CN108102977B - Degradation strain JN2 for petroleum hydrocarbons in oily sludge and application thereof - Google Patents
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Abstract
The invention discloses a strain JN2 for efficiently degrading petroleum hydrocarbons in oily sludge and application thereof. The degrading bacteria JN2 are preserved in China general microbiological culture collection center with the preservation number of CGMCC NO.14973 and the classification number of Rheinheimera sp. The strain JN2 with the petroleum hydrocarbon degradation function is used for treating the oily sludge in a petroleum hydrocarbon degradation mode, the degradation rate of the bacteria on Total Petroleum Hydrocarbons (TPH) in the oily sludge within 30 days is 93.3%, the optimal degradation condition of the bacteria on petroleum pollutants is 15-50 ℃, and the pH value is 7-11. The biological method is used for the technology for restoring the oily sludge, and has the advantages of high efficiency, low cost, environmental friendliness and the like.
Description
Technical Field
The invention relates to the technical field of bioremediation, in particular to a bacterial strain for degrading and restoring oily sludge and application thereof in degrading the oily sludge.
Background
Since the 20 th century, petroleum has become one of the most important energy sources for human beings, and while being largely exploited and used, the damage of petroleum and its processed products to the environment and human beings has become more and more serious. Since the annual yield of crude oil exceeds 1 hundred million t since the innovation of China, China which is one of ten major oil producing countries in the world has more than 400 oil fields and oil and gas fields which are explored and developed and are distributed in 25 provinces (cities) and autonomous regions in the country according to the proportion of occupying about 3 percent of the area of the national soil. However, in the process of rapid development of economic technology and continuous increase of petroleum demand, the technical process for extracting oil from an oil field area is still relatively lagged behind, an environmental impact evaluation system is relatively incomplete, and meanwhile, a remediation technology for petroleum pollution is relatively lacked, so that the petroleum pollution degree of China is far higher than that of developed countries, and the petroleum pollution degree is in a state of accumulating and aggravating year by year.
The oil sludge is a main waste pollution problem commonly existing in various oil fields in China, and the oil sludge is classified as a dangerous waste (waste category HW08) by the national environmental protection Bureau of 1998, and the oil sludge and other wastes are required to be subjected to harmless treatment. The oil-containing sludge has different components and properties due to different sources. Through sampling and analyzing various kinds of oily sludge, the oily sludge generally contains 15 to 50 percent of oil. Has great harm to the environment. The oily sludge must be treated in a harmless way according to the requirements of the promotion of clean production of the people's republic of China. At present, a large amount of manpower and material resources are used for research on effective treatment of oily sludge at home and abroad, and methods such as solidification and landfill, incineration, leaching, chemical oxidation, bioremediation and the like are successively tried to treat the oily sludge. The bioremediation technology has the advantages of relatively low cost, high safety, simple operation, difficult secondary pollution, capability of implementing in-situ treatment and the like, and is considered as the most promising remediation means. But because the components of the oily sludge are complex and difficult to degrade, the research on the microbial degradation repair of the oily sludge at home and abroad is relatively less at present.
CN104928220A discloses a marine pollution source water riemerella juncea strain (Rheinheimera aquimaris) QSI02 and application thereof in bacterial quorum sensing inhibition screening. The invention reports that the strain of Rheinheimera has the bacterial quorum sensing inhibition activity for the first time. The metabolite prepared by the strain fermentation liquor has double inhibition effects on a quorum sensing system of the purple bacillus and the pseudomonas aeruginosa, and does not have inhibition effect on the thallus generation of the purple bacillus CV026 of the screened model in an effective concentration range. The strain extract can obviously reduce the yield of purple bacillus purpurin and the cluster movement of pseudomonas aeruginosa, and weaken the drug resistance of pathogenic bacteria, thereby having wide application prospect in the aspects of solving the problem of drug resistance of bacteria and researching and developing novel antibacterial drugs.
