CN114058527B - Oil-containing pollutant degradation strain XT2, microbial agent and application - Google Patents
Oil-containing pollutant degradation strain XT2, microbial agent and application Download PDFInfo
- Publication number
- CN114058527B CN114058527B CN202010750313.4A CN202010750313A CN114058527B CN 114058527 B CN114058527 B CN 114058527B CN 202010750313 A CN202010750313 A CN 202010750313A CN 114058527 B CN114058527 B CN 114058527B
- Authority
- CN
- China
- Prior art keywords
- achromobacter
- degradation
- strain
- oil
- sludge
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000015556 catabolic process Effects 0.000 title claims abstract description 73
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 73
- 230000000813 microbial effect Effects 0.000 title claims abstract description 19
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 14
- 239000003344 environmental pollutant Substances 0.000 title claims abstract description 12
- 231100000719 pollutant Toxicity 0.000 title claims abstract description 12
- 239000010802 sludge Substances 0.000 claims abstract description 71
- 241000590020 Achromobacter Species 0.000 claims abstract description 45
- 239000003921 oil Substances 0.000 claims description 57
- 239000001963 growth medium Substances 0.000 claims description 23
- 230000001580 bacterial effect Effects 0.000 claims description 18
- 239000000356 contaminant Substances 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000010779 crude oil Substances 0.000 claims description 14
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- 235000015278 beef Nutrition 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 9
- 239000000725 suspension Substances 0.000 claims description 9
- 230000000593 degrading effect Effects 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 230000008439 repair process Effects 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims 1
- 239000003209 petroleum derivative Substances 0.000 abstract description 30
- 241001250076 Achromobacter piechaudii Species 0.000 abstract description 25
- 238000012360 testing method Methods 0.000 abstract description 16
- 230000000694 effects Effects 0.000 abstract description 12
- 238000004321 preservation Methods 0.000 abstract description 7
- 239000002689 soil Substances 0.000 description 20
- 239000003208 petroleum Substances 0.000 description 19
- 241000894006 Bacteria Species 0.000 description 13
- 244000005700 microbiome Species 0.000 description 10
- 229930195733 hydrocarbon Natural products 0.000 description 9
- 150000002430 hydrocarbons Chemical class 0.000 description 9
- 239000000126 substance Substances 0.000 description 8
- 230000008569 process Effects 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 229910017053 inorganic salt Inorganic materials 0.000 description 4
- 239000004033 plastic Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000005067 remediation Methods 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- YBYIRNPNPLQARY-UHFFFAOYSA-N 1H-indene Chemical compound C1=CC=C2CC=CC2=C1 YBYIRNPNPLQARY-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- NNBZCPXTIHJBJL-UHFFFAOYSA-N decalin Chemical compound C1CCCC2CCCCC21 NNBZCPXTIHJBJL-UHFFFAOYSA-N 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241001478312 Comamonas sp. Species 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 241000588754 Klebsiella sp. Species 0.000 description 1
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000589614 Pseudomonas stutzeri Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000002283 diesel fuel Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- FPAFDBFIGPHWGO-UHFFFAOYSA-N dioxosilane;oxomagnesium;hydrate Chemical compound O.[Mg]=O.[Mg]=O.[Mg]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O FPAFDBFIGPHWGO-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000002920 hazardous waste Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000003345 natural gas Substances 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 0.000 description 1
- 244000000000 soil microbiome Species 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- PXXNTAGJWPJAGM-UHFFFAOYSA-N vertaline Natural products C1C2C=3C=C(OC)C(OC)=CC=3OC(C=C3)=CC=C3CCC(=O)OC1CC1N2CCCC1 PXXNTAGJWPJAGM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/343—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of grease, fat, oil
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/344—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of mineral oil
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Water Supply & Treatment (AREA)
- Environmental & Geological Engineering (AREA)
- Hydrology & Water Resources (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Treatment Of Sludge (AREA)
Abstract
The invention provides an oily pollutant degradation strain XT2, a microbial agent and application. The pollutant-containing degradation strain is Achromobacter freundii (Achromobacter piechaudii) XT2, the preservation number of the strain is CGMCC NO.17160, and the invention degrades thick oil sludge by using a single strain of Achromobacter freundii (Achromobacter piechaudii) XT2, thereby achieving the effect of greatly reducing the petroleum hydrocarbon content of the sludge. In the indoor test, the degradation rate of the total petroleum hydrocarbon content in the thick oil sludge inoculated with the Achromobacter pekinensis strain for 21 days is 15.17 percent. In the outdoor test, the degradation rate of the total petroleum hydrocarbon content in the thick oil sludge inoculated with the Achromobacter freundii (Achromobacter piechaudii) strain for 52 days is 51.60%.
