacillus complex microbial inoculant and application of the bacillus complex microbial inoculant in promoting anaerobic degradation of petroleum hydrocarbon by microbial flora, and relates to the technical field of petroleum microorganisms. The bacillus composite bacterial agent is prepared from bacillus tequilensis (Bacillus tequilensis) and bacillus subtilis (Bacillus subtilis), and the bacillus tequilensis (Bacillus subtilis) is prepared from bacillus tequilensis (Bacillus tequilensis) and bacillus subtilis (Bacillus subtilis). The bacillus complex microbial inoculant is simple and convenient to prepare, the anaerobic degradation efficiency of microorganisms on petroleum hydrocarbon is improved by regulating and controlling microbial communities, and the bacillus complex microbial inoculant has application value in oil and gas resource exploitation and petroleum pollution remediation.

Description

Technical Field

[0001] The present invention relates to the technical field of petroleum microorganisms, in particular to a Bacillus composite bacterial agent and its use as a synergist to improve the anaerobic degradation efficiency of microbial flora on petroleum hydrocarbons. Background Art

[0002] Oil-containing environments are home to a large number of microbial communities capable of degrading petroleum hydrocarbons. Oil reservoirs and deep, oil-contaminated environments are anoxic or even completely anaerobic. Both residual oil biogasification in abandoned reservoirs and microbial remediation of oil pollution involve the anaerobic biodegradation of petroleum hydrocarbons.

[0003] Because there is no participation of oxygen electron acceptors, anaerobic degradation of petroleum hydrocarbons by microorganisms has the problems of low degradation efficiency and long degradation time compared with aerobic degradation.

[0004] Developing microbial strains or communities capable of efficiently degrading petroleum hydrocarbons under anaerobic conditions can improve the efficiency of anaerobic degradation of petroleum hydrocarbons to a certain extent. However, developing efficient anaerobic degradation bacteria for petroleum hydrocarbons is difficult, with a low success rate, and the process is cumbersome, time-consuming, and labor-intensive. Currently, there is a need for new technologies that can easily and conveniently improve the efficiency of anaerobic microbial degradation of petroleum hydrocarbons. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for promoting the anaerobic degradation of petroleum hydrocarbons by microbial flora using a Bacillus composite agent. Bacillus tequilensis ) and Bacillus subtilis ( Bacillus subtilis ) is used as the bacterial strain to prepare a Bacillus composite agent, and the anaerobic degradation of petroleum hydrocarbons is promoted by adding the Bacillus composite agent to the anaerobic hydrocarbon decomposition microbial enrichment to solve the problem of low efficiency of anaerobic degradation of petroleum hydrocarbons by microorganisms.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A Bacillus composite bacterial agent comprising Bacillus tequilensis and Bacillus subtilis Two Bacillus species.

[0007] described Bacillus tequilensis and Bacillus subtilis They are all conventional Bacillus, which can be purchased or screened and isolated from environmental samples.

[0008] The preparation method of the above-mentioned Bacillus composite agent is as follows: culturing the Bacillus tequilensis and Bacillus subtilis After 8 to 12 hours, the bacteria in the bacterial solution were counted and the bacterial concentration was higher than 2.0×10 8CFU / mL of two Bacillus seed liquids; the two bacterial seed liquids are mixed in a volume ratio of 1:1, the bacterial precipitates are collected by centrifugation at 4000-6000 rpm, and the bacterial precipitates are resuspended with an equal volume of sterile physiological saline to obtain a Bacillus composite bacterial agent.

[0009] The liquid LB medium comprises 1% peptone, 0.5% yeast extract, 1% sodium chloride, and the remainder is distilled water; the pH is adjusted to 6.8-7.2 and sterilized at 121° C. for 20 minutes.

[0010] The sterile physiological saline solution comprises 0.9% sodium chloride and distilled water and is prepared by sterilizing at 121° C. for 20 minutes.

[0011] A method for promoting anaerobic degradation of petroleum hydrocarbons by microbial flora using a Bacillus composite agent is carried out according to the following steps: (1) Prepare crude oil inorganic salt culture medium and anaerobic enrichment: Take 0.2%~0.6% nitrate, 0.1%~0.3% sulfate, 0.03%~0.08% ferric chloride, 0.05%~0.15% fumaric acid, 0.2%~0.5% potassium dihydrogen phosphate, 0.3%~0.6% dipotassium hydrogen phosphate, 0%~0.8% trace element solution, 1%~5% crude oil, and the rest is water, prepare crude oil inorganic salt culture medium, and dispense it into closed anaerobic culture devices such as anaerobic tubes or anaerobic bottles. Add 2%~10% of oil reservoir produced fluid, petroleum-contaminated soil, or petroleum wastewater, add sodium sulfide with a final concentration of 5~20 mg / L to remove oxygen from the culture system, and enrich and culture at a temperature of 25~40℃ and a rotation speed of 60~100 rpm for 8~20 days to obtain microbial enrichment that can anaerobicly degrade petroleum hydrocarbons.

[0012] (2) Adding Bacillus composite bacterial agent to promote anaerobic degradation of petroleum hydrocarbons by enriched microorganisms: Add the prepared Bacillus composite bacterial agent in an amount of 5% to 15% by volume to the anaerobic enriched material in (1), and culture it anaerobically for 20 to 200 days to enhance the anaerobic degradation of petroleum hydrocarbons.

[0013] The method of promoting the anaerobic degradation of petroleum hydrocarbons by the Bacillus composite agent has application value in oil and gas resource exploitation and petroleum pollution remediation.

[0014] The application of the above-mentioned Bacillus composite bacterial agent in regulating anaerobic hydrocarbon decomposition microbial communities.

[0015] Furthermore, the Bacillus complex agent regulated the decrease in the abundance of Proteobacteria in the bacterial community and the increase in the abundance of Firmicutes and Actinobacteria.

[0016] Furthermore, the Bacillus complex agent regulated the increase in the abundance of hydrocarbon-degrading bacteria in the microbial community.

[0017] Furthermore, the Bacillus complex agent regulated the increase in the abundance of anaerobic hydrocarbon decomposition functional genes in the bacterial community.

[0018] Furthermore, the anaerobic hydrocarbon decomposition functional gene is masD and bamA .

[0019] The beneficial effects of the present invention are: The Bacillus sp. composite inoculant used in the present method acts as a synergist, improving the efficiency of anaerobic degradation of petroleum hydrocarbons by microbial communities through ecological regulation of the microbial community. The Bacillus sp. composite inoculant of the present invention exhibits vigorous growth, high biomass, and easy expansion. Compared with other methods, the composite inoculant of the present invention is readily available, low-cost, and amenable to industrial production. The method of using the Bacillus sp. composite inoculant to promote anaerobic degradation of petroleum hydrocarbons by microbial communities offers advantages such as high efficacy, high stability, and a simple process. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The community composition of anaerobic enrichment bacteria at the phylum level; Figure 2 Community composition of anaerobic enrichment bacteria at the genus level. DETAILED DESCRIPTION

[0021] The present invention will be described in detail with reference to the following specific examples to enable those skilled in the art to more fully understand the present invention, but the present invention is not limited in any way. In the following examples, unless otherwise specified, the materials and reagents used can be purchased from biochemical reagent material companies. Bacillus tequilensis ) and Bacillus subtilis ( Bacillus subtilis ) are all conventional Bacillus, which can be purchased or screened and isolated from environmental samples.

[0022] Example 1: Preparation of Bacillus sp. composite bacterial agent Use an electronic balance to weigh the required reagents and prepare liquid LB medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, and add distilled water to 1 L, pH 7.0. To prepare solid LB medium, add 20 g / L agar powder to the liquid LB medium. Sterilize all media at 121°C for 20 min.

[0023] In this example, Bacillus subtilis ( Bacillus subtilis ) CGMCC NO. 26136 and Bacillus tequila ( Bacillus tequilensis) SL9 (Su et al., Rapid biosynthesis of biosurfactants by Bacillus tequilensis SL9 isolated from oily sludge: Characterization, optimization, and potential applications. Journal of Surfactants and Detergents. 2024, 27(2): 211-222) is the bacterial strain.

[0024] Use an inoculation loop to pick up 1 loop of bacterial slant and inoculate it into a triangular flask containing 100 mL of liquid LB medium. Cultivate at 35°C and 170 rpm for 10 hours to obtain seed solutions of two Bacillus strains, CGMCC NO.21276 and SL9, respectively.

[0025] The bacteria in the two Bacillus seed solutions were counted by dilution plating method using solid LB medium, and the bacterial concentration of Bacillus CGMCC NO.21276 was 2.62×10 9 CFU / mL, the strain concentration of Bacillus SL9 was 2.34×10 9 CFU / mL.

[0026] The seed liquid of two Bacillus species was mixed in a volume ratio of 1:1, 20 mL of seed liquid of each was taken into a sterilized 50 mL centrifuge tube, centrifuged at 5000 rpm for 10 min, the bacterial precipitate was collected, and the bacterial precipitate was resuspended in 40 mL of sterile saline to obtain a Bacillus composite agent.

[0027] Example 2: Preparation of anaerobic enrichment Weigh the required reagents using an electronic balance to prepare an inorganic salt medium: 3.0 g sodium nitrate, 1.5 g magnesium sulfate, 0.5 g ferric chloride, 1.0 g fumaric acid, 3.0 g potassium dihydrogen phosphate, 4.0 g potassium hydrogen phosphate, and 2.0 mL of trace elements. Add distilled water to 1 L, pH 7.0. Boil the solution for 15 minutes to remove oxygen. Add crude oil by gravimetric method to an anaerobic flask to a crude oil concentration of 20 g / L. Add 100 mL of inorganic salt medium to the anaerobic flask. Trace element solution (g / L): MnCl2·4H2O 0.03, H3BO3 0.06, CoCl2·6H2O 0.2, ZnSO4·7H2O 0.1, CuSO4 0.01, (NH4)6Mo7O 24 ·4H2O0.02.

[0028] In this example, oil reservoir produced fluid was used as the bacterial source. 7 mL of produced fluid was added to the anaerobic flask containing the crude oil inorganic salt culture medium described above using a syringe. 0.1 mL of filter-sterilized sodium sulfide solution (10 g / L) was also added to the anaerobic flask using a syringe to remove oxygen from the system. The anaerobic flask was then cultured at 35°C and 80 rpm for 12 days to obtain an enrichment of microorganisms capable of anaerobic degradation of petroleum hydrocarbons derived from the produced fluid.

[0029] Example 3: Effect of Bacillus composite agent on the efficiency of anaerobic degradation of crude oil by enriched bacterial population Following the method of Example 2, an anaerobic enrichment was obtained by anaerobically culturing in a crude oil inorganic salt medium. The anaerobic enrichment was divided into two groups. One group continued to undergo anaerobic culture for 30 days, while the other group was added with 10 mL of the Bacillus complex inoculum prepared in Example 1 using a sterile syringe. All cultures were anaerobically cultured at 35°C and 80 rpm for 30 days.

