CN113957018A

CN113957018A - Flora with petroleum degrading function under low temperature condition and application thereof

Info

Publication number: CN113957018A
Application number: CN202111448925.9A
Authority: CN
Inventors: 郑立; 孙莹莹; 高伟; 韩彬; 李倩; 王帅
Original assignee: First Institute of Oceanography MNR
Current assignee: First Institute of Oceanography MNR
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-01-21
Anticipated expiration: 2041-11-30
Also published as: CN113957018B

Abstract

The invention provides a flora with the function of degrading petroleum pollutants under the condition of low temperature, wherein the flora comprises rhodococcus fan-sheng with the preservation number of CGMCC No.23403 and lactococcus lactis subspecies with the preservation number of CGMCC No. 23403. The composite flora provided by the invention can be used for treating petroleum pollution environment. The flora provided by the invention can efficiently degrade petroleum pollutants at low temperature, and the degradation rate of the flora is remarkably improved compared with that of natural weathering degradation; compared with single bacteria in the composition, the petroleum degradation rate is also obviously improved. Especially has higher degradation effect on high molecular weight alkane and polycyclic aromatic hydrocarbon which have high toxicity and are difficult to degrade.

Description

Flora with petroleum degrading function under low temperature condition and application thereof

Technical Field

The invention belongs to the technical field of bioremediation of petroleum and petroleum product polluted environments, and particularly relates to a flora with a function of degrading petroleum pollutants under a low-temperature condition and application thereof.

Background

In recent years, with the exploitation and transportation of petroleum resources by human beings, oil spill accidents frequently occur, which causes serious ecological environment destruction and economic loss. Therefore, the search for a friendly and low-cost oil spill removal strategy is an urgent problem to be solved by current environmental governance. Since the 80's of the 20 th century, humans began using various methods to remediate petroleum pollution.

At present, the international methods for treating petroleum pollution can be mainly divided into three methods, namely a physical method, a chemical method and a biological method. Physical methods and chemical methods in practical application usually only play a good role in removing oil at the initial stage of oil spilling, but have high cost and are easy to cause secondary pollution, and biological methods have the advantages of cleanness, high efficiency and low cost compared with other methods, so the method is considered as the most potential petroleum pollution remediation means.

According to statistics, there are more than 100 genera, more than 200 kinds of microorganisms that can degrade petroleum in nature, and the microorganisms belong to bacteria, molds, actinomycetes, etc., and mainly include Rhodococcus (Rhodococcus), Pseudomonas (Pseudomonas), Lactococcus (Lactococcus), Rahnella (Rahnella), etc. The separation and screening of the bacterial strains with high-efficiency petroleum degradation capability is the premise for carrying out the microbial remediation of petroleum pollution.

The efficiency of oil degradation by microorganisms was found to be affected by temperature. The influence of normal temperature, 40, 50 and 60 ℃ on the petroleum degradation by microorganisms is researched by Liyingli and the like, and the highest petroleum degradation efficiency by the microorganisms is found at 40 ℃; the method comprises the following steps of (1) screening a dominant petroleum degrading bacterium Bacillus sp.SW-1 from oil-containing mud by Gong Hanyi and the like, researching the influence of the dominant petroleum degrading bacterium Bacillus sp.SW-1 on petroleum degradation of a strain at the temperature of 25-40 ℃, and finding that the strain reaches the maximum degradation rate at the temperature of 30 ℃; the crude oil degrading bacteria Acinetobacter sp.YQJ-1 are screened from oil contaminated soil near abandoned wells of Tianjin Hongkong oil fields by the skilful and the like, the petroleum degrading efficiency of the strains at 20-40 ℃ is researched, and the highest crude oil degrading efficiency is found at 35 ℃. The researches show that the petroleum degrading bacteria can exert higher degrading effect in medium-high temperature environment, but in practical application, the process of repairing petroleum pollution by microorganisms needs to pass through a long medium-low temperature period, particularly the process of repairing petroleum in cold regions at medium and high latitudes. Under the condition of low temperature, the metabolism of the microorganism can be inhibited, the degradation efficiency of the high molecular weight alkane and the aromatic hydrocarbon is reduced, and the relative concentration of the high molecular weight component is increased, so that the microorganism can generate stronger toxic effect, thereby reducing the microbial remediation effect of the petroleum pollutants. Therefore, how to obtain the low-temperature high-efficiency petroleum degrading bacteria resource is the key of the application of the petroleum pollution bioremediation technology.