Description
Technical Field
The invention relates to the technical field of microbial remediation, in particular to an oily pollutant degradation strain XT2, a microbial agent and application.
Background
The oily sludge is listed in the national hazardous waste list in China, and the national requirements of the oily sludge are that the oily sludge must be subjected to harmless treatment to realize recycling. The capacity of reducing treatment and recycling of the oily sludge not only restricts the survival and development of enterprises, but also has great hidden environmental pollution. At present, the treatment of the oily sludge mainly adopts a chemical heat washing method, an extraction separation method, a biological treatment method, an incineration method and the like, but the traditional chemical heat washing method is not clean and thorough for the oily sludge with relatively complex components, especially for the thick oil sludge, and has poor reduction effect; the extraction separation method has high treatment cost and low recovery and reuse efficiency of the extractant; the incineration method has higher cost, serious secondary pollution and larger one-time investment. At present, the treatment methods such as chemical heat washing, extraction and the like have certain limitations, and the microbial degradation of the oily sludge has the advantages of high safety, no secondary pollution, low cost, environmental friendliness and the like, and is considered as an economic and environmental-friendly method for treating the oily sludge, so how to screen and obtain the efficient petroleum degradation strain is a key for treating the oily sludge by using a biological method.
Chinese patent application 201810115869.9 discloses a strain of efficient degradation bacteria JN6 of petroleum hydrocarbons in oily sludge and application thereof, wherein the strain is Comamonas sp. The strain JN6 with petroleum hydrocarbon degradation function is used for treating the oily sludge in a petroleum hydrocarbon degradation mode, the degradation rate of the strain on Total Petroleum Hydrocarbon (TPH) in the oily sludge within 30 days is 62.16%, and the optimal degradation condition of the strain on petroleum pollutants is 15-40 ℃ and the pH value is 7-11. Chinese patent application 201810115860.8 discloses a strain of efficient degradation bacteria JN4 for petroleum hydrocarbon in oily sludge and application thereof, wherein the strain is Pseudomonas stutzeri Pseudomonas stutzeri. The strain JN4 with the petroleum hydrocarbon degradation function is used for treating the oil-containing sludge in a petroleum hydrocarbon degradation mode. The bacterium has a degradation rate of 71.05% for Total Petroleum Hydrocarbon (TPH) in oil-containing sludge within 30 days, and has obvious degradation capability for n-C10-n-C30, and GC-MS analysis shows that the bacterium has strong degradation capability for naphthene decalin, indene and homoandrostane, and the optimal degradation condition for petroleum pollutants is 25-40 ℃ and pH value is 5-11. Chinese patent application 201710022605.4 discloses a Klebsiella (Klebsiella sp.) bacterium CGMCC13201 which can effectively degrade crude oil in crude oil contaminated soil such as oil-containing sludge, oil-containing silt and the like. The invention discloses a Chinese patent application 201410072228.1, which relates to a petroleum degrading bacterium in oily sludge, wherein the strain is corynebacterium glutamicum separated from the oily sludge of an oil field of Bohai sea. The preservation number of the bacterium CGMCC8647 proves that the degradation rate of RS1 to saturated hydrocarbon is higher, reaching 20.74 percent, the degradation rate of the bacterium 30d to Total Petroleum Hydrocarbon (TPH) in oil-containing sludge is 39.69 percent, and the optimal degradation condition of the bacterium to petroleum is 37 ℃, the salinity is 0.5-5 percent and the pH is 6-8.
However, the degradation bacteria mentioned above have limited degradation range for oily contaminants, and do not perform well for oily sludge, especially residual thick oil sludge after treatment by thick oil sludge chemical fine heat washing treatment technology, so that it is highly demanded to find a microbial strain for degrading oily contaminants better.
Disclosure of Invention
In order to improve the limitation of the existing microorganisms on the degradation range of oil-containing pollutants, the invention aims to provide a microorganism Achromobacter Pisi-L XT2 for degrading the oil-containing pollutants, which can effectively degrade the residual thick oil sludge after being treated by a thick oil sludge chemical fine heat washing treatment technology.
