CN118006497A - Rhodococcus and application thereof in petroleum pollution remediation - Google Patents
Rhodococcus and application thereof in petroleum pollution remediation Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to rhodococcus and application thereof in petroleum pollution restoration. The strain OS36 is preserved in China Center for Type Culture Collection (CCTCC) at 12 and 22 days 2023, and the preservation number is M20232625. The strain OS36 disclosed by the invention can be used for repairing petroleum pollution, and has the advantages of high repairing efficiency and short repairing time in repairing petroleum pollution.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to rhodococcus and application thereof in petroleum pollution restoration.
Background
Rhodococcus is a gram-positive bacterium which can be isolated from various environments such as soil, deep sea and the like, and belongs to the families of actinomycota, actinomycetes and nocardiaceae; the cell wall structure, large genome and circular plasmid are unique, can secrete great amount of active enzyme, and can utilize organic compound as energy source and carbon source fully.
Petroleum and its derivative product have very important roles in daily life, industrial production and other fields. However, the emission of petroleum during exploitation, smelting, storage, transportation and use causes great damage to the environment, and frequent oil leakage accidents around the world are aggravated by this damage. The world has attracted a great deal of attention to the pollution of most waters on earth, especially the ocean, which is contaminated with petroleum throughout the year.
Petroleum pollution refers to pollution caused by the entry of crude oil and various petroleum products into the environment during exploitation, refining, storage and use. Pollution of the ocean by petroleum is currently mainly a serious problem worldwide.
The treatment method of petroleum pollutants mainly comprises a physical method, a chemical method and a biological method. The biodegradation method has the advantages of low cost, strong adaptability, high efficiency, safety and no secondary pollution, and becomes a research hot spot.
The ability of microorganisms to degrade petroleum hydrocarbons depends on a combination of factors such as the composition of the various components in the petroleum, the complexity of the carbon chains and polycyclic aromatic hydrocarbons, the environmental conditions, the type of strain, the presence or absence of surfactants, the presence or absence of surfactant-producing bacteria, and the physicochemical properties of the environment (pH, temperature, ratio of C: N: P). At present, microorganisms degrading petroleum are mainly concentrated on Acinetobacter, bacillus, arthrobacter and the like. However, the existing microorganisms capable of degrading petroleum have the problems of weak environmental adaptability, low repair efficiency and long repair time in the petroleum degradation process. For example, chinese patent CN 105907675A discloses a Rhodococcus strain with low temperature petroleum degrading function and its application, the Rhodococcus strain is a Rhodococcus strain (Rhodococcus sp.qy-2) of degradable crude oil separated from the surface layer of petroleum polluted seawater, and the petroleum polluted wastewater with crude oil concentration of 0.5% is continuously repaired for 60 days, and the crude oil degradation rate reaches 53%. Chinese patent CN 107893047A discloses a strain of Bacillus laterosporus (Brevibacillus laticosporus. G-40) separated from petroleum contaminated soil, the crude oil concentration is 0.6%, and the degradation rate of the crude oil is 53.02% after continuous 30 days of restoration. Chinese patent CN 104017747B discloses an invention for degrading oily sludge by using corynebacterium glutamicum (Corynebacterium glutamicum), and the petroleum degradation rate is 39.69% after 30 days of treatment at 37 ℃. The strains disclosed in the above patents all require a longer time (1 month to several months) to achieve a higher degradation efficiency of petroleum hydrocarbons. Therefore, the existing microorganism capable of degrading petroleum has the problems of low repairing efficiency and long repairing time in repairing petroleum pollution, and a new microorganism capable of degrading petroleum is needed to repair petroleum pollution.
Disclosure of Invention
In order to solve the problems of low restoration efficiency and long restoration time of the existing microorganism capable of degrading petroleum in the prior art in restoration of petroleum pollution, the invention provides rhodococcus and application thereof in restoration of petroleum pollution wastewater. In order to achieve the above purpose, the invention adopts the following technical scheme:
The invention provides a rhodococcus strain OS36, wherein the rhodococcus strain OS36 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20232625 at 12 and 22 of 2023.
The invention also provides application of the rhodococcus strain OS36 in petroleum pollution restoration.
Preferably, the rhodococcus strain OS36 is used for remediation of petroleum contaminated soil or petroleum contaminated wastewater.
Preferably, the rhodococcus strain OS36 is used for degradation of petroleum in petroleum contaminated soil or petroleum contaminated wastewater.
The invention also provides a culture comprising the rhodococcus strain OS 36.
The invention also provides a microbial inoculum for repairing petroleum pollution, which comprises one or a mixture of a plurality of strains OS36 and a culture of the strains OS 36.
Preferably, the culture comprises a bacterial strain broth, a bacterial strain fermentation broth, an ERM enrichment medium.
Preferably, the ERM enrichment medium consists of the following final concentrations of material: petroleum oil 9.8～10.2g/L、(NH4)2SO40.8～1.2g/L、NaNO31.8～2.2g/L、KH2PO44.8～5.2g/L、MgSO4·7H2O0.25～0.35g/L、NaCl 4.8～5.2g/L、FeSO4·7H2O 0.0005～0.0015g/L.
Wherein the petroleum is conventionally collected petroleum. The petroleum used in the present invention is petroleum collected from a well in pacify the border region county, elm, shanxi province.
Preferably, the microbial agent is processed into an agriculturally acceptable formulation.
The invention also provides a method for repairing petroleum pollution, which comprises the step of adding the rhodococcus strain OS36, the culture and the petroleum pollution repairing microbial inoculum into petroleum pollution soil or petroleum pollution wastewater to repair the petroleum pollution soil or petroleum pollution wastewater.
Compared with the prior art, the invention has the following beneficial effects:
1. The rhodococcus OS36 used in the method has the characteristics of high degradation efficiency and high degradation rate in the petroleum degradation process, and can quickly restore petroleum pollution in water. Therefore, the rhodococcus provided by the invention can be used for repairing petroleum pollution, and has the advantages of high repairing efficiency and short repairing time in repairing petroleum pollution.
2. The rhodococcus provided by the invention is rhodococcus strain OS36, and the strain OS36 is obtained by enrichment, separation and purification from petroleum-polluted soil. The strain is preserved in China Center for Type Culture Collection (CCTCC) at present, and the preservation number is CCTCC NO: M20232625.
Drawings
FIG. 1 is a photograph of strain OS36 of the present invention on PYG liquid medium and an electron microscope photograph; wherein A is a picture of strain OS36 on PYG liquid medium; b is an electron microscope photograph of the strain OS 36;
FIG. 2 is a phylogenetic tree constructed based on the 16S rRNA gene according to the present invention;
FIG. 3 is a gas chromatogram of a portion of petroleum hydrocarbons (C10-C40) in a water body before and after 7 days of remediation of the petroleum-contaminated water body with OS36 in accordance with the present invention;
FIG. 4 is a graph showing the effect of using rhodococcus OS36 to treat simulated 1% oily wastewater in the present invention; wherein A is a blank control without bacteria; b is a picture of the simulated 1% oily wastewater after 1 week of treatment with strain OS 36.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
The media and reagents used in the following examples were as follows:
(1) Culture medium
The formula of the PYG liquid culture medium is as follows: 5.0g/L peptone, 0.2 g/L yeast powder, 5.0g/L glucose, 3.0g/L, naCl 0.5.5 g/L, mgSO 4·7H2 O1.5 g/L beef extract, pH 7.5, and sterilizing at 121deg.C for 15min.
The formula of the ERM enrichment medium is as follows: petroleum 10.0g/L、(NH4)2SO41.0 g/L、NaNO32.0g/L、KH2PO45.0 g/L、MgSO4·7H2O 0.3g/L、NaCl 5.0g/L、FeSO4·7H2O 0.001g/L,pH is 7.5, and the sterilization is carried out for 15min at 121 ℃.
The petroleum used in the invention is petroleum collected from a certain oil well in pacify the border region county of elm of Shaanxi province.
(2) Reagent(s)
Peptone, yeast powder, glucose, beef extract were purchased from Shanghai microphone Biochemical technologies Co., ltd; the inorganic salt reagent in the culture medium is purchased from Shanghai Ala Biochemical technology Co., ltd; oil is collected from a well in the city pacify the border region county of elm, shaanxi province.
