Geobacillus stearothermophilus and application thereof

Abstract

The invention belongs to the technical field of microbial engineering, and particularly relates to geobacillus stearothermophilus and application thereof. The geobacillus stearothermophilus is preserved in the China General Microbiological Culture Collection Center on February 20, 2025, and the preservation number of the geobacillus stearothermophilus is CGMCC (China General Microbiological Culture Collection Center) NO.33577. The invention further discloses a preparation method of the geobacillus stearothermophilus. The strain can stably grow at the temperature of 80 DEG C and survive for 48 hours at the temperature of 85 DEG C. The method has the advantages of high genome stability and high metabolic activity, and the crude oil recovery rate can be remarkably improved; the method can also be applied to bioremediation of a high-temperature polluted environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial engineering, and particularly relates to a Geobacillus stearothermophilus strain and its uses. Background Art

[0002] Petroleum is a non-renewable energy source. The oil extraction technologies include primary oil recovery, secondary oil recovery, and tertiary oil recovery. After primary and secondary oil recoveries, 60%-70% of the oil still remains in the formation. Therefore, the tertiary oil recovery technology has emerged. The tertiary oil recovery technology, namely enhanced oil recovery (EOR) technology, is mainly divided into thermal recovery, chemical flooding, gas flooding, and microbial flooding. Microbial enhanced oil recovery (MEOR) uses the movement of microorganisms themselves and their metabolites to change the chemical and physical properties of reservoir rocks and crude oil, so as to achieve the effect of displacing and stripping oil from depleted and high water cut reservoirs.

[0003] Microorganisms feed on crude oil and activators in porous media, maintain activity at the oil-water interface under simulated reservoir conditions, while degrading residual oil, changing the wettability of the pore wall. After displacement, the residual oil in the membrane is reduced by 51.86% compared with water flooding, and the oil recovery factor is increased by 17.44%. Geobacillus thermophilus strains that produce biosurfactants and their metabolites can effectively emulsify the crude oil in the reservoir, reduce the oil-water interfacial tension, and the formed emulsion has a high viscosity, which can increase the oil flow rate, expand the volume of the injected fluid wave, and the clustered and columnar residual oils are reduced by 64% and 68% respectively. In the microchannel test tank, the in-situ cultured microorganisms rely on unique life activities (such as interface tropism and in-situ metabolism) to strip the residual oil on the pore wall deep in the blind end, which cannot be achieved by the exogenous injection method. After the in-situ microbial cultivation process, the deep blind end residual oil is reduced by about 47%, and the oil production is increased by about 15% compared with the exogenous injection method.

[0004] Microbial enhanced oil recovery (MEOR) technology relies on thermotolerant microorganisms, but the heat resistance limit (70°C) of existing thermophilic bacteria (such as the wild type of G. stearothermophilus SL-1) is not sufficient to adapt to the high temperature environment (≥80°C) of shale oil reservoirs. The genetic manipulation system of thermophilic bacteria is imperfect, and the stability of molecular components (such as promoters, plasmids) at high temperatures is insufficient, which limits the efficiency of genetic engineering modification. The existing defective wild type strains have insufficient heat resistance and cannot survive and play a role in high temperature reservoirs for a long time. Traditional genetic engineering methods are limited in the application of thermophilic bacteria, lacking an efficient electrotransformation system and stable expression elements. Summary of the Invention

[0005] To make up for the deficiencies in the existing technology, the present invention provides a Geobacillus stearothermophilus strain with significantly improved heat resistance. This strain has excellent high-temperature resistance, and its heat resistance limit reaches above 80°C. This strain can be applied to microbial enhanced oil recovery in high-temperature reservoir environments.

[0006] A Geobacillus stearothermophilus strain provided by the present invention Geobacillus stearothermophilus was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 20, 2025, with the deposit number CGMCC NO. 33577.

[0007] The present invention also provides the use of the above-mentioned strain, which is applied to microbial enhanced oil recovery or bioremediation in high-temperature environments.

[0008] The present invention also provides a composition for microbial enhanced oil recovery or bioremediation in high-temperature environments, which contains the above-mentioned strain or its metabolites.

[0009] The present invention further provides a method for microbial enhanced oil recovery, in which the above-mentioned strain or its metabolites are injected into the oil reservoir.

[0010] The present invention also further provides a method for bioremediation in high-temperature environments, in which the above-mentioned strain or its metabolites are added to petroleum-contaminated soil or industrial wastewater.

[0011] The Geobacillus stearothermophilus strain provided by the present invention Geobacillus stearothermophilus is obtained by means of adaptive laboratory evolution (ALE) and overexpression of heat-resistant related genes through genetic engineering. This strain can grow stably at 80°C and survive for 48 hours at 85°C (the wild type dies within 24 hours at 85°C). It has the advantages of high genomic stability and strong metabolic activity, can significantly improve the crude oil recovery rate, and can also be applied to the bioremediation of high-temperature polluted environments. Description of the Drawings

[0012] Figure 1 is the temperature range for the growth and reproduction of the wild-type strain; Figure 2 is the comparative genomics analysis of the heat-resistant mutant strain and the wild-type strain in the examples of the present invention; Figure 3 is the difference comparison of the distribution of gene islands in the genome; Figure 4 is the distribution in the genome of the changes at the DNA level between the heat-resistant mutant strain SL-1-80 and the wild-type strain; among them, a. wild-type strain; b. heat-resistant mutant strain SL-1-80; Figure 5 is the change at the gene level in the metabolic pathway between the heat-resistant mutant strain SL-1-80 and the wild-type strain; Figure 6 Comparison of the growth status of the heat-resistant mutant strain SL-1-80 and the wild-type strain under different hydrothermal conditions; Figure 7 Comparison of the growth status of the heat-resistant mutant strain SL-1-80 and the wild-type strain under different dry-heat conditions; a. 65 °C; b. 80 °C; Figure 8 Comparison of the growth status of the heat-resistant mutant strain SL-1-80 and the wild-type strain under different hydrothermal conditions. Specific implementation mode

[0013] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0014] Example 1. Heat-resistant mutant G. stearothermophilus Construction of SL-1-80 1. Determination of the growth temperature range of the wild type Inoculate the wild-type strain G. stearothermophilus SL-1 monoclonal into 1.8 mL of LB medium and culture it at 60 °C with shaking at 960 rpm for 12 h. Then take 30 μL of the culture solution and inoculate it into fresh 1.8 mL of LB medium. Set a series of temperature gradients of 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, and 75 °C. At different temperatures, after culturing with shaking at 960 rpm for 12 h, measure OD 600 , to determine the temperature range in which the wild-type Geobacillus stearothermophilus SL-1 can grow.

[0015] Through a single-factor experiment with the culture temperature as the variable, it was observed that within the established growth temperature range, the growth level of the wild-type strain showed a trend of first increasing and then decreasing, and reached the highest growth level at 65 °C. Therefore, it can be determined that the optimal growth temperature of this strain is 65 °C, and it can be judged that the limiting temperatures for the growth and reproduction of the wild-type strain under experimental conditions are 45 °C and 70 °C. When the culture temperature is lower than 45 °C or higher than 70 °C, the strain cannot grow vigorously, as Figure 1 shown.

[0016] 2. Induction of heat-resistant mutants To induce the strain to mutate towards heat resistance, the ALE experiment under high temperature was applied. The development of heat-resistant mutants included heat acclimation to the maximum survival temperature and the selection of single colonies with heat resistance. The wild-type Geobacillus stearothermophilus SL-1 was cultured at the optimal temperature for 16 h and then spread on an LB plate to prepare the strain for heat acclimation. A wild-type single colony was inoculated into 1.8 mL of LB medium and placed at its maximum growth temperature, and cultured at 960 rpm for 18 h. After the culture was completed, the OD of the bacterial solution was measured. 600 The mutant strain with the optimal phenotype in this round was selected, and the remaining bacterial solution was centrifuged and concentrated. One part was used for glycerol preservation of bacteria, and the other part was spread on an LB solid plate and cultured at the corresponding temperature. Subsequently, the culture temperature was increased by 2 °C and the same process was repeated until no colonies survived on the plate after the temperature was increased again. The mutant strain preserved in glycerol during the same period was the mutant strain with the highest temperature tolerance.

[0017] This heat-resistant mutant strain was named Geobacillus stearothermophilus Geobacillus stearothermophilus SL-1-80, and was deposited on February 20, 2025 at the China General Microbiological Culture Collection Center, with the deposit number CGMCC NO. 33577. The address of the deposit unit is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0018] 3. Whole-genome sequencing was performed on the wild-type strain and the mutant strain with the highest temperature tolerance obtained by adaptive evolution. The GeneMarkS software was used to predict the coding genes of the newly sequenced genome, the RepeatMasker software was used to search for tandem repeat sequences in the sequence, and the IslandPath-DIOMB software was used to predict gene islands. The antiSMASH was used to analyze the gene clusters of stimulatory metabolites in the genome, and tools such as SignalP, TMHMM, and EffectiveT3 were used to predict signal peptides and transmembrane structures, and the secreted proteins and secretion systems were comprehensively predicted. The Geneious Prime software was used to carefully compare the size, gene number, and differences in gene sequences between the wild-type and the mutant strain with the highest temperature tolerance of Geobacillus stearothermophilus SL-1 to determine the types of mutations that are beneficial to improving the heat resistance of the SL-1 strain, such as Figure 2 shown.

[0019] This invention confirmed that the total length of the coding region of the genome of the heat-resistant mutant strain G. stearothermophilus SL-1-80 did not change, and the genome size decreased by 515 bp compared with the wild-type strain. Interestingly, the total number of genes in the SL-1-80 strain did not show consistency with the total length of the coding region or the genome size, but instead increased by 4, as shown in Table 1.