Another object of the present invention is to provide a microbial agent containing the above Achromobacter Pitussilago XT2.
It is a further object of the present invention to provide the use of the microorganism as described above and microbial agents thereof for the degradation and remediation of oily contaminants.
The aim of the invention is achieved by the following technical scheme:
achromobacter freundii (Achromobacter piechaudii) XT2, which Achromobacter freundii XT2 has been deposited in the deposit unit under the name: china general microbiological culture Collection center (CGMCC) with the address: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no.1, no. 3, north Chen West Lu, the Korean region of Beijing; preservation date: the collection number of the strain is CGMCC NO.17160 and the classification is Achromobacter piechaudii on 1 month 11 2019.
According to some embodiments of the present invention, the nucleotide sequence of the 16S rDNA of the above Achromobacter freundii XT2 is shown in SEQ ID NO. 1.
The bacterial colony morphology of the Achromobacter freundii XT2 is as follows: light white, round, translucent, smooth, moist, clean-edged, gram-negative bacilli. According to some embodiments of the present invention, the above-mentioned Achromobacter freundii XT2 is degraded to petroleum hydrocarbons at a temperature of 20-30℃and a pH of 7-8.
On the other hand, the invention also provides a microbial agent which contains the achromobacter Pityrosporum XT2, and other conventional auxiliary components such as talcum powder, tween and the like can be added, and the preparation method of the microbial agent can be obtained through conventional experiments.
In yet another aspect, the invention also provides the use of the above Achromobacter freundii XT2 in degradation remediation of oily contaminants.
According to some embodiments of the invention, the oil-containing contaminants include oil-containing sludge, water bodies contaminated with crude oil, and crude oil contaminants.
According to some embodiments of the invention, the oil-containing sludge comprises thickened oil sludge which remains after treatment by a thickened oil sludge chemical fine heat-wash treatment technique.
According to some embodiments of the invention, the above application comprises the steps of: inoculating Achromobacter freundii XT2 into a culture medium for culture to prepare bacterial suspension; and step two, mixing the bacterial suspension with the oil-containing pollutant, repeatedly inoculating after a proper time, and then preserving for a proper time to finish degradation and repair.
According to a specific embodiment of the invention, in the first step, the culture medium is beef extract peptone liquid culture medium.
According to a specific embodiment of the present invention, in the first step, the Achromobacter freundii XT2 is inoculated to a culture medium and cultured at 30℃for 180r/min with shaking for 3 days.
According to a specific embodiment of the invention, in step two, the inoculation is repeated after mixing the bacterial suspension with the oily contaminants for 13-15 days.
According to some embodiments of the invention, the beef extract peptone liquid medium is prepared from the following raw materials: each 1000mL of water contains 5g of beef extract, 10g of peptone and 5g of sodium chloride, and the pH value is 7.0-7.4.
According to some embodiments of the invention, the oleaginous contaminant is from a LiaoHe field.
According to some embodiments of the invention, the pH of the oleaginous contaminant is from 8 to 9 (soil: water=1:5).
According to some embodiments of the invention, the above application comprises the steps of: step one, the Achromobacter suis XT2 is selected and inoculated into a beef extract peptone liquid culture medium (without agar), and is cultured, and the culture medium is oscillated for 3 days at the temperature of 30 ℃ and at the speed of 180r/min to prepare bacterial suspension; collecting the same batch of oily sludge in a container as a treatment, adding 300ml of the bacterial suspension into the container, stirring, repeating inoculation after 2 weeks, and setting a control treatment without adding a bacterial agent; step three, stirring (manual stirring) is performed every two weeks during the test to provide oxygen required for microbial degradation of petroleum. In the experimental process, part of water is properly added according to the dry and wet conditions of the crude oil containing the silt, so that the crude oil containing the silt is in a wet state in the experimental process, a plastic box is covered by a weather such as heavy rain, the total petroleum hydrocarbon content and the water content of the soil are measured once every half month in the first month of the test, and the total petroleum hydrocarbon content and the water content of the soil are measured once every month from the second month, and the statistical result is obtained.
According to some embodiments of the invention, the volume of the vessel in which the oleaginous contaminants are collected is 40cm by 55cm by 6cm.
The invention further provides application of the Achromobacter freundii XT2 in degradation and restoration of oily pollutants.
The invention has the beneficial effects that:
1. achromobacter freundii (Achromobacter piechaudii) XