CN117165483A - Petroleum degrading bacterium and application thereof in petroleum pollution repairing field - Google Patents
Petroleum degrading bacterium and application thereof in petroleum pollution repairing field Download PDFInfo
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- CN117165483A CN117165483A CN202311143242.1A CN202311143242A CN117165483A CN 117165483 A CN117165483 A CN 117165483A CN 202311143242 A CN202311143242 A CN 202311143242A CN 117165483 A CN117165483 A CN 117165483A
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a strain of petroleum degrading bacteria and application thereof in the field of petroleum pollution restoration. The invention discloses a high-efficiency petroleum degrading bacterium Rhodococcus sp.NBL-B0739, the colony of which is round, beige, opaque, smooth and moist in surface and regular in edge; the degradation rate of the Rhodococcus sp.NBL-B0739 is determined to be stable at 86% by anaerobic and light-proof static culture at 25 ℃ and continuous culture for 46d with 5% diesel concentration; according to the invention, 3% of petroleum degrading bacteria are inoculated into oil-containing soil (the initial oil content is 15000 mg/kg), a pot experiment of the cucumber is carried out, the germination rate of the cucumber reaches 61.33% after 16d, and the effect of promoting the growth is obvious.
Description
Technical Field
The invention belongs to the technical field of petroleum hydrocarbon degrading microorganisms, and particularly relates to a high-efficiency petroleum degrading bacterium Rhodococcus sp.NBL-B0739, a microbial inoculum and application thereof in the field of bioremediation of petroleum contaminated soil.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Worldwide demand for petroleum products is steadily increasing, making petroleum leakage more and more common. Causing the environmental pollution problem which is becoming serious at present. Because of their biological amplification, they can cause extensive and permanent damage to the ecosystem. Petroleum-based contaminants are widely released by underground storage tanks, well demolitions, leaks and leaks from abandoned refineries, resulting in serious pollution of soil, atmosphere and water (Asadzadeh and souza, 2020,Chaima et al, 2021,Sabarathinam et al, 2019). In addition, crop growth is also affected. Mainly shows that the germination rate of crops is reduced, the survival rate is reduced, the resistance is weakened, the growth period is prolonged, the fruiting rate is low, and the like. Is harmful to human health. Petroleum pollutants in the soil can be immersed into underground water along with surface water or enter human bodies through plant enrichment. When petroleum components (such as PAHs) with stronger toxicity enter a human body, the petroleum components can cause injury to organs of the body, the light people can reduce the immunity of the human body, and the heavy people can induce canceration, fetal malformation and the like.
Currently, petroleum pollution remediation processes mainly include physical, chemical and biological processes (Ayol and yurdakos.,2019, ke et al, 2021). However, the physical and chemical methods are limited by high economic cost, production of toxic intermediates, difficulty in scale, damage to the attached ecological environment, and the like. The eye of the learner is then gradually turning to more environmentally friendly and sustainable, cost-effective bioremediation techniques. Microorganisms are considered as the ultimate scavenger in nature because of their ability to extract carbon and energy from a wide variety of compounds. The petroleum pollution environment provides an ideal resource library for separating bacteria with petroleum degradation capability. This is because the presence of petroleum hydrocarbons in the environment selectively promotes the proliferation of hydrocarbon-resistant, toxic bacteria, thereby affecting the diversity of bacterial communities (Kim et al, 2019,Mehrizi et al, 2019).
Patent CN 105907675A discloses Rhodococcus sp.QY-2 which is a strain of degradable crude oil and is screened out by a seawater layer polluted by crude oil at 0 ℃, and the degradation rate of the crude oil reaches 53% after 60 days of continuous culture on the concentration of the crude oil of 0.5%; chinese patent CN 107893047A discloses a strain of Bacillus laterosporus (Brevibacillus laticosporus G-40) screened from petroleum polluted soil in oil extraction area of Guanghua oil field in Zhijiang, the degradation rate of the strain on petroleum aromatic hydrocarbon reaches 53.02% after continuous 30 days of culture for the oil concentration of 0.6%; chinese patent CN 104017747B discloses a petroleum degrading bacterium and application thereof, wherein corynebacterium glutamicum (Corynebacterium glutamicum) is utilized to degrade oily sludge, and the petroleum hydrocarbon degradation rate reaches 39.69% after 30 days of culture at 37 ℃; chinese patent CN 105483037B discloses a strain QPH-9 (Haliea mediterranea genus) for degrading diesel oil, the diesel oil concentration is about 366mg/kg, and the degradation rate of the diesel oil reaches 49.1% after 7 days of shaking culture.
The strain provided in the above patent has high degradation efficiency in a long-term environment with low oil content. However, for a short period of time, the petroleum degradation strain with good economic benefit has no remarkable result, and the influence of petroleum pollution soil treated by the strain on the growth of crops is not researched in the above patent.
Disclosure of Invention
Based on the technical background, the bacterial strain Rhodococcus sp.NBL-B0739 with the effect of efficiently degrading petroleum hydrocarbon is obtained through screening, can effectively repair petroleum-polluted soil and promote crop growth in the polluted soil, and is expected to be used as a biological treatment agent for petroleum pollution.
According to the technical effects obtained, the invention provides the following technical scheme:
in a first aspect, a strain of petroleum degrading bacterium Rhodococcus sp.NBL-B0739, which has been preserved in China center for type culture collection, abbreviated as CCTCC, at the address of 2023, 8 months and 17 days: china, university of Wuhan, and its biological preservation number is: cctccc M20231505.
According to the invention, the oil degrading bacteria are screened by taking the soil of the Kramam oil field in Xinjiang as a sample, and the strain can degrade diesel oil in liquid and soil environment by taking the diesel oil as a unique carbon source. The invention adopts a 16SrDNA gene sequence analysis method to identify, the strain has 16S rDNA shown in SEQ ID NO.1, has 99.86 percent of homology with Rhodococcus sequences in NCBI database, is identified as Rhodococcus and is named as Rhodococcus sp.NBL-B0739.
The morphological characteristics of the above strains are as follows:
characteristics of the cells: in a state where the rod-like spheres coexist.
Colony characteristics: the colonies were round, off-white, opaque, smooth in surface, moist in texture and regular in edges.
In a second aspect, there is provided a microbial inoculum comprising the highly efficient petroleum degrading bacterium Rhodococcus sp.NBL-B0739 of the first aspect and/or a culture of said bacterium.
The above culture means a culture product obtained by inoculating the strain Rhodococcus sp.NBL-B0739 to a medium for a certain period of time, including the strain itself and a metabolite of the strain. The culture medium comprises a liquid culture medium and a solid culture medium, and the metabolites comprise primary metabolites and/or secondary metabolites generated in the microbial fermentation process, wherein the primary metabolites comprise monomers such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids and the like, and various macromolecular polymers consisting of the monomers such as proteins, nucleic acids, polysaccharides, lipids and the like; specific types of secondary metabolites such as antibiotics, hormones, alkaloids, toxins etc.
In the microbial inoculum, the highly efficient petroleum degrading bacterium Rhodococcus sp.NBL-B0739 and/or a culture of the bacterium is used as a main active ingredient. Besides the active ingredient, the strain comprises a carrier which is feasible in the agro-pharmacy and biology, and the carrier is in a solid form or a liquid form. Wherein the solid form is selected from one of mineral material, plant material or polymer compound, and specific examples thereof are clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, diatomaceous earth, corn flour, bean flour, starch, polyvinyl alcohol or polyglycol. The liquid form is selected from an organic solvent, which may be decane and/or dodecane, vegetable oil, mineral oil or water. Accordingly, viable dosage forms of the microbial inoculum include solid dosage forms such as emulsions, powders, granules, wettable powders or water dispersible granules, liquid dosage forms such as solutions, suspensions.
In a third aspect, the high-efficiency petroleum degrading bacterium Rhodococcus sp.NBL-B0739 in the first aspect and the application of the microbial inoculum in the field of bioremediation of petroleum contaminated soil in the second aspect are provided.