Example 1
1. Isolation and purification of strains
Petroleum-polluted soil collected from the northern Shaanxi region is taken as a sample, added into ERM enrichment medium according to the amount of 1g/100mL, subjected to shaking culture at 180rpm and 30 ℃ for enrichment for 7 days, then 1mL of enrichment culture solution is sucked and added into 9mL of sterile water to prepare diluent with gradient dilution of 10 -1, and the diluent is sequentially diluted according to the steps to obtain 10 -2、10-3、10-4、10-5. 100 mu L of each gradient dilution is respectively absorbed and coated into an ERM enrichment medium plate without petroleum, the coating is uniform, and the super clean bench is dried. Then, 3mL of petroleum solution dissolved in petroleum ether was added, and the plate was turned and spread. And opening a dish cover in an ultra-clean workbench, volatilizing petroleum ether, culturing at 30 ℃ for 7 days, and picking bacterial colonies on the culture dish into 100 mu l of sterile water to obtain the potential petroleum degradation bacterial strain. Purifying the potential petroleum degradation strain on a PYG culture medium plate by using a three-wire method, picking single bacterial colonies, and transferring the single bacterial colonies on a blank PYG culture medium plate to obtain the pure culture of the strain. The above strain is referred to as strain OS36.
Wherein, the oil is collected from a certain oil well in pacify the border region county of Ulmin of Shaanxi province.
2. Identification of Strain OS36
2.1 Morphology and culture characterization observations of Strain OS36
After inoculating strain OS36 on a plate of PYG medium and culturing for 3 days at 30℃obvious single colonies of the strain were grown on the plate, as shown in FIG. 1. It can be observed that: the colony is light pink, round, moist in surface and smooth in edge. The electron micrograph shows that the bacteria are mainly spherical, partially short-rod-shaped and have a large amount of surface secretion.
2.2 Sequencing and analysis of the Strain OS3616S rRNA Gene
Fresh thalli of the strain OS36 are taken, a bacterial genome DNA extraction kit (DP 302) is used for extracting genome total DNA of the strain OS36, after electrophoresis identification is carried out on the extracted genome total DNA, the PCR amplification is carried out by using 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) universal primers, and amplified products are sent to Beijing qing department biotechnology Co Ltd (Beijing Tsingke Biotech Co., ltd.) for sequencing, and the sequencing result is shown in SEQ ID No. 1:
GGTAAGGCCTTTCGGGGTACACGAGCGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCTCCTATCGCATGGTGGGTGGTGGAAAGATTTATCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGACGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGAGTTCGTAGGCGGTTTGTCGCGTCGTTTGTGAAAACCAGCAGCTCAACTGCTGGCTTGCAGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAACGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTCCTTCCACGGAATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATATACCGGAAAGCTGCAGAGATGTGGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTATGTTGCCAGCACGTTATGGTGGGGACTCGTAAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAGACCGTGAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCTTAACCCCTTGTGGGAGGGAGCCGTCGAAGG;
Among them, the bacterial genomic DNA extraction kit (DP 302) was purchased from the astronomical root biochemical technology (beijing) limited.
The sequences obtained were aligned in EzBioCloud Database (https:// www.ezbiocloud.net /), and 16S rRNA phylogenetic tree was constructed using MEGA software. The results are shown in FIG. 2.
As can be seen from FIG. 2, the similarity between the strain OS36 and Rhodococcus qingshengii JCM 15477 (Rhodococcus celebratus) is 100%, and the similarity between the strain OS36 and the 16S rRNA gene sequence of Rhodococcus baikonurensis GTC 1041 (Rhodococcus berkovictoriae) is 99.77%, and the OS36, the Rhodococcus celebratus JCM 15477 and the Rhodococcus berkovictoriae GTC 1041 are gathered on the same phylogenetic branch in the phylogenetic tree.
Combining morphological characteristics and molecular identification results, the strain OS36 was initially identified as Rhodococcus (Rhodoco ccus sp.OS36) which had been deposited with: china center for type culture Collection. Preservation address of the strain: the preservation date of Wuhan is 2023, 12 months and 22 days, and the preservation number of Wuhan is CCTCC NO: M20232625.
3. Study of Strain OS36 for treating oily wastewater
According to the invention, the strain OS36 is found to be capable of degrading petroleum, and the components and the content of petroleum hydrocarbon after degradation are analyzed by utilizing a gas chromatography technology. The specific studies were as follows:
(1) Culturing strain OS363 in a shaking table at 30deg.C and 180rpm with PYG liquid culture medium, collecting thallus, and re-suspending in ERM enrichment medium without petroleum, and adjusting OD 600 to about 1 to obtain bacterial preparation.
(2) 0.2G of petroleum collected from pacify the border region was added to 20mL of sterile water to prepare simulated oily wastewater containing 1% petroleum concentration.
(3) The microbial inoculum is added into the oily wastewater according to the addition amount of 1% by volume, KNO 3 is added simultaneously, so that the nitrogen content (calculated as N) in the oily wastewater reaches 80g/L, and the oily wastewater solution after restoration is obtained after treatment for 7 days at 30 ℃ and 180 rpm. Meanwhile, the same oily wastewater was prepared and the same KNO 3 was added but no microbial inoculum was added as a blank according to the above procedure. And (5) extracting and measuring the residual petroleum content in the repaired oily wastewater solution by referring to the national standard HJ 637-2018. The specific detection steps are as follows:
firstly, adding 100mL of repaired oily wastewater solution into a separating funnel, acidifying a water sample solution to pH less than or equal to 2 by using a hydrochloric acid solution, then adding 10mL of tetrachloroethylene (special for infrared), fully oscillating for 2min, standing and layering; opening a tap of a separating funnel, putting the lower organic phase into a 20mL volumetric flask through a glass funnel filled with anhydrous sodium sulfate, rinsing the glass funnel with a proper amount of tetrachloroethylene, combining the rinsing liquid into an extract, and finally fixing the volume to 20mL with tetrachloroethylene; 10mL of the extract was introduced into a 50mL triangular flask containing 2g of magnesium silicate, placed on a horizontal shaker, shaken for 20min, left to stand, and glass wool was placed in a glass funnel, and the extract was introduced into the glass funnel and filtered into a 25mL cuvette for oil content measurement by an infrared oil meter.
Meanwhile, 1mL of the residual petroleum extracted according to the above steps is put into a gas chromatography vial, and is tightly covered by a polytetrafluoroethylene cover, and is sent to Beijing and Ci technology Co., ltd. For measuring the components and the content of the residual petroleum by using the gas chromatography technology, and the result is shown in figure 3.
The result shows that after 1 week of restoration, the petroleum in the oily wastewater added with the microbial inoculum is obviously emulsified, the solution is dark brown, and the surface of the solution is free from oil slick; whereas the petroleum from the blank group was pooled together to form a distinct oil bolus (see fig. 4). The residual petroleum in the water body of the oil-containing wastewater is 0.17+/-0.1 g, and the petroleum degradation rate can reach 91.7+/-0.5%. At the same time, the gas chromatography results also show that the OS36 can degrade most of alkanes in petroleum, but has a slightly weaker degradation effect on C 15、C16、C17. In chinese patent CN 105907675A, rhodococcus sp.qy-2 was used for 60 days of continuous remediation of petroleum-contaminated wastewater with petroleum concentration of 0.5% and the petroleum degradation rate was only 53%. Bacillus laterosporus (Brevibacillus latiosporrus. G-40) published in China patent CN 107893047A, the petroleum concentration is 0.6%, and the degradation rate of petroleum aromatic hydrocarbon can reach 53.02% after continuous 30 days of restoration.
4. Research on petroleum-polluted soil restoration by strain OS36
The invention researches the repair of the simulated petroleum polluted soil by the strain OS36, and the specific researches are as follows:
(1) Farmland soil was collected from the Yanan area of Shaanxi, simulated petroleum contaminated soil with final petroleum concentration of 10000mg/kg was prepared using petroleum collected from pacify the border region, the simulated petroleum contaminated soil was charged into flowerpots, each pot was charged with 1kg, and left standing at room temperature for 7 days to equilibrate petroleum contaminants in the soil.
(2) Culturing strain OS363 in a shaking table at 30deg.C and 180rpm with PYG liquid culture medium, collecting thallus, and re-suspending in ERM enrichment medium without petroleum, and adjusting OD 600 to about 1 to obtain bacterial preparation.