[0020] Table 1 Comparison of parameters at the genome level between the wild-type and the mutant strain Sample Name Genome Size (bp) Number of Genes (pcs) Coding Region Length (bp) Average Gene Length (bp) Wild Type 3,611,261 3,850 3,107,286 807 SL-1-80 3,610,746 3,854 3,107,286 806

[0021] In the analysis based on genomic components, the heat-resistant mutant strain G. stearothermophilus There was no observed difference in non-coding RNAs between strain SL-1-80 and the wild-type strain. However, there was a 1366-bp shortening in the length of the genomic island of strain SL-1-80. Obvious differences were also observed when examining the distribution characteristics of genes in 21 genomic islands. The total number of genes increased by 2, as Figure 3 shown. Through alignment, it was found to be the insertion element IS5377 and the transposon IS4. These sequences are exactly the genetic factors related to biological functions such as pathogenic mechanisms and organism adaptability that are integrated into the microbial genome through horizontal gene transfer from bacteria, phages, or plasmids.

[0022] 4. Differential comparison of the distribution of genomic islands Further alignment and analysis of the genomic sequences of the heat-resistant mutant strain and the wild-type strain found 2 position changes of transposons in strain SL-1-80, both of which were the co-movement of the insertion element IS5377 and the transposon IS4. The occurrence positions are shown in Figure 4 a and b in it. In addition, 1 gene deletion was found, which was the deletion of the transposon IS110. The translocation and deletion of the transposon reduced the genome by 515 bp.

[0023] In the effector-based comparison, no significant differences were observed between the strains in secreted effector proteins and secondary metabolite gene clusters. Through KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathway analysis based on gene annotation, it was found that the number of genes encoding cell motility proteins and factors related to signal transduction in the genome of strain SL-1-80 changed, as Figure 5 shown. By sequence alignment to find these changes, it was found that the gene encoding the BglG family transcriptional antiterminator in strain SL-1-80 terminated prematurely, and a frameshift mutation occurred within the gene encoding the FliA / whiG family RNA polymerase σ factor, and these 2 genes are both related to DNA transcription. In addition, there were 3 amino acid differences caused by single-base mutations, a Ser to Pro mutation occurred within the gene encoding the flagellar motility switch phosphatase FliY, a Ser to Ala mutation occurred within the gene encoding the toxic anion resistance protein, and the 206th amino acid in the UPF0111-like protein YkaA gene changed from Glu to Ala. These results were basically consistent with the changes predicted by KEGG. The mutations occurred in signal transduction and motility proteins during DNA transcription, and their metabolic and synthetic pathways did not change. This indicates that the heat-resistant mutant strain SL-1-80 provided by the present invention has further improved the ability to transduce environmental signals and respond to them on the basis of maintaining the physiological functions of the existing wild-type Geobacillus stearothermophilus, including the improvement of heat resistance to high-temperature environments.

[0024] The present invention compares the growth conditions of the heat-resistant mutant strain SL-1-80 and the wild-type strain under different humid heat and dry heat conditions, and the results are as Figure 6 and Figure 7 shown. The heat resistance of the SL-1-80 strain is increased to 80 °C, which is significantly better than that of the existing wild-type strain.

[0025] The present invention also identifies the survival rates of the wild-type strain G. stearothermophilus SL-1 and the heat-resistant mutant strain SL-1-80 at 85 °C, and the results are as Figure 8 shown. The heat-resistant mutant strain SL-1-80 survives for 48 hours at 85 °C, while the wild-type strain dies within 24 hours at 85 °C. This may be due to enhanced genomic stability (reduction of transposons), reduced gene replication burden, and improved high-temperature adaptability.

[0026] II. Uses of the heat-resistant mutant strain G. stearothermophilus SL-1-80 1. Application in microbial enhanced oil recovery (MEOR): Inject the heat-resistant mutant strain SL-1-80 into the oil reservoir, and use its metabolites (such as biosurfactants, organic acids) to reduce the viscosity of crude oil and improve the oil recovery rate.

[0027] 2. Bioremediation in high-temperature environments: Add the heat-resistant mutant strain SL-1-80 or its metabolites to petroleum-contaminated soil or industrial wastewater for high-temperature degradation treatment of petroleum-contaminated soil or industrial wastewater.

Claims

1. A strain of Geobacillus stearothermophilus Geobacillus stearothermophilus , characterized in that: The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on February 20, 2025, with the deposit number CGMCCNO.33577.

2. The use of the strain according to claim 1, characterized in that: The strain is used for microbial enhanced oil recovery or bioremediation in high temperature environments.

3. A composition for microbial enhanced oil recovery or high temperature environment bioremediation, characterized in that: The composition comprises the strain according to claim 1 or its metabolites.

4. A method for microbial enhanced oil recovery, characterized in that: The strain or its metabolites according to claim 1 are injected into an oil reservoir.

5. A high temperature environment bioremediation method, characterized in that: The strain or its metabolites according to claim 1 are added to petroleum-contaminated soil or industrial wastewater.

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