Specific forms of the above application include, but are not limited to, any of the following:
(1) The application in the preparation of petroleum degradation agents;
(2) The application in preparing the petroleum-polluted soil restoration agent;
(3) The application of the composition in preparing crop growth promoters.
In the above applications, the petroleum mainly means light petroleum, medium petroleum, heavy petroleum, and further petroleum hydrocarbons such as diesel oil or engine oil in consideration of the condition of petroleum contaminated soil.
The beneficial effects of the above technical scheme are:
the invention provides a high-efficiency petroleum degrading bacterium Rhodococcus sp.NBL-B0739, which has high-efficiency petroleum degrading effect, has good decomposing effect on common soil pollution sources such as petroleum hydrocarbon of diesel oil, engine oil and the like, and can reach 86.64% on the degradation effect on field polluted soil 30 d. Furthermore, the research of the invention also discovers that the strain can effectively promote the growth of crops in the polluted soil and has important significance for bioremediation of the petroleum polluted soil.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is an evaluation of the tolerance of different petroleum degrading bacteria to diesel fuel described in example 1;
wherein, FIG. 1A shows degradation of each group of strains when the diesel oil content is 3%; FIG. 1B shows degradation of each group of strains at a diesel content of 10%; "739" in the figure is Rhodococcus sp.NBL-B0739 provided in the present invention;
FIG. 2 is a colony morphology of Rhodococcus sp.NBL-B0739 described in example 2;
FIG. 3 is a diagram showing the bacterial morphology of Rhodococcus sp.NBL-B0739 described in example 2;
FIG. 4 is a graph showing the results of determining the total petroleum hydrocarbon degradation rate of Rhodococcus sp.NBL-B0739 described in example 4;
wherein, FIG. 4A is the measurement result of the total petroleum hydrocarbon degradation rate at 16 d;
FIG. 4B shows the results of the total petroleum hydrocarbon degradation rate measurement at 26 d;
FIG. 4C is a graph showing the results of measurement of the total petroleum hydrocarbon degradation rate at 36 d;
FIG. 4D is a graph showing the results of total petroleum hydrocarbon degradation rate measurement at 46 f;
"739" in the drawing is Rhodococcus sp.NBL-B0739 provided in the present invention;
FIG. 5 shows the total petroleum hydrocarbon degradation rate of three high-efficiency petroleum degrading bacteria in example 5;
"739" in the drawing is Rhodococcus sp.NBL-B0739 provided in the present invention;
FIG. 6 shows the germination rate results of three high-efficiency petroleum degrading bacteria in example 5;
"739" in the drawing is Rhodococcus sp.NBL-B0739 provided in the present invention;
FIG. 7 shows the petroleum hydrocarbon degradation rate for different treatment groups in the field trial of example 6.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In order to further illustrate the technical means and effects adopted by the invention to achieve the preset aim, the invention provides a high-efficiency petroleum degrading bacterium Rhodococcus sp.NBL-B0739, and specific screening and identifying modes, characteristics and effects thereof are shown in the examples.
Example 1 screening of strains
1. Experimental materials
Soil sample: an oil-containing soil sample was taken from somewhere in the oil field of Kramam oil, stored at 4℃and returned to the laboratory.
Diesel oil: is obtained from Shandong Taian China Petroleum Shandong petrochemical company.
Test medium: inorganic salt culture medium: mgSO (MgSO) 4 0.20g；CaCl 2 0.02g；KH 2 PO 4 1.00g；K 2 HPO 4 1.00g；NH 4 Cl 0.70g；FeCl 3 0.08g, distilled water 1L, natural pH, 18-20 g agar added into the solid culture medium, and high pressure sterilization at 121 ℃ for 25min.
0.9% physiological saline: naCl 0.9g, distilled water 99.1mL,121 ℃ autoclave for 20min.
NB liquid medium: 18g of the product, 1L of distilled water and autoclaving at 121 ℃ for 20min.
NA solid plate medium and slant preservation medium: NB product 18g, agar 20g, distilled water 1L, natural pH, autoclaving at 121deg.C for 20min, and taking out at 60-70deg.C.
2. Apparatus and device
An ultra-clean workbench, a constant temperature shaking table, an oscillator, an electric heating constant temperature incubator, an autoclave and an electron microscope.
3. Screening of strains
(1) And (3) primary screening: putting 1g of soil sample into a conical flask filled with 100mL of 0.9% physiological saline, oscillating for 30min at a constant temperature of a shaking table at 30 ℃ and 180r/min, then sucking 100uL of oscillating culture solution, adding the oscillating culture solution into an inorganic salt culture medium with 2% diesel oil as a unique carbon source for enrichment, carrying out oscillating culture at 30 ℃ and 180r/min for 4d, then taking 100uL of oscillating culture solution, coating the oscillating culture solution onto an inorganic salt solid culture medium with diesel oil as a unique carbon source, carrying out constant temperature culture at 30 ℃ for 2-4 d, picking colonies with different forms, continuing to purify until purified colonies with single forms are obtained, and storing single colonies on an inclined-surface storage culture medium at 4 ℃ for later use.
Results: the primary screening is carried out to obtain 9 petroleum degrading bacteria with degradation capability, namely AP62, NBL-B0739, 617, 1N064, 1N071, 1N073, BS214, petroleum-3 and petroleum-4.
(2) And (3) re-screening: adding a proper amount of diesel oil into a glass centrifuge tube containing 10mL of inorganic salt culture medium, enabling the diesel oil content to be 3% and 10% respectively, sterilizing at 121 ℃ for 20min under high pressure, then inoculating the strain obtained by primary screening into the centrifuge tube, placing the strain into a constant temperature shaking table for shaking culture at 30 ℃ and 180r/min, and carrying out tolerance experiments of the strain subjected to primary screening in the 3% and 10% diesel oil culture medium, as shown in figure 1.
Results: 6 petroleum degrading bacteria (AP 62, NBL-B0739, 1N064, 1N071, 1N073, BS 214) were selected for emulsification experiments.
Example 2: identification of species (identification of thallus, colony morphology and molecule)
1. Morphological characteristics of strains
Single colonies were streaked into NA solid plate medium, the plates were inverted into a constant temperature incubator, and incubated at 30 ℃ for 24h, and the colonies were round, off-white, opaque, smooth in surface, moist in texture, and regular in edge, see fig. 2.
2. Cell morphology (microscopic image)
The coexistence of the strain NBL-B0739 in the form of a rod was observed by a microscope, see FIG. 3.
3. Molecular characterization
The genus species of the isolated strain NBL-B0739 was identified by 16S rDNA gene sequence analysis. The homology of the 16rDNA gene sequence (SEQ ID NO. 1) with the rhodococcus sequence in NCBI database reaches 99.86%, and the strain NBL-B0739 is identified as rhodococcus and named Rhodococcus.
Rhodococcus sp.NBL-B0739 Gene full sequence:
GGGGGGTGGGGGGTGCTACACATGCAGTCGAACGGTAAGGCCCCTTCGGG
GGTACACGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCA
CTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGAGCTCCTGT
CGCATGGCGGGGGTTGGAAAGGTTTACTGGTGCAGGATGGGCCCGCGGCC
TATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCG
GCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAAACTC
CTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATG
CAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCA
GCAGGGACGAAGCGAGAGTGACGGTACCTGCAGAAGAAGCACCGGCCAA
CTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAAT
TACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCCGTGAAAACT
TGGGGCTCAACCCCAAGCTTGCGGGCGATACGGGCAGACTTGAGTACTGC
AGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGA
GGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGG
AGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCC
GTAAACGGTGGGCGCTAGGTGTGGGTTTCCTTCCACGGGATCCGTGCCGTA
GCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAAC
TCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAAT
TCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATATACCGGAAAGCCG
TAGAGATACGGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCG
TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT
TGTCCTGTGTTGCCAGCGCGTAATGGCGGGGACTCGCAGGAGACTGCCGG
GGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTAT
GTCCAGGGCTTCACACATGCTACAATGGCCGGTACAGAGGGCTGCGATACC
GTGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTG
CAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACG
CTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGA
AAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGAGGGAGCCGTCGAACGGGGGT(SEQ ID NO.1)。
EXAMPLE 3 identification of emulsifying Capacity of Strain
1. Materials and instruments
Materials: diesel oil, emulsifying agent Tween 80, inorganic salt culture medium, NB liquid culture medium and glass tube;
instrument and apparatus: a constant temperature shaking table, an electric heating constant temperature incubator, an autoclave and an ultra-clean workbench.
2. The method comprises the following steps: adding 4mL NB liquid medium and emulsifier Tween 80 into glass tube containing 6mL diesel oil, and taking the same as control group CK 2 、CK 1 . Adding the primarily screened petroleum degrading bacteria into NB liquid culture medium, shaking and culturing at 30deg.C and 180r/min for 24 hr, adding shaking culture solution of each petroleum degrading bacteria into glass tube containing 6mL diesel oil, shaking vigorously for 2min, standing at room temperature for 24 hr, and measuring emulsion layer height to obtain stable emulsion layer. At an emulsion index EI 24 Judging the emulsification effect. Emulsion index EI 24 =emulsion layer height/total liquid height). See table 1.
Results: emulsification index EI of treated group Rhodococcus sp.NBL-B0739 24 Is 0.41 compared with the CK in the control group 2 The emulsification effect of 6 petroleum degrading bacteria is stable, namely BS214, 1N064, NBL-B0739, 1N073, 1N071 and AP62.
Table 1 emulsifying index of each treatment group
Example 4: soil simulated degradation experiment 1, material and instrument of high-efficiency petroleum degrading bacterium Rhodococcus sp.NBL-B0739
Materials: greenhouse soil, diesel oil, conical flask, anhydrous sodium sulfate and n-hexane;
instrument and apparatus: weighing balance, superclean bench, fume hood, oven, electrothermal constant temperature incubator, muffle furnace, separating funnel and iron stand.
2. The method comprises the following steps: (1) Putting 300g of soil in a shed area into a 500mL conical flask filled with 15mL of diesel oil, uniformly mixing to ensure that the total petroleum hydrocarbon content in the soil is 15000mg/kg, and marking as W 0 Standing diesel polluted soil for standby; (2) The treatment groups are 7, including a control group and a treatment group, petroleum degrading bacteria (BS 214, 1N064, NBL-B0739, 1N073, 1N071 and AP 62) with stable effects in an emulsification experiment are used as the treatment groups, the treatment groups are inoculated into diesel polluted soil by 3mL of inoculation amount, and the diesel polluted soil without inoculation is usedSoil was used as a control group. And (3) continuously repairing for 46 days at room temperature, and measuring the degradation effect of petroleum degrading bacteria on diesel oil in soil in 16, 26, 36 and 46 days by adopting a gravimetric method.
(3) Total petroleum hydrocarbon extraction and content determination: 50mL Erlenmeyer flask was dried at 60℃to constant weight and designated as W 1 . Taking 4g of experimental soil sample, naturally airing at a fume hood, mixing air-dried soil with equal amount of anhydrous sodium sulfate, adding 30mL of normal hexane, transferring into a 250mL separating funnel together, fully oscillating, standing, collecting an extract liquid, drying at 60 ℃ in a 50mL conical flask, weighing, and marking as W 2 Weight difference of two times, namely W 2 -W 1 Is the weight of total petroleum hydrocarbons. The content and degradation rate of the residual total petroleum hydrocarbon in the sample are calculated according to the formula, and the calculation formula is as follows:
total petroleum hydrocarbon content (mg/kg) = (W) 2 -W 1 )×10 6 /2
Total petroleum hydrocarbon degradation rate (%) = { W 0 -[(W 2 -W 1 )×10 6 /2]}/W 0 ×100％
In which W is 0 Mg/kg is the original content of total petroleum hydrocarbon in the greasy dirt soil;
W 1 drying the mixture to the mass of constant weight g for a 100mL conical flask;
W 2 is the mass g of the conical flask and the residual total petroleum hydrocarbon after n-hexane extraction
See fig. 4 for specific results.
3. Results: the results of 4 times of measurement of the actual total petroleum hydrocarbon content and degradation rate in the petroleum polluted soil show that: the petroleum degradation rate of the AP62 and NBL-B0739 in the 6 strains of petroleum degradation bacteria is high, the petroleum degradation rate is stable to 86%, and the petroleum degradation rate is stable to 71% in the 6 strains of petroleum degradation bacteria, namely 1N 064.
Example 5: efficient petroleum degradation Rhodococcus sp.NBL-B0739 for petroleum pollution restoration and influence of petroleum degradation Rhodococcus sp.NBL-B0739 on crop growth
1. Materials and instruments
Materials: greenhouse soil, cucumber seeds, diesel oil and flowerpots;
instrument and apparatus: slide caliper, superclean bench, chlorophyll content tester.
2. The method comprises the following steps: (1) preparation of greasy dirt soil: screening the soil in the greenhouse which is not polluted by the diesel oil by a 4-mesh sieve, adding the diesel oil, fully and uniformly mixing the soil, enabling the pollution level of the diesel oil to be 15000mg/kg, standing the soil for 3d, and carrying out a repairing test after the soil is fully mixed to reach a stable state.
(2) Test treatment: setting 6 treatments including 3 control groups, respectively adding clear water and oil-free soil (CK) 1 ) Mixing NB liquid culture medium with greasy dirt soil (CK) 2 ) Mixing clear water with greasy dirt soil (CK) 3 ) The method comprises the steps of carrying out a first treatment on the surface of the The 3 experimental groups are high-efficiency petroleum degrading bacteria (AP 62, NBL-B0739 and 1N 064) screened by adding soil simulated degradation experiments into greasy dirt soil, and each group is provided with 3 repetitions; taking 18 flowerpots, each potted with 3kg of oil-free soil or oil-free soil, sowing cucumber with 11 seeds per potted, measuring germination rate of cucumber after sowing for 4d, inoculating 90mL of bacteria with inoculation concentration of 10% quantitatively 6 Inoculation frequency: the application frequency is 2 d-3 d once in the early period of repair, and the application frequency is reduced to 5d once after 1 week.
(3) Detecting the index:
1) And (3) measuring the total petroleum hydrocarbon content before spraying the microbial inoculum, and measuring the total petroleum hydrocarbon content and the degradation rate in the soil after 4 times of inoculation in a potting experiment.
Total petroleum hydrocarbon extraction and content determination: 50mL Erlenmeyer flask was dried at 60℃to constant weight and designated as W 1 . Taking 4g of experimental soil sample, naturally airing at a fume hood, mixing air-dried soil with equal amount of anhydrous sodium sulfate, adding 30mL of normal hexane, transferring into a 250mL separating funnel together, fully oscillating, standing, collecting an extract liquid, drying at 60 ℃ in a 50mL conical flask, weighing, and marking as W 2 Weight difference of two times, namely W 2 -W 1 Is the weight of total petroleum hydrocarbons. The content and degradation rate of the residual total petroleum hydrocarbon in the sample are calculated according to the formula, and the calculation formula is as follows:
total petroleum hydrocarbon content (mg/kg) = (W) 2 -W 1 )×10 6 /2
Total petroleum hydrocarbon degradation rate (%) = { W 0 -[(W 2 -W 1 )×10 6 /2]}/W 0 ×100％
In which W is 0 Is oil stainRaw content of total petroleum hydrocarbon in soil, mg/kg;
W 1 drying the mixture to the mass of constant weight g for a 100mL conical flask;
W 2 is the mass g of the conical flask and the residual total petroleum hydrocarbon after n-hexane extraction
Results: the actual value of total petroleum hydrocarbon in the petroleum polluted soil measured before spraying the microbial inoculum is 15000mg/kg and recorded as W 0 After 4 times of inoculation, the results show that the petroleum degradation rate of NBL-B0739 in 3 high-efficiency petroleum degradation bacteria (AP 62, 1N064 and NBL-B0739) is higher, and the petroleum degradation rate is 66 percent compared with the control group CK 2 (NB) was increased by about 4-fold, followed by AP62 and 1N064, with petroleum degradation rates of 62% and 55%, respectively, see FIG. 5.