(3) The microbial inoculum is added into the simulated petroleum polluted soil with the application amount of 50mL/kg and stirred uniformly, and the mixture is kept stand at room temperature for repairing for 2 weeks. After the repair test is finished, the soil after repair is collected by a 5-point sampling method by using a soil sampler (each sampling point is collected from the surface layer to the deep layer soil), and the residual petroleum concentration in the soil is extracted and measured according to the national standard HJ 1051-2019 operation steps, wherein the concrete steps are as follows:
And uniformly mixing soil samples collected by a 5-point sampling method, drying in the shade, weighing 10g of samples, adding anhydrous sodium sulfate, grinding and homogenizing to form a quicksand shape, and transferring to a triangular flask with a plug. 20.0mL of tetrachloroethylene was added to the flask, sealed, and placed in an oscillator, and extracted with shaking at a frequency of 200 times/min for 30min. After 10min of rest, the extract was filtered into a 50mL cuvette using a glass funnel with a glass fiber filter. The extract and the sample were all transferred and filtered again after re-extraction with 20.0mL of tetrachloroethylene. The plug bottle, membrane, glass funnel and soil sample were rinsed with 10.0mL tetrachloroethylene and the extracts combined. Pouring the extract into a magnesium silicate adsorption column, discarding the first 5mL effluent, retaining the remaining effluent, measuring the petroleum concentration by using an infrared oil meter, and calculating the petroleum content in the soil.
The result shows that after 2 weeks of repair, the residual petroleum concentration in the petroleum-polluted soil is 1416.4 +/-89.7 mg/kg, and the petroleum degradation rate is 85.8+/-0.9%. Chinese patent CN117165483A discloses a high-efficiency petroleum degrading bacterium Rhodococcus sp.NBL-B0739, and the degrading effect of the high-efficiency petroleum degrading bacterium on field polluted soil 30d can reach 86.64%. Chinese patent CN116515675A discloses a petroleum degrading fungus Pseudocercospora diplusodonii LJD-5, and after the fungus is cultured in an inorganic salt culture solution of 500 mg.L -1 petroleum hydrocarbon for 14 days, the degradation rate of the petroleum hydrocarbon can reach 76.6 percent.
The results show that the rhodococcus OS36 has high-efficiency restoration capability on petroleum-polluted wastewater and petroleum-polluted soil.
The rhodococcus OS36 provided by the invention has the characteristics of high degradation efficiency and high degradation rate in the petroleum degradation process, and can quickly repair petroleum pollution in water. Therefore, the rhodococcus provided by the invention can be used for repairing petroleum pollution, and has the advantages of high repairing efficiency and short repairing time in repairing petroleum pollution.
It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. The rhodococcus strain OS36 is characterized in that the rhodococcus strain OS36 is preserved in China Center for Type Culture Collection (CCTCC) No. M20232625 at 12 and 22 of 2023.
2. Use of rhodococcus strain OS36 according to claim 1 for remediation of petroleum pollution.
3. The use according to claim 2, characterized in that the rhodococcus strain OS36 is used for the remediation of petroleum contaminated soil or petroleum contaminated wastewater.
4. The use according to claim 3, characterized in that the rhodococcus strain OS36 is used for the degradation of petroleum in petroleum contaminated soil or petroleum contaminated wastewater.
5. A culture comprising rhodococcus strain OS36 of claim 1.
6. A microbial agent for the remediation of petroleum pollution, the microbial agent comprising one or more of strain OS36, a culture of strain OS36, and a mixture mixed together.
7. The microbial agent for repairing petroleum pollution according to claim 6, wherein the culture comprises a bacterial strain broth, a bacterial strain fermentation broth, an ERM enrichment medium.
8. The microbial agent for remediation of petroleum pollution of claim 7, wherein the ERM enrichment medium consists of the following final concentrations of materials: petroleum oil 9.8～10.2g/L、(NH4)2SO40.8～1.2g/L、NaNO31.8～2.2g/L、KH2PO44.8～5.2g/L、MgSO4·7H2O 0.25～0.35g/L、NaCl 4.8～5.2g/L、FeSO4·7H2O 0.0005～0.0015g/L.
9. The microbial agent for restoration of petroleum pollution according to claim 6, wherein said microbial agent is processed into an agriculturally acceptable formulation.
10. A method for repairing petroleum pollution, characterized in that rhodococcus strain OS36 of claim 1, the culture of claim 5, and the microbial inoculum for repairing petroleum pollution of claim 6 are added to petroleum-contaminated soil or petroleum-contaminated wastewater to repair petroleum-contaminated soil or petroleum-contaminated wastewater.
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