The inventor screens out a high-efficiency petroleum hydrocarbon degrading bacterium, namely Rheinheimera sp (CGMCC NO. 14973), from the oil-containing sludge in Liaohe oilfield, and further researches the degrading capability of the bacterial strain on the petroleum hydrocarbon in the oil-containing sludge so as to be applied to microbial remediation of the oil-containing sludge.
Disclosure of Invention
The invention aims to provide a petroleum hydrocarbon degrading strain and application thereof in a degradation and restoration process of oily sludge.
The invention relates to a rhinestone bacteria strain and application thereof in degrading oily sludge, and the technical scheme is as follows:
the petroleum hydrocarbon degrading strain is characterized in that the strain is Rheinheimeriras (Rheinheimeriras sp.), is preserved in China general microbiological culture Collection center (CGMCC for short) in 2017, 12 and 1 days, and is addressed to the microbial research institute of China academy of sciences No. 3, West Lu No.1 Hopkins, Tokyo-Yang-oriented areas, and the preservation number is CGMCC NO. 14973.
The method for separating the efficient degrading strain from the oil-containing sludge in the Liaohe oil field to degrade the petroleum hydrocarbon pollutants in the oil-containing sludge is characterized by comprising the following steps:
10g of certain oily sludge in the Liaohe oil field is put into a conical flask filled with 100m L L B liquid culture medium, and is shaken on a constant temperature shaking table at 30 ℃ and 120rpm for enrichment culture for 24 h.
The L B culture medium comprises 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000m of distilled water L.
And inoculating the settled upper layer bacterial liquid into a screening culture medium after 24 hours, wherein the initial addition amount of the n-octadecane in the screening culture medium is 1 g/L, and continuously increasing the content of the n-octadecane in the screening culture medium until the aseptic colony grows within 5 days.
The inorganic salt culture medium comprises the following components: NaCl 30g, NH4NO33g，KH2PO41g，K2HPO41g，CaCl20.02g,MgSO40.5g, 20g of agar powder, 1L parts of deionized water and 10ml of trace element solution.
Solution of trace elements: CuSO40.05g，MnSO40.05g，FeSO4·7H20.05g of O and 50ml of deionized water.
Screening medium (alkane medium): adding n-octadecane as the only carbon source in the inorganic salt culture medium.
Observing the colony morphology after the colony grows out, selecting the colony with large morphological difference from the screening culture medium with the previous concentration without colony growth by using an aseptic inoculating loop, inoculating the colony on L B solid culture medium by adopting a plate streaking separation method, placing the colony in a constant temperature incubator for inverted culture for 24h at 30 ℃, selecting a single colony after the colony grows out, inoculating the single colony to L B solid culture medium by the same method, repeating the streaking separation process until a purified colony with single morphology is formed, and inoculating the purified colony on a storage culture medium inclined plane for storage in a refrigerator at 4 ℃ for later use.
The colony morphology of the strain is as follows: the colony is light yellow, the surface is smooth and wet, and the edge is neat, as shown in figure 1. The thallus is rod-shaped with different lengths, and the shape of the bacterium is shown in a scanning electron microscope picture 2.
The sequence of the 16S ribosomal RNA gene of the strain is compared with a database by an analytical method for analysis, the strain is found to belong to the genus Rheinheimer (Rheinheimer sp.), and a DNA sequence table of the strain is shown in a sequence table.
Before use, the strain is activated in L B liquid culture medium for 24h to prepare a strain liquid, and the strain liquid is inoculated into the oily sludge in an inoculation amount of 10%.
Preferably, the optimal conditions for degradation are as follows: the 10% inoculation amount, the temperature of 40 ℃, and the pH of 11 can lead the best degradation effect.
Compared with the prior art, the invention has the following advantages:
1. the Rheinheimera sp JN2 is derived from oily sludge, so that the strain becomes dominant flora after entering the oily sludge, occupies a certain ecological position, effectively promotes the degradation of petroleum hydrocarbons, optimizes a soil microbial system, improves the physical properties of soil and enhances the biological activity of the soil.
2. The Rheinheimera sp JN2 is a dominant strain screened by using octadecane as a unique carbon source, and the strain can change the properties of petroleum hydrocarbon substances in the oily sludge in the growth and propagation process so as to change the components of the petroleum hydrocarbon substances in the oily sludge.
3. The Rheinheimera sp JN2 can degrade petroleum pollutants in the oil-containing sludge in a short time, and the degradation rate of Total Petroleum Hydrocarbon (TPH) in the oil-containing sludge in 30 days is more than 90%.
Drawings
FIG. 1 shows the colony morphology of strain JN 2.
FIG. 2 is a scanning electron micrograph of strain JN 2.
FIG. 3 shows the degradation curve of Rheinheimera sp.JN2 on petroleum hydrocarbons in oily sludge.
Fig. 4 shows the degradation curve of Rheinheimera sp.jn2 for petroleum hydrocarbons in oily sludge at different pH.
FIG. 5 shows the degradation curve of Rheinheimera sp.JN2 for petroleum hydrocarbons in oily sludge at different temperatures.
Detailed Description
For better understanding of the present invention, the following examples are given as examples to illustrate the present invention, but the present invention is not limited to the following examples.
A bacterial strain for degrading petroleum hydrocarbon in oily sludge is of the genus Rheinheimer.
(I) Material preparation
The oily sludge is taken back from a certain place of the Liaohe oil field, stored at 4 ℃, transferred back to a laboratory and inoculated into L B culture medium for activation.
2. Culture medium:
the inorganic salt culture medium comprises the following components: 1g of n-octadecane, 30g of NaCl, NH4NO33g，KH2PO41g，K2HPO41g，CaCl20.02g，MgSO40.5g, 20g of agar powder, 1L parts of deionized water and 10ml of trace element solution.
Solution of trace elements: CuSO40.05g，MnSO40.05g，FeSO4·7H20.05g of O and 50ml of deionized water.
L B solid culture medium comprising tryptone 10g, yeast extract 5g, NaCl 10g, agar powder 20 and distilled water 1000m L, and adjusting pH to 7
3. Laboratory apparatus and device
Constant temperature oscillator
Electric heating constant temperature incubator
A high-pressure sterilization pot is used for sterilizing,
UV-1750 ultraviolet-visible spectrophotometer- -Shanghai Ringzhi optical technology Co., Ltd.,
the ultra-clean workbench is provided with a workbench,
OI L480 infrared spectroscopic oil tester- -Beijing Huaxia scientific Co., Ltd.,
a PCR instrument.
(II) separation, screening and domestication of strains
Taking 10g of oily sludge, putting the oily sludge into a conical flask filled with 100ml of L B liquid culture medium, shaking the oily sludge on a constant-temperature bed at 30 ℃ and 120rpm, and carrying out enrichment culture for 24 hours;
after 24 hours, taking 1ml of culture solution, coating the culture solution into a solid inorganic salt culture medium containing n-octadecane by using a coating rod, and putting the culture solution into a constant-temperature incubator for culture for 7 days;
observing the colony morphology after the colony grows out, selecting the colony with large morphological difference from the solid inorganic salt culture medium by using an aseptic inoculating loop, inoculating the colony on L B solid culture medium by adopting a plate streaking separation method, placing the colony in a constant-temperature incubator for inverted culture for 24h at 30 ℃, selecting a single colony after the colony grows out, inoculating the single colony to L B solid culture medium by the same method, repeating the streaking separation process until a purified colony with single morphology is formed, propagating the single colony in a liquid L B culture medium, storing the colony at 4 ℃, and activating the stored strain in a L B liquid culture medium before use.
The following is a detailed description of the embodiments.
Example 1: degradation repair test of Liaohe oil field oil-containing sludge
The method comprises the steps of taking oil-containing sludge in Liaohe oil field, carrying out air drying and grinding, sieving by a 1mm sieve for later use, picking a single colony in a sterilized L B liquid culture medium, sealing the single colony by using a breathable sealing film, placing a conical flask in a shaking table, activating the conical flask at 120rpm and 30 ℃ for 24h, weighing 40g of the oil-containing sludge after air drying, grinding and sieving, adding 10% of inoculum size into Rheinheimera sp.JN2 activated bacterial liquid with OD600 ═ 1, adjusting the water-soil ratio to be 2:5 by using distilled water, sealing the conical flask by using the breathable sealing film, placing all the conical flasks in a 30 ℃ constant temperature incubator for standing and culturing, sampling every 5d, measuring the content of total petroleum hydrocarbons in the oil-containing sludge by using an infrared spectrophotometer, wherein the degradation curve is shown in figure 3.