[0030] After the incubation period, the anaerobic degradation rate of crude oil by the microorganisms was determined gravimetrically, using n-hexane as the solvent for extracting the remaining crude oil. The microbial culture medium and the remaining crude oil were transferred in batches to 50 mL centrifuge tubes. The crude oil adhered to the rubber stopper was rinsed with n-hexane. The crude oil was then extracted by adding n-hexane. The tubes were centrifuged at 4°C and 3000 rpm for 10 min, and the upper n-hexane organic phase containing the crude oil was transferred to a weighed Petri dish. The n-hexane solvent was allowed to evaporate naturally in a fume hood (>6 hours). The Petri dish and the remaining crude oil were weighed to calculate the remaining crude oil weight. The crude oil degradation rate was calculated using the following formula: Crude oil degradation rate (%) = (initial crude oil weight - remaining crude oil weight) / initial crude oil weight × 100%. The anaerobic degradation rate of crude oil by the anaerobic enrichment was 26.88% ± 4.11%, while that of the anaerobic enrichment supplemented with the Bacillus sp. complex inoculum reached 44.35% ± 2.99%. The results show that the method of the present invention can significantly promote the degradation of crude oil by anaerobic enrichment bacteria under anaerobic conditions.

[0031] Example 4 Anaerobic enrichment was obtained by anaerobic culture in a crude oil inorganic salt medium according to the method of Example 2. The anaerobic enrichment was divided into two groups. One group continued to undergo anaerobic culture for 180 days, while the other group was added with 10 mL of the Bacillus composite inoculum prepared in Example 1 using a sterile syringe and anaerobically cultured for 180 days.

[0032] After the incubation period, the anaerobic degradation rate of crude oil by the microorganisms was measured gravimetrically, using n-hexane as the solvent for extracting the remaining crude oil. The anaerobic degradation rate of crude oil by the anaerobic enrichment was 45.94% ± 3.93%, while that by the anaerobic enrichment supplemented with the Bacillus sp. composite inoculant reached 72.91% ± 3.64%. The results demonstrate that the method of the present invention significantly promotes the anaerobic degradation of crude oil by microbial flora, with good and stable results. The addition of the Bacillus sp. composite inoculant may have altered the microbial community in the anaerobic enrichment, thereby improving the anaerobic degradation efficiency of crude oil by the microorganisms.

[0033] Example 5: Regulation of the enrichment bacterial community by a Bacillus complex agent Anaerobic enrichment culture fluids and anaerobic enrichment culture fluids supplemented with a Bacillus complex inoculum were collected and centrifuged at 10,000 rpm for 15 minutes at 4°C to obtain bacterial pellets. Total DNA was extracted from the collected bacterial cells using a soil genomic DNA extraction kit. DNA samples were analyzed for concentration and purity using a Nanodrop ultraviolet spectrophotometer and stored at -20°C until use. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified by PCR using the barcoded bacterial universal primers 338F (5'-Barcode-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGG TWTCTAAT-3'). Libraries of the amplified 16S rDNA V3-V4 region fragments were constructed and sequenced using a high-throughput sequencer. After the raw sequencing data is downloaded from the machine, data quality control processing is performed. The optimized sequence is obtained through sequence splicing, filtering and removal of chimeras. Then, OTU (Operational Taxonomic Units) clustering and annotation are performed, and diversity analysis is performed based on the clustering results.

[0034] like Figure 1As shown, the dominant bacterial phyla in the anaerobic enrichment and the anaerobic enrichment supplemented with the Bacillus complex were Proteobacteria, Actinobacteriota, Firmicutes, Bacteroidota, Gemmatimonadota, Desulfobacterota, and Chloroflexi. Bacillus belongs to the phylum Firmicutes. Proteobacteria, Actinobacteria, and Firmicutes dominated the bacterial communities in the anaerobic enrichment, regardless of whether the Bacillus complex was added. The relative abundance of Proteobacteria in the anaerobic enrichment was 63.82%, while that in the anaerobic enrichment supplemented with the Bacillus complex was 53.70%, a decrease of 10.12%. The relative abundance of Actinobacteria in the anaerobic enrichment was 13.21%, while that in the anaerobic enrichment with the Bacillus complex increased to 19.74%, a 6.53% increase. The relative abundance of Firmicutes in the anaerobic enrichment was 8.24%, while that in the anaerobic enrichment with the Bacillus complex increased to 11.49%, a 3.25% increase. The abundance of Proteobacteria decreased in the anaerobic enrichment with the Bacillus complex, while the abundance of Firmicutes and Actinobacteria increased.

[0035] like Figure 2 As shown in the figure, at the genus level, the abundance of Bacillus genus was significantly increased in the anaerobic enrichment after the addition of Bacillus complex inoculant compared with the anaerobic enrichment, indicating that the added Bacillus can colonize in the anaerobic system. Roseovarius (relative abundance 16.30%), Stenotrophomonas (relative abundance 16.3%), Pseudomonas (Relative abundance 7.44%), Nocardioides (relative abundance 5.61%), Ochrobactrum (relative abundance 3.41%), Achromobacter (relative abundance 2.46%), Dietzia (relative abundance 1.53%), Saccharomonospora (relative abundance 1.50%) became the dominant bacterial genera in the system. The results showed that the added Bacillus complex inoculant indirectly promoted the anaerobic degradation of crude oil by microorganisms by regulating the anaerobic enrichment bacterial community, especially increasing the abundance of hydrocarbon-degrading bacterial genera, thereby enhancing the anaerobic degradation of crude oil.

[0036] Example 6: Quantitative PCR analysis of anaerobic hydrocarbon decomposition functional genes Gene masD and bamAThe quantitative PCR method was used to study the genes in the anaerobic enrichment microbial community and the anaerobic enrichment with the addition of Bacillus sp. masD and bamA The abundance information of Bacillus complex was used as the control. The quantitative PCR primers for the genes were: masD-F (5'-KGAYTTTGAGSASCTTTTCS-3') masD-R (5'-TCGTCCACRTARTCGTCGTC-3') bamA-F (5'-GCAGTACAAYTCCTACACSACYGABATGGT-3') bamA-R (5'-CCRTGCTTSGGRCCVGCCTGVCCGAA-3') Quantitative PCR system: 10 µL of 2× UltraSYBR Mixture, 0.5 µL of 10 µM upstream primer and 0.5 µL of downstream primer, water added to a total volume of 18 µL, then 2 µL of DNA sample was added. Quantitative PCR was performed according to the following protocol: 95°C for 10 minutes, followed by 40 cycles of PCR (95°C for 15 seconds, 60°C for 1 minute), with fluorescence collected. After the amplification reaction, a melting curve of the PCR product was constructed: 95°C for 15 seconds, 60°C for 1 minute, 95°C for 30 seconds, 60°C for 15 seconds, and then slowly heated from 60°C to 99°C. A plasmid containing the corresponding target hydrocarbon-dissolving gene was used as a quantitative PCR standard, starting from 10 1 -10 5 Perform a 10-fold serial dilution, using 2 µL of each dilution as template. Plot a standard curve for the target gene using the logarithmic value of the target gene copy number as the horizontal axis and the initial cycle number (Ct value) at which the fluorescence signal threshold is reached during the PCR reaction as the vertical axis. Calculate the target gene copy number in the sample based on the standard curve.

[0037] Anaerobic hydrocarbon decomposition functional genes were detected in both anaerobic enrichment and anaerobic enrichment with Bacillus complex inoculum. masD and bamA No anaerobic hydrocarbon decomposition functional genes were detected in anaerobic cultures containing only Bacillus sp. masD and bamA . Functional genes for anaerobic hydrocarbon degradation in anaerobic enrichment masD and bamA The abundance is 1.33×10 4 copies / mL and 6.41×10 5 Copies / mL, anaerobic hydrocarbon decomposition functional genes in anaerobic enrichment with Bacillus complex inoculummasD and bamA The abundance of 5 copies / mL and 2.57×10 6 The increase in the abundance of anaerobic hydrocarbon-degrading functional genes indicates that the added Bacillus complex agent promotes the anaerobic degradation of crude oil by microorganisms by regulating the increase in the abundance of anaerobic hydrocarbon-degrading functional bacteria in the anaerobic enrichment microbial community.

[0038] The composite bacterial agent of the present invention is readily available, grows vigorously, produces high biomass, is easily scalable, is low-cost, and is readily industrially prepared. The Bacillus sp. composite bacterial agent of the present invention acts as a synergist, improving the anaerobic degradation efficiency of petroleum hydrocarbons by microbial communities through ecological regulation. It has advantages such as good efficacy, high stability, and a simple process, and has application value in oil and gas resource extraction and petroleum pollution remediation.

Claims

1. Application of a Bacillus composite agent to improve the efficiency of petroleum hydrocarbon degradation under anaerobic conditions in oil and gas resource exploitation and oil pollution remediation, characterized in that: The Bacillus composite bacterial agent includes Bacillus tequila ( Bacillus tequilensis ) and Bacillus subtilis ( Bacillus subtilis ).

2. A method for promoting anaerobic degradation of petroleum hydrocarbons by microbial flora using a Bacillus composite inoculant, characterized in that: The Bacillus composite bacterial agent includes Bacillus tequila ( Bacillus tequilensis ) and Bacillus subtilis ( Bacillus subtilis ); The steps to enhance efficiency are: (1) Prepare crude oil inorganic salt culture medium and anaerobic enrichment: take 0.2%~0.6% nitrate, 0.1%~0.3% sulfate, 0.03%~0.08% ferric chloride, 0.05%~0.15% fumaric acid, 0.2%~0.5% potassium dihydrogen phosphate, 0.3%~0.6% dipotassium hydrogen phosphate, 0%~0.8% trace element solution, 1%~5% crude oil, and the rest is water, prepare crude oil inorganic salt culture medium, add 2%~10% of oil reservoir produced fluid or petroleum contaminated soil or petroleum wastewater, and enrich and culture under anaerobic conditions to obtain microbial enrichment for anaerobic degradation of petroleum hydrocarbons; Anaerobic enrichment culture parameters were as follows: sodium sulfide was added to a closed anaerobic culture system to remove oxygen at a final concentration of 5-20 mg / L, the enrichment time was 8-20 days, the temperature was 25-40°C, and the rotation speed was 60-100 rpm; (2) Preparation of Bacillus complex agent: activation Bacillus tequilensis and Bacillus subtilis Obtain seed solutions of two Bacillus species, mix the two seed solutions in a volume ratio of 1:1, collect bacterial precipitates by centrifugation at 4000-6000 rpm, and resuspend the bacterial precipitates in an equal volume of sterile physiological saline to obtain a Bacillus composite agent; The concentrations of the two Bacillus species in the composite bacterial agent are both higher than 1.0×10 8 CFU / mL; (3) Bacillus composite agent as a synergist to promote the anaerobic degradation of petroleum hydrocarbons by the enriched microbial community: the Bacillus composite agent prepared in (2) was added to the anaerobic enriched material in (1) at a volume ratio of 5% to 15%, and anaerobic culture was carried out for 20 to 200 days to enhance the anaerobic degradation of petroleum hydrocarbons.

3. Application of Bacillus complex inoculants in regulating anaerobic hydrocarbon-degrading microbial communities.

4. The use according to claim 3, characterized in that The Bacillus complex inoculant decreased the abundance of Proteobacteria in the bacterial community, while increased the abundance of Firmicutes and Actinobacteria.

5. The use according to claim 3, characterized in that The Bacillus complex agent regulates the increase in the abundance of hydrocarbon-degrading bacteria in the microbial community.

6. The use according to claim 3, characterized in that The Bacillus complex agent regulates the increase in the abundance of anaerobic hydrocarbon-decomposing functional genes in the bacterial community.

7. The use according to claim 6, characterized in that The anaerobic hydrocarbon decomposition functional gene is masD and bamA .

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