Research shows that compared with a single pure culture strain, the microbial flora obtained by co-culture of multiple strains has better degradation efficiency on petroleum pollutants, on one hand, the positive effect between functions is driven by the high complementarity between species, namely, microorganisms in the flora depend on the activities of other microorganisms to grow, adapt and reproduce. On the other hand, various bacteria in the flora contain more metabolic pathways and wider enzyme types, so that the metabolic range of hydrocarbon degradation is expanded, and the petroleum degradation rate is improved due to the synergistic degradation effect. Therefore, the flora has more efficient petroleum degradation potential than a single strain, and in recent years, researchers have focused on the construction of petroleum degradation flora in the bioremediation research of petroleum pollution so as to solve the problem of low degradation rate of single strain bacteria.

The average temperature in the south Pole area in summer is 0 ℃, and the average temperature in inland and winter is-30 ℃. Despite the harsh environmental conditions of the south pole, the south pole possesses a very abundant microbial resource and has a unique metabolic mechanism that adapts to low temperatures. In recent years, human activities cause certain petroleum pollution to the Antarctic region, which not only changes the microbial living environment of soil, but also provides a new idea for screening low-temperature petroleum degradation microorganisms from the soil and applying the microorganisms to bioremediation.

Disclosure of Invention

The invention provides a flora with the function of degrading petroleum under the low-temperature condition and application thereof, wherein the provided flora comprises two strains of low-temperature petroleum degrading bacteria and has the characteristic of efficiently degrading high-molecular-weight oil stains under the low-temperature condition (not higher than 10 ℃); can be used for bioremediation of petroleum polluted environments (including soil, sediment and water) in high latitude areas.

The invention firstly provides a flora with the function of degrading petroleum pollutants under the condition of low temperature, wherein the flora comprises Rhodococcus rhodochrous (Rhodococcus guinshengii) NJ-XFW-6-A and Lactococcus lactis subsp. lactis (Lactococcus lactis) NJ-CCZ-10-A;

the fan Qingsheng Rhodococcus (Rhodococcus qinshengii) NJ-XFW-6-A is preserved in China general microbiological culture Collection center on 14 th 09 month 2021, the address is No. 3 of Beijing Wenyu No. 1 Hospital in the sunward area, and the preservation number is CGMCC No. 23403;

the Lactococcus lactis subsp.lactis NJ-CCZ-10-A is preserved in China general microbiological culture Collection center on 14 days 09 and 14 months in 2021, and the address is No. 3 of the Xilu No. 1 Hospital of the Korean district in Beijing, and the preservation number is CGMCC No. 23406;

the invention also provides an application of the flora, which is an application in treating petroleum polluted environment;

the petroleum polluted environment comprises petroleum polluted soil, sediment or water body.

In another aspect, the invention provides a method for treating petroleum pollution, which uses the flora for treating.

The flora provided by the invention can efficiently degrade petroleum pollutants at low temperature, and the degradation rate of the flora is remarkably improved compared with that of natural weathering degradation; compared with single bacteria in the composition, the petroleum degradation rate is also obviously improved. Especially has higher degradation effect on high molecular weight alkane and polycyclic aromatic hydrocarbon which have high toxicity and are difficult to degrade.

Drawings

FIG. 1: a photo picture of a culture of Rhodococcus fangii;

FIG. 2: a picture of a lactococcus lactis subspecies lactis NJ-CCZ-10-A culture;

FIG. 3: determining a petroleum degradation rate graph of the strain by a gravimetric method;

FIG. 4: a degradation rate graph of petroleum degrading bacteria to alkane;

FIG. 5: and (3) a graph of the content of alkanes in the residual petroleum after degradation, wherein NC: natural efflorescence, NC09 to NC35 represent normal alkanes of different carbon chain lengths, PR: pristane, PY: phytane;

FIG. 6: a degradation rate graph of the petroleum degrading bacteria on the polycyclic aromatic hydrocarbon;

FIG. 7: and (3) content diagram of polycyclic aromatic hydrocarbon in residual petroleum after degradation, wherein NC: natural weathering; NAP, FLU, PHE, DBT, CHR refer to naphthalene, fluorene, phenanthrene, dibenzothiophene, chrysene, respectively; C1-C4 represent alkyl groups modified at the corresponding carbon atom.

Detailed Description

The invention obtains the soil sample polluted by petroleum in Antarctic field in China scientific investigation, separates out low-temperature petroleum degrading strains, constructs degrading strains to improve the petroleum degrading rate, and is expected to be used for bioremediation of petroleum polluted environment in high-latitude cold areas.

The specific components of the culture medium used in the present invention are described below:

1. inorganic salt culture medium: deionized water 1L, NaCl 5g, K₂HPO₄ 1g、KH₂PO₄ 1g、(NH₄)₂SO₄1g of 0.1% of trace element solution SL-6, adjusting the pH value to 7.2-7.4, performing steam sterilization (121 ℃, 15min), adding 1% of trace element solution SL-4 and 0.5% of M after sterilizationgSO₄Solution for preparing various experimental culture media.

2. SL-4 formulation: deionized Water 1L, FeSO₄·7H₂O0.02 g, and filtering the mixture through a 0.22 mu m filter membrane for sterilization.

3. SL-6 formulation: ZnSO₄·7H₂O 0.1g、MnCl₂·4H₂O 0.03g、H₃BO₃ 0.3g、CoCl₂·6H₂O 0.2g、CuSO₄·5H₂O 0.1g、NiCl₂·6H₂O 0.02g、Na₂WO₄·2H₂O0.03 g, steam sterilization (121 ℃, 15 min).

MgSO₄The solution formula is as follows: deionized Water 100mL, MgSO₄20g, steam sterilized (121 ℃, 15 min).

4. Liquid culture medium: adding a carbon source on the basis of an inorganic salt culture medium, wherein the carbon source content in the 1L inorganic salt culture medium is as follows: 2g of sodium acetate, 0.5g of peptone, 0.5g of yeast extract, 0.5g of potato extract powder, 0.2g of glucose, 0.2g of sucrose, 0.05g of sodium malate, 0.05g of sodium citrate and 0.05g of sodium tartrate, adjusting the pH to 7.2-7.4, and performing steam sterilization (121 ℃, 15min) for culturing single bacteria.

5. Solid medium: on the basis of liquid medium, 1.5% agar was added and steam sterilized (121 ℃, 15min) for plating and isolation of single bacteria.

6. Petroleum culture medium: 1% (m/V) of petroleum is added into a sterilized inorganic salt culture medium for measuring the petroleum degradation rate of single bacteria and flora of petroleum degrading bacteria.

The experiment uses petroleum as medium crude oil, which is sourced from the south-sea spurge oil platform.

The present invention will be described in detail with reference to examples.

Example 1: screening of strains capable of degrading petroleum under low temperature condition

The sample for screening the strain is a soil sample obtained from Antarctic field Fei-Er-Si peninsula of 36 th Antarctic scientific in China during 12 months in 2019 to 1 month in 2020.

1. The bacteria screening process is as follows:

5g of soil sample is added into a conical flask filled with 100mL of petroleum culture medium, the blank control selects the petroleum culture medium without the added sample, and 3 parallels are arranged in each group. The temperature condition of the shaking table is set to be 10 ℃, and the rotating speed is 150 r/min. After enrichment culture for 14d, 1mL of the enrichment culture was taken into fresh 100mL of petroleum medium, and enrichment culture for 14d was continued under the same conditions. After 3 times of enrichment, gradient dilution is carried out by adopting a 10-fold dilution method, and the strain is coated on a flat plate (solid culture medium) for strain separation. Culturing at constant temperature of 10 ℃ for 7-14 days, after bacterial colonies grow out, selecting single bacterial colonies with different forms, carrying out streak purification in a fresh plate, and culturing at corresponding temperature for 7-14 days. The purified single strain is subjected to activated culture in a liquid culture medium at 10 ℃ and is used for evaluating petroleum degradation characteristics.

2. Evaluation standard of the high-efficiency petroleum degrading bacteria:

after the purified single strain is cultured in a liquid culture medium to an exponential phase (OD600 is 0.8), the single strain is inoculated into 100ml of petroleum culture medium according to the inoculation amount of 3 percent, the petroleum culture medium without adding bacterial liquid is selected as a blank control, and three strains are arranged in each group in parallel. The culture was carried out at 10 ℃ and 150rpm for 28 days, and the degradation rate of the oil by the single bacterium was measured by a gravimetric method after 28 days.

The total residual oil after 28 days of degradation was extracted with 50mL of dichloromethane solution, 20mL of extract from the dichloromethane phase was taken in a glass plate, evaporated at normal temperature and weighed, and the petroleum degradation capacity of the individual bacteria was analyzed gravimetrically. And selecting the strains with the petroleum degradation rate which is improved by more than 40 percent relative to the blank. Finally, two high-efficiency petroleum degrading bacteria NJ-XFW-6-A and NJ-CCZ-10-A are obtained by screening.

3. Identification of strains based on molecular biology methods:

16S rRNA gene amplification is carried out on the high-efficiency petroleum degradation strain, and sequencing primers are respectively 7F: 5'-CAGAGTTTGATCCTGGCT-3' and 1540R: 5'-AGGAGGTGATCCAGCCGCA-3', or 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' are provided. PCR System (50L): 2L of extract, 1L of forward and reverse primers, 1L of dNTP 4L, 1L of DNA polymerase, buffer 5L, and ddH2O 36L. PCR conditions were as follows: pre-denaturation at 98 ℃ for 3min, 10s at 98 ℃, 15s at 55 ℃, 20s at 72 ℃, 35 cycles, and extension at 72 ℃ for 5 min. And (3) carrying out gel electrophoresis on the amplified product, and sending the product to a Qingdao anthiological organism for sequencing. Subsequently, the 16S rRNA gene sequences obtained by sequencing were aligned using the 16S-basedID tool in EzBioCloud. After comparison and de-duplication, MEGA 7.0 software is used for multiple sequence comparison, an adjacent phase joining Method (NJ) is selected to construct a phylogenetic tree, confidence detection is carried out through bootstrap analysis, the bootstrap number is selected to be 1000, the similarity with known strains reaches more than 98%, and the same level is determined.

The sequence information of the 16S rRNA gene of the determined high-efficiency petroleum degrading bacteria NJ-XFW-6-A is as follows:

GGTGCATCATTCTACGCTTCGTCCAATCGCCGATCCCACCTTCGACGGCTCCCTCCCACAAGGGGTTAAGCCACCGGCTTCGGGTGTTACCGACTTTCATGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCCGACTTCACGGGGTCGAGTTGCAGACCCCGATCCGAACTGAGACCAGCTTTAAGGGATTCGCTCCACCTCACGGTCTCGCAGCCCTCTGTACTGGCCATTGTAGCATGTGTGAAGCCCTGGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCTTACGAGTCCCCACCATAACGTGCTGGCAACATAAGATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTATACCGACCACAAGGGGGGCCACATCTCTGCAGCTTTCCGGTATATGTCAAACCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCGCTTAATGCGTTAGCTACGGCACGGATTCCGTGGAAGGAACCCACACCTAGCGCCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTACCCACGCTTTCGTTCCTCAGCGTCAGTTACTGCCCAGAGACCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCAGTCTCCCCTGCAGTACTCAAGTCTGCCCGTATCGCCTGCAAGCCAGCAGTTGAGCTGCTGGTTTTCACAAACGACGCGACAAACCGCCTACGAACTCTTTACGCCCAGTAATTCCGGACAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTCTTCTGCAGGTACCGTCACTTGCGCTTCGTCCCTGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCACCCTCTCAGGTCGGCTACCCGTCGTCGCCTTGGTAGGCCATTACCCCACCAACAAGCTGATAGGCCGCGGGCCCATCCTGCACCGATAAATCTTTCCACCACCCACCATGCGATAGGAGGTCATATCCGGTATTAGACCCAGTTTCCCAGGCTTATCCCGAAGTGCAGGGCAGATCACCCACGTGTTACTCACCCGTTCGCCGCTCGTGTACCCCGAAAGGCCTTACCGCTCGACTGCATAGTAGACAGCATCTCACCTTGGT(SEQ ID NO:1)

by comparison, NJ-XFW-6-A was determined to have the highest similarity to a strain of Rhodococcus, and was finally named Rhodococcus fasciatus (Rhodococcus guinshengii) NJ-XFW-6-A strain.

The sequence information of the 16S rRNA gene of the high-efficiency petroleum degrading bacteria NJ-CCZ-10-A is determined as follows:

ATTGAATTTAGCGGCCGCGAATTGGCCCTTAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTTGAGCGCTGAAGGTTGGTACTTGTACCGACTGGATGAGCAGCGAACGGGTGAGTAACGCGTGGGGAATCTGCCTTTGAGCGGGGGACAACATTTGGAAACGAATGCTAATACCGCATAAAAACTTTAAACACAAGTTTTAAGTTTGAAAGATGCAATTGCATCACTCAAAGATGATCCCGCGTTGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGATGATACATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGAAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGGTAGAGAAGAACGTTGGTGAGAGTGGAAAGCTCATCAAGTGACGGTAACTACCCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGTGGTTTATTAAGTCTGGTGTAAAAGGCAGTGGCTCAACCATTGTATGCATTGGAAACTGGTAGACTTGAGTGCAGGAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGCCTGTAACTGACACTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGATGTAGGGAGCTATAAGTTCTCTGTATCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATACTCGTGCTATTCCTAGAGATAGGAAGTTCCTTCGGGACACGGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTGGTTGCCATCATTAAGTTGGGCACTCTAACGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGACAGTGATGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTGGGAGTACCCGAAGTAGGTTGCCTAACCGCAAGGAGGGCGCTTCCTAAGGTAAGACCGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAAAGGGCCAATTCGTTTAAACCTGCAGGACTAGTCCCTTTAGTG(SEQ ID NO:2)；

by comparison, NJ-CCZ-10-A was determined to be most similar to a strain of the genus Lactococcus, and was finally designated as Lactococcus lactis subsp.lactis (strain NJ-CCZ-10-A).

The Rhodococcus rhodochrous (Rhodococcus qinshengii) NJ-XFW-6-A is preserved in China general microbiological culture Collection center at 14 days 09 and 14 years 2021, with the address of No. 3 Beijing Hongyang district North West Lu No. 1, and the preservation number of CGMCC No. 23403;

lactococcus lactis subsp.lactis (Lactococcus lactis) NJ-CCZ-10-A is preserved in China general microbiological culture center on 14 days 09 and 14 years 2021, and the address is No. 3 of Xilu No. 1 of Beijing Korean district north Chen, and the preservation number is CGMCC No. 23406.

Example 2: detection of petroleum degrading effect of single strain and flora

Strains NJ-XFW-6-A and NJ-CCZ-10-A were cultured in liquid medium to logarithmic growth phase (OD)₆₀₀0.8), inoculating into 100ml of petroleum culture medium according to the inoculation amount of 3 percent respectively, wherein 3ml of single bacterium is inoculated into each single bacterium group NJ-XFW-6-A and NJ-CCZ-10-A respectively; the bacterial group NJ-XFW-6-A + NJ-CCZ-10-A is as follows: the inoculation ratio of NJ-CCZ-10-A is 1-9: 9-1, and the total inoculation amount is 3 ml; the blank control (natural weathering) selects a petroleum culture medium without added bacterial liquid, and three groups are arranged in parallel.

1. Gravimetric method for calculating petroleum degradation rate

The above single and flora experimental groups were cultured at 10 ℃ and 150rpm for 28 days, and the total residual oil after 28 days of degradation was extracted with 50mL of dichloromethane solution. 20mL of the extract was taken up from the methylene chloride phase in a glass plate, volatilized at ordinary temperature and weighed, and the petroleum-degrading ability was calculated by the following formula.

In the formula (1), m₀The weight of petroleum in the culture medium is initially added, m is the weight of residual oil stain after extraction, 2.5 is 20mL of 50mL of extractAnd (4) proportion.

2. Degradation rate of total alkane and total arene

In order to detect the degradation characteristics of the single bacterium and the flora on the n-alkane in the petroleum, the extraction liquid is analyzed by GC-MS. The deuterated n-tetracosane solution is used as an internal standard for normal alkane quantification with the concentration of 100 mu g/mL, and the deuterated terphenyl solution is used as an internal standard for polycyclic aromatic hydrocarbon quantification with the concentration of 10 mu g/mL. The sample was connected to a 5973 mass spectrometer equipped with a quadrupole detector using a 6890A gas chromatograph (Agilent) and an HP-5MS capillary column (30 m.times.250 μm inner diameter, 0.22 μm thickness). The specific parameters are as follows:

gas chromatography conditions: column HP-5MS (30 m.times.0.25 mm.times.0.25 μm); the injection port temperature is 260 ℃, the carrier gas is high-purity He (purity 99.999%), the flow rate is 1.0 mL/min < -1 >, the constant-flow mode is adopted, and the sampling is performed in a non-shunting manner by 1 mu L. The column oven adopts a temperature rise program: the temperature is initially kept at 50 ℃ for 5min, and then raised to 300 ℃ at 6 ℃ and min-1 for 20 min.

Mass spectrum conditions comprise interface temperature of 280 ℃, Electron Impact (EI) ion source, electron energy of 70eV, ion source of 230 ℃, quadrupole of 150 ℃ and solvent delay of 3 min. The alkane and polycyclic aromatic hydrocarbon analyses were performed using a selective ion scanning mode (SIM) with a characteristic ion mass fragment (m/z) of 85.

The total n-alkane degradation rate and the total polycyclic aromatic hydrocarbon degradation rate of the petroleum are calculated by the formulas (2) to (3).

In the formula, D_alkane、D_PAHRespectively the degradation rates of total n-alkanes and total polycyclic aromatic hydrocarbons of petroleum, (C)_alkane)_controlAnd (C)_PAH)_controlThe concentrations of the residual normal alkane and polycyclic aromatic hydrocarbon in the blank group of petroleum respectively (C)_alkane)_sampleAnd (C)_PAH)_sampleThe concentrations of the residual normal paraffin and polycyclic aromatic hydrocarbon in the petroleum of the experimental group are respectively.

3. Results of the experiment

3.1 evaluation of degradation Effect of Low-temperature Petroleum-degrading bacteria by gravimetric method

The petroleum degradation effect is shown in fig. 3, after 28 days of degradation at 10 ℃, compared with a blank group (natural weathering), the petroleum degradation rates of single bacteria NJ-XFW-6-A and NJ-CCZ-10-A are respectively 44% and 36.6%, while the petroleum degradation rate of the flora NJ-XFW-6-A + NJ-CCZ-10-A is 64.4%, and the blank control is only about 10%, so that the degradation rate is improved by nearly one time after two single bacteria are combined into the flora. Because the petroleum used in the invention is medium crude oil, the heavy components in the oil product are higher than those in light crude oil and are closer to weathered crude oil. Therefore, under the low temperature condition (10 ℃), the petroleum degradation rate of the flora in the surrounding time is improved by 54.4 percent compared with that of a blank control, and compared with the reported research results, the degradation capability is greatly improved.

3.2 evaluation of degradation characteristics of Low-temperature Petroleum-degrading bacteria by GC-MS method

3.2.1 characteristics of Petroleum-degrading bacteria to degrade alkanes

GC-MS analysis is carried out on the extract liquid obtained by the gravimetric method, the content of all normal alkanes is counted, the degradation rate is calculated, the result is shown in figure 4, the degradation rates of the total normal alkanes of single bacteria NJ-XFW-6-A and NJ-CCZ-10-A are respectively 72 percent and 76.38 percent, the degradation rate of the total normal alkanes of the flora NJ-XFW-6-A and NJ-CCZ-10-A reaches 92.62 percent, and the normal alkanes can be almost completely degraded.

The results of analyzing the degradation conditions of main n-alkanes (NC 09-35, numbers of C are represented by 09-35) are shown in FIG. 5, except NC09, NC14, Pristone (PR), Phytane (PY) and NC19, from NC10-NC28, the degradation rate of single bacterium NJ-XFW-6-A on single alkane is 31% -90%, the degradation rate of single bacterium NJ-CCZ-10-A on single alkane is 60% -89%, and the degradation rate of bacterial colony NJ-XFW-6-A + NJ-CCZ-10-A on single alkane is more than 90%. And with the increase of the number of C, the degradation rate of single alkane of single bacterium and flora is gradually reduced, for high molecular weight alkane NC29-NC35, the degradation rate of single bacterium NJ-XFW-6-A is between 10% and 24%, the degradation rate of single bacterium NJ-CCZ-10-A is between 23% and 53%, and the degradation rate of flora NJ-XFW-6-A + NJ-CCZ-10-A is up to between 42% and 82%. In addition, the degradation rates of the colonies on the two degradation-resistant biomarkers of the Pristine (PR) and the Phytane (PY) are respectively 27.17% and 32.71%, and a single bacterium has no degradation effect on the two substances.

3.2.2 characteristics of Petroleum-degrading bacteria to degrade polycyclic aromatic hydrocarbons

GC-MS analysis is carried out on the extract obtained by the gravimetric method, the content of all polycyclic aromatic hydrocarbons is counted, the degradation rate is calculated, the result is shown in figure 6, the degradation rates of the total polycyclic aromatic hydrocarbons of single bacteria NJ-XFW-6-A and NJ-CCZ-10-A are respectively 27.72 percent and 27.40 percent, and the degradation rate of the total polycyclic aromatic hydrocarbons of the bacteria NJ-XFW-6-A + NJ-CCZ-10-A is 47.56 percent.

From the content of single polycyclic aromatic hydrocarbon (figure 7), the degradation rate of the flora NJ-XFW-6-A + NJ-CCZ-10-A to the naphthalene series, the fluorene series, the phenanthrene series, the dibenzothiophene series and the flexor series is between 45% and 57%, the degradation rate of the single flora NJ-XFW-6-A to the naphthalene series, the fluorene series, the phenanthrene series, the dibenzothiophene series and the flexor series is between 24% and 40%, and the degradation rate of the single flora NJ-CCZ-10-A to the naphthalene series, the fluorene series, the phenanthrene series, the dibenzothiophene series and the flexor series is between 13% and 30%. On the whole, the degradation rate of the flora NJ-XFW-6-A + NJ-CCZ-10-A to C1-NAP and C1-PHE is below 40%, and the degradation rate to other polycyclic aromatic hydrocarbon series is above 40%.

Example 3: capability of petroleum degrading bacteria to produce surfactant

The surfactant production capacity of single bacterium NJ-XFW-6-A, NJ-CCZ-10-A and flora NJ-XFW-6-A + NJ-CCZ-10-A is determined by adopting an oil drain circle method: 10mL of liquid paraffin was added to the flask, followed by 1g of Sudan III dye. Adding 20mL of distilled water into a glass culture dish, then adding 100 mu L of liquid paraffin added with dye into the center of the liquid surface, standing until the liquid paraffin is uniformly distributed in the center of the water surface to form an oil film with the diameter of more than 30mm, then slowly dripping 50 mu L of strain culture solution into the center of the paraffin to form an oil discharge ring in the center of the oil film. If an oil drain ring is generated, the size of the oil drain ring formed after the culture medium is added to the culture dish is measured. The blank control was liquid medium without inoculation.

The results show that both single strains have the capability of generating the surfactant (Table 1), the diameters of oil discharge rings of the strains NJ-CCZ-10-A and NJ-XFW-6-A reach 35mm and 15mm respectively, and the diameters of the oil discharge rings of the constructed strains NJ-XFW-6-A + NJ-CCZ-10-A reach 47mm, so that the capability of generating the surfactant is the superposition of the capabilities of the two single strains, and the surfactant has high capability of generating the surfactant, so that the medium crude oil can be quickly emulsified at low temperature and is easy to biodegrade.

Table 1: surfactant producing ability of bacteria

The low-temperature petroleum degrading flora can be used for bioremediation of soil, near-shore sediments and water bodies polluted by petroleum in high-latitude cold regions.

Sequence listing

<110> department of natural resources first oceanographic institute

<120> a flora having petroleum degradation function under low temperature condition and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1453

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggtgcatcat tctacgcttc gtccaatcgc cgatcccacc ttcgacggct ccctcccaca 60