Results of GC-MS analysis of oily sludge before and after 30 days of Rheinheimeria sp.JN2 degradation (Table 1), the main components of the original oily sludge were decalin, N-tetradecane, octadecene, tetramethylpentadecane, tetramethylhexadecane, indane, homoandrostane, N- (2-trifluoromethylbenzene) -3-pyridinecarboxamide oxime (C)13H10F3N3O), hexacosene and hopane. The oily sludge after the Rheinheimera sp.JN2 microbial degradation contains anthracene (C)14H10) Pregnane (C)21H36) And C18H18And O, the molecular structure of petroleum hydrocarbon substances in the oily sludge is changed under the action of microorganisms, the pollutants with the macromolecular structure are oxidized and decomposed into small molecular structures under the action of the microorganisms, pentadecane is generated after the microorganisms are degraded, the Rheinheimer sp.JN2 microorganisms react with methyl of the tetramethylpentadecane firstly, and the methyl is removed and then is further degraded. JN2 to tetramethyl hexadecane, octadecene, hexacosane and decalin (C) in oily sludge10H18) Indene alkyl (C)19H36) And highly androstane (C)21H36O2) Has strong degradation capability, and the degradation rate reaches 100 percent. JN2 is degraded for 30 days, part of petroleum hydrocarbon substances in the oil-containing sludge still remain, and the degradation can be further degraded in a mode of prolonging the microbial degradation time, changing the microbial degradation condition and the like.
TABLE 1 GC-MS results of oily sludge before and after 30 days of Rheinheimera sp JN2 degradation
Example 2: degradation effect of Rheinheimera sp.JN2 on petroleum hydrocarbon in oil-containing sludge under different pH values
Taking oil-containing sludge of Liaohe oil field, air-drying, grinding and sieving by a 1mm sieve. Weighing 40g of the oily sludge in a conical flask, and adding diluted H2SO4Adjusting the pH of the soil to 1, 3, 5, 7, 9 and 11 respectively by NaOH, adding 10% of inoculum size of Rheinheimera sp.JN2 activated bacteria solution with OD600 ═ 1, adjusting the water-soil ratio to 2:5 by distilled water, and sealing by a breathable sealing film. Placing all the conical flasks in a constant-temperature incubator at 30 ℃ for static culture, sampling after 25 days, and determining the content of total petroleum hydrocarbons in the oily sludge by using an infrared spectrophotometer. The degradation effect of the strain Rheinheimera sp.JN2 on petroleum hydrocarbons in the oily sludge under different pH conditions is shown in FIG. 4. As can be seen from FIG. 4, the strain of the invention has good removal effect on petroleum hydrocarbons in the oily sludge within the pH range of 7-11, and the degradation effect is best under the condition of pH 11.
Example 3: JN2 degrading effect of Rheinheimera sp on petroleum hydrocarbon in oil-containing sludge at different temperatures
Taking oil-containing sludge of Liaohe oil field, air-drying, grinding and sieving by a 1mm sieve. Weighing 40g of the oily sludge in an erlenmeyer flask, adding 10% of inoculum size into Rheinheimera sp.JN2 activated bacterial liquid with OD600 ═ 1, adjusting the water-soil ratio to be 2:5 by using distilled water, and sealing by using a breathable sealing film. Placing the conical flasks in constant temperature incubator at 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C and 50 deg.C respectively, standing for 25 days, sampling, and measuring total petroleum hydrocarbon content in the oily sludge with infrared spectrometer. The effect of strain Rheinheimera sp.jn2 on the degradation of total petroleum hydrocarbons in oily sludge at different temperatures is shown in fig. 5. As can be seen from FIG. 5, the strain of the invention has certain degradation effect on petroleum hydrocarbons in the oily sludge within the range of 15-50 ℃, the degradation rate is not large within the range of 15-50 ℃, and the degradation effect is optimal under the condition of 40 ℃.
Example 4: JN2 repair of actual oil-containing sludge by Rheinheimera sp
On the basis of the above embodiment, Rheinheimera sp.jn2 of Rheinheimera is used for repairing actual oily sludge, and the experiment specifically comprises the following steps:
(1) preparing a bacterial suspension of the strain Rheinheimera sp.JN2, namely selecting a single colony of the strain JN2 in a L B liquid culture medium, placing the single colony in a shaking table, culturing for 24h under the conditions of 40 ℃ and 120rpm, carrying out fermentation culture by adopting a fermentation tank, inoculating the cultured seed liquid into a L B culture medium according to the inoculation amount of 5%, aerating by adopting an air compressor, stirring at the rotating speed of 200r/min, sampling at regular time to detect the OD600 value, and growing to the logarithmic growth period for later use.
(2) Test oily sludge: the oily sludge collected from a certain place of the Liaohe oil field is naturally dried and sieved by a sieve with 8 meshes (a sieve with 3 mm). Loading the oily sludge sample into an organic glass container, adding a 10% inoculation amount of a fungus liquid activated by Rheinheimerisp.JN 2 with OD600 ═ 1, regulating the liquid-soil ratio to be 2:5 by using an inorganic salt culture medium, and sealing by using a breathable sealing film.
(3) And (3) placing the oily sludge added with the bacterial liquid and the inorganic salt culture medium in a constant-temperature incubator for static culture, adjusting the pH value of the soil to 11 for achieving the optimal degradation effect, and maintaining the temperature at 40 ℃.
After 30 days of biodegradation treatment, the oily sludge is detected, and the degradation rate of Total Petroleum Hydrocarbon (TPH) in the oily sludge is 93.3%, so that the oily sludge has a good degradation effect on petroleum hydrocarbon in the oily sludge.
Sequence listing
<110> Beijing university
<120> degrading strain JN2 for petroleum hydrocarbons in oily sludge and application thereof
<130>2018
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1417
<212>DNA
<213> Salmonella reinhardtii (Rheinheimeria sp.)
<400>1
acacatgcag tcgagcgaat gaggggagct tgcttcctga tttagcggcg gacgggtgag 60
taatgtatag ggagctgccc gatagagggg gataccagtt ggaaacgact gttaataccg 120
cataatgtct acggaccaaa gtgtgggaccttcgggccac atgctatcgg atgcacctat 180
atgggattag ctagttggtg gggtaacggc tcaccaaggc gacgatccct agctggtttg 240
agaggatgat cagccacact ggaactgaga cacggtccag actcctacgg gaggcagcag 300
tggggaatat tggacaatgg gcgcaagcct gatccagcca tgccgcgtgt gtgaagaagg 360
ccttcgggtt gtaaagcact ttcagcgagg aggaagggtg ttgtgttaat agcacagcat 420
tttgacgtta ctcgcagaag aagcaccggc taactccgtg ccagcagccg cggtaatacg 480
gagggtgcaa gcgttaatcg gaattactgg gcgtaaagcg cacgtaggcg gtgtgttaag 540
ttggatgtga aagccccggg ctcaacctgg gaattgcatt caaaactggc acgctagagt 600
atgtgagagg ggggtagaat tccaagtgta gcggtgaaat gcgtagagat ttggaggaat 660
accagtggcg aaggcggccc cctggcacaa tactgacgct caggtgcgaa agcgtgggga 720
gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgtctact agctgttcgt 780
ggtcttgtac tgtgagtagc gcagctaacg cactaagtag accgcctggg gagtacggtc 840
gcaagattaa aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt 900
aattcgacgc aacgcgaaga accttaccta ctcttgacat ctagcgaaga ttgcagagat 960
gcagttgtgc cttcgggaac gctaagacag gtgctgcatg gctgtcgtca gctcgtgttg 1020
tgaaatgttg ggttaagtcc cgcaacgagc gcaaccctta tccttagttg ccagcacgta 1080
atggtgggaa ctctagggag actgccggtg ataaaccgga ggaaggtggg gacgacgtca 1140
agtcatcatg gcccttacga gtagggctac acacgtgcta caatggtacg tacagaggga 1200
ggcaagctgg cgacagtgag cggatctctt aaagcgtatc gtagtccgga tcgcagtctg 1260
caactcgact gcgtgaagtc ggaatcgcta gtaatcgcaa atcagaatgt tgcggtgaat 1320
acgttcccgg gccttgtaca caccgcccgt cacaccatgg gagtgggttg caaaagaagt 1380
aggtagctta accttcggga gggcgctacc acttgga 1417
Claims (6)
1. The strain JN2 for degrading and repairing the oily sludge is characterized by being Rheinheimera sp, the name of a preservation unit is China general microbiological culture preservation management center, and the preservation number is CGMCCNO.14973.
2. The application of the strain JN2 for degrading and remediating the oily sludge according to claim 1, wherein the strain JN2 comprises the following components in percentage by weight: the strain JN2 is used for degrading and repairing oily sludge in a petroleum hydrocarbon degrading mode.
3. Use according to claim 2, characterized in that: the strain JN2 has the degradation condition of 15-50 ℃ and the pH value of 7-11 to petroleum hydrocarbons.
4. Use according to claim 2, characterized in that it comprises the following steps:
(1) selecting Rheinheimera sp.JN2 single colony in a sterilized conical flask of L B liquid culture medium, sealing with a breathable sealing film, placing the conical flask in a shaking table at 120rpm and 30 ℃, and activating for 24h to prepare a seed culture solution;
(2) inoculating the cultured seed solution into a fermentation tank containing L B culture medium according to the inoculation amount of 5% for culture, aerating by adopting an air compressor, and culturing to logarithmic phase at the stirring speed of 200 r/min;
(3) air-drying and grinding the oily sludge in the Liaohe oil field, sieving the oily sludge by a 1mm sieve, adding 10 percent of inoculum size into Rheinheimera sp.JN2 activated bacterial liquid with OD600 ═ 1, regulating the liquid-soil ratio to be 2:5 by using an inorganic salt culture medium, and sealing the opening by using a breathable sealing film;
(4) and (3) placing the oily sludge added with the bacterial liquid and the inorganic salt culture medium in a constant-temperature incubator for static culture, adjusting the pH value of the soil to 11 for achieving the optimal degradation effect, and maintaining the temperature at 40 ℃.
5. The use as claimed in claim 4, wherein the L B medium comprises tryptone 10g, yeast extract 5g, NaCl 10g, distilled water 1000m L, and has a pH of 7.
6. Use according to claim 4, characterized in that: the inorganic salt culture medium comprises the following components: NaCl 30g, NH4NO33g，KH2PO41g，K2HPO41g，CaCl20.02g，MgSO40.5g of agar powder, 20g of agar powder, 1L parts of deionized water and 10ml of trace element solution, wherein the trace element solution comprises CuSO40.05g，MnSO40.05g，FeSO4·7H20.05g of O and 50ml of deionized water.
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4807799B2 (en) * | 2007-09-25 | 2011-11-02 | イビデン株式会社 | Method for treating waste liquid containing fat, alpha starch and beta starch |
Non-Patent Citations (2)
| Title |
|---|
| Microcosm-based interaction studies between members of two ecophysiological groups of bioemulsifier producer and a hydrocarbon degrader from the Indian intertidal zone;Markande,AR and Nerurkar,AS;《ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH》;20160630;第23卷(第14期);14462-14471 * |
| Oil-degrading bacteria from a membrane bioreactor (BF-MBR) system for treatment of saline oily waste: Isolation, identification and characterization of the biotechnological potential;Cappello,S. et al.;《INTERNATIONAL BIODETERIORATION & BIODEGRADATION》;20160531;第110卷;235-244 * |
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| CN108102977A (en) | 2018-06-01 |
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