2) Cucumber growth index determination: plant height was measured using a ruler; chlorophyll was measured using a chlorophyll content meter, measured in the morning, 3 sub-leaves with relatively consistent growth conditions were selected for each treatment, and each leaf was repeatedly measured 3 times, and averaged.
Results: the maximum germination rate of cucumber of NBL-B0739 in 3 experimental groups reaches 61.33 percent, which is obviously higher than that of the control group CK 3 The plant height is 2.75cm, which is obviously higher than that of the CK in the control group 1 、CK 2 、CK 3 Compared with experimental groups AP62 and 1N064, the chlorophyll content of the plant is 27.63SPAD, which is obviously higher than that of a control group CK 2 、CK 3 From this, it can be seen that NBL-B0739 can meet the growth requirement of crops after 16d planting, and has plant growth promoting effect, see Table 2 and FIG. 6.
TABLE 2
Example 6: petroleum degrading microbial inoculum field effect experiment
Experiment design and method:
(1) Each cell area is 18m 2 Ploughing the soil in the earlier stage, adding 22kg of engine oil into each district, standing for one week, fully mixing the oil and the soil to reach a stable state, and then applying a microbial inoculum to carry out a repair test, wherein the theory is thatThe petroleum hydrocarbon pollution value was 50000mg/kg, and the initial petroleum hydrocarbon content was measured.
The experimental setup was as follows: CK-petroleum hydrocarbon pollutes soil, and no microbial inoculum is added; t1: petroleum hydrocarbon contaminated soil, the concentration of the additive bacteria is 1 hundred million/ml; the concentration of the added microbial inoculum of the soil polluted by the T2-petroleum hydrocarbon is 1 million/ml; the T3-petroleum hydrocarbon pollutes the soil, and the concentration of the additive bacteria is five million/ml. The microbial inoculum is prepared by mixing 3 efficient petroleum degrading bacteria (AP 62, 1N064 and NBL-B0739) fermentation liquor 1:1:1, and the microbial inoculum is added into the T1, T2 and T3 treatment groups once after 7 days.
(2) And (3) collecting soil samples of soil layers of 0-5cm by adopting a five-point sampling method, uniformly mixing, measuring the content of petroleum hydrocarbon by using a weight method, and calculating the degradation rate of the petroleum hydrocarbon.
(3) Total petroleum hydrocarbon extraction and content determination: 50mL Erlenmeyer flask was dried at 60℃to constant weight and designated as W 1 . Taking 4g of soil sample, naturally airing at a fume hood, mixing air-dried soil with equal amount of anhydrous sodium sulfate, adding 30mL of normal hexane, transferring into a 250mL separating funnel together, fully oscillating, standing, collecting the extract liquid, drying at 60 ℃ in a 50mL conical flask, weighing, and marking as W 2 Weight difference of two times, namely W 2 -W 1 Is the weight of total petroleum hydrocarbons. The content and degradation rate of the residual total petroleum hydrocarbon in the sample are calculated according to the formula, and the calculation formula is as follows:
total petroleum hydrocarbon content (mg/kg) = (W) 2 -W 1 )×10 6 /2
Total petroleum hydrocarbon degradation rate (%) = { W 0 -[(W 2 -W 1 )×10 6 /2]}/W 0 ×100％
In which W is 0 Mg/kg is the original content of total petroleum hydrocarbon in the greasy dirt soil;
W 1 drying the mixture to the mass of constant weight g for a 100mL conical flask;
W 2 is the mass g of the conical flask and the residual total petroleum hydrocarbon after n-hexane extraction
The degradation rate of petroleum hydrocarbon after various times of microbial inoculum treatment is shown in figure 7.
As can be seen from fig. 7, the degradation effect of different microbial inoculum concentrations on petroleum hydrocarbons in soil is significantly different. The degradation rate of petroleum hydrocarbon in the soil of each treatment group is as follows: t2> T1> T3> CK. Overall, the degradation rate of petroleum hydrocarbon in each treatment group is in an ascending trend along with the extension of time, which indicates that the addition of the exogenous strain and the nutrient substances is supplemented, so that the degradation of petroleum hydrocarbon is promoted, and the degradation capability of indigenous petroleum degrading bacteria is possibly enhanced at the same time, thereby improving the degradation rate of petroleum hydrocarbon. The degradation rate of petroleum hydrocarbon in the T2 treatment group (millions of microbial inoculum) can reach 86.64% at 30d, which is obviously improved by 152% compared with the control group (34.42%), and the petroleum pollution repair period is obviously shortened.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. Petroleum degrading bacterium Rhodococcus sp.NBL-B0739 is preserved in China Center for Type Culture Collection (CCTCC) at the address of 2023, 8 and 17 days: china, university of Wuhan, and its biological preservation number is: cctccc M20231505.
2. The petroleum degrading bacterium Rhodococcus sp.NBL-B0739 according to claim 1, wherein the strain has 16S rDNA as shown in SEQ ID NO. 1.
3. The petroleum degrading bacterium Rhodococcus sp.NBL-B0739 according to claim 1, wherein the strain has the following morphological characteristics:
characteristics of the cells: in a state where the rod-like spheres coexist.
Colony characteristics: the colonies were round, off-white, opaque, smooth in surface, moist in texture and regular in edges.
4. A microbial agent comprising a petroleum degrading bacterium Rhodococcus sp.nbl-B0739 and/or a culture of said bacterium according to any one of claims 1 to 3.
5. The microbial inoculum of claim 4 wherein the culture represents a culture product obtained by inoculating the strain rhodococcus sp. The culture medium comprises a liquid culture medium and a solid culture medium, the metabolites comprise primary metabolites and/or secondary metabolites generated in the microbial fermentation process, and the primary metabolites comprise monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids and various macromolecular polymers consisting of the monosaccharides or the monosaccharide derivatives, the nucleotides, the vitamins, the amino acids, the fatty acids and the macromolecular polymers comprise proteins, nucleic acids, polysaccharides and lipids; the secondary metabolite is selected from antibiotics, hormones, alkaloids and toxins.
6. The microbial inoculum according to claim 4, wherein the microbial inoculum comprises as a main active ingredient a petroleum degrading bacterium Rhodococcus sp.NBL-B0739 and/or a culture of said bacterium; besides the active ingredient, the strain comprises a carrier which is feasible in the agro-pharmacy and biology, and the carrier is in a solid form or a liquid form.
7. The microbial agent according to claim 6, wherein the carrier is selected from the group consisting of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, diatomaceous earth, corn flour, soy flour, starch, polyvinyl alcohol or polyglycol, decane and/or dodecane.
8. The microbial inoculum of claim 6, wherein the dosage form of the microbial inoculum comprises a solid dosage form, a liquid dosage form, wherein the solid dosage form is an emulsion, a powder, a granule, a wettable powder or a water dispersible granule, and the liquid dosage form is a solution or a suspension.
9. Use of a bacterium of the type described in any one of claims 1 to 3, rhodococcus sp.nbl-B0739, or a bacterium of the type described in any one of claims 4 to 8 in the field of bioremediation of petroleum contaminated soil.
10. The application of claim 9, wherein the specific form of the application includes, but is not limited to, any of the following:
(1) The application in the preparation of petroleum degradation agents;
(2) The application in preparing the petroleum-polluted soil restoration agent;
(3) The application of the composition in preparing crop growth promoters.
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