aggggttaag ccaccggctt cgggtgttac cgactttcat gacgtgacgg gcggtgtgta 120

caaggcccgg gaacgtattc accgcagcgt tgctgatctg cgattactag cgactccgac 180

ttcacggggt cgagttgcag accccgatcc gaactgagac cagctttaag ggattcgctc 240

cacctcacgg tctcgcagcc ctctgtactg gccattgtag catgtgtgaa gccctggaca 300

taaggggcat gatgacttga cgtcgtcccc accttcctcc gagttgaccc cggcagtctc 360

ttacgagtcc ccaccataac gtgctggcaa cataagatag gggttgcgct cgttgcggga 420

cttaacccaa catctcacga cacgagctga cgacagccat gcaccacctg tataccgacc 480

acaagggggg ccacatctct gcagctttcc ggtatatgtc aaacccaggt aaggttcttc 540

gcgttgcatc gaattaatcc acatgctccg ccgcttgtgc gggcccccgt caattccttt 600

gagttttagc cttgcggccg tactccccag gcggggcgct taatgcgtta gctacggcac 660

ggattccgtg gaaggaaccc acacctagcg cccaccgttt acggcgtgga ctaccagggt 720

atctaatcct gttcgctacc cacgctttcg ttcctcagcg tcagttactg cccagagacc 780

cgccttcgcc accggtgttc ctcctgatat ctgcgcattt caccgctaca ccaggaattc 840

cagtctcccc tgcagtactc aagtctgccc gtatcgcctg caagccagca gttgagctgc 900

tggttttcac aaacgacgcg acaaaccgcc tacgaactct ttacgcccag taattccgga 960

caacgcttgc accctacgta ttaccgcggc tgctggcacg tagttagccg gtgcttcttc 1020

tgcaggtacc gtcacttgcg cttcgtccct gctgaaagag gtttacaacc cgaaggccgt 1080

catccctcac gcggcgtcgc tgcatcaggc tttcgcccat tgtgcaatat tccccactgc 1140

tgcctcccgt aggagtctgg gccgtgtctc agtcccagtg tggccggtca ccctctcagg 1200

tcggctaccc gtcgtcgcct tggtaggcca ttaccccacc aacaagctga taggccgcgg 1260

gcccatcctg caccgataaa tctttccacc acccaccatg cgataggagg tcatatccgg 1320

tattagaccc agtttcccag gcttatcccg aagtgcaggg cagatcaccc acgtgttact 1380

cacccgttcg ccgctcgtgt accccgaaag gccttaccgc tcgactgcat agtagacagc 1440

atctcacctt ggt 1453

<210> 2

<211> 1584

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

attgaattta gcggccgcga attggccctt agagtttgat cctggctcag gacgaacgct 60

ggcggcgtgc ctaatacatg caagttgagc gctgaaggtt ggtacttgta ccgactggat 120

gagcagcgaa cgggtgagta acgcgtgggg aatctgcctt tgagcggggg acaacatttg 180

gaaacgaatg ctaataccgc ataaaaactt taaacacaag ttttaagttt gaaagatgca 240

attgcatcac tcaaagatga tcccgcgttg tattagctag ttggtgaggt aaaggctcac 300

caaggcgatg atacatagcc gacctgagag ggtgatcggc cacattggga ctgagacacg 360

gcccaaactc ctacgggagg cagcagtagg aaatcttcgg caatggacga aagtctgacc 420

gagcaacgcc gcgtgagtga agaaggtttt cggatcgtaa aactctgttg gtagagaaga 480

acgttggtga gagtggaaag ctcatcaagt gacggtaact acccagaaag ggacggctaa 540

ctacgtgcca gcagccgcgg taatacgtag gtcccgagcg ttgtccggat ttattgggcg 600

taaagcgagc gcaggtggtt tattaagtct ggtgtaaaag gcagtggctc aaccattgta 660

tgcattggaa actggtagac ttgagtgcag gagaggagag tggaattcca tgtgtagcgg 720

tgaaatgcgt agatatatgg aggaacaccg gtggcgaaag cggctctctg gcctgtaact 780

gacactgagg ctcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc 840

gtaaacgatg agtgctagat gtagggagct ataagttctc tgtatcgcag ctaacgcaat 900

aagcactccg cctggggagt acgaccgcaa ggttgaaact caaaggaatt gacgggggcc 960

cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct 1020

tgacatactc gtgctattcc tagagatagg aagttccttc gggacacggg atacaggtgg 1080

tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1140

cccctattgt tggttgccat cattaagttg ggcactctaa cgagactgcc ggtgataaac 1200

cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt 1260

gctacaatgg atggtacaac gagtcgcgag acagtgatgt ttagctaatc tcttaaaacc 1320

attctcagtt cggattgtag gctgcaactc gcctacatga agtcggaatc gctagtaatc 1380

gcggatcagc acgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1440

acgggagttg ggagtacccg aagtaggttg cctaaccgca aggagggcgc ttcctaaggt 1500

aagaccgatg actggggtga agtcgtaaca aggtagccgt aaagggccaa ttcgtttaaa 1560

cctgcaggac tagtcccttt agtg 1584

Claims

1. The composite flora is characterized by comprising Rhodococcus fangciensis with the preservation number of CGMCC No.23403 and lactococcus lactis subspecies lactis with the preservation number of CGMCC No. 23406.

2. The use of complex bacteria as claimed in claim 1 in the treatment of petroleum-contaminated environments.

3. The use of claim 2, wherein the petroleum-contaminated environment comprises petroleum-contaminated soil, sediment or water.

4. A method for treating petroleum pollution, which comprises treating petroleum pollution with the complex microbial population of claim 1.

5. The method of claim 4, wherein the method is a treatment of a petroleum-contaminated soil, sediment or water body using the complex microbial population of claim 1.

6. An article for treating petroleum-polluted environment, wherein the article comprises the complex microbial flora of claim 1.

7. The article of claim 6, wherein the article is a liquid microbial inoculum article.

8. The Rhodococcus erythropolis is characterized in that the preservation number of the Rhodococcus erythropolis is CGMCC No. 23403.

9. The lactococcus lactis subspecies lactis is characterized in that the preservation number of the lactococcus lactis subspecies lactis is CGMCC No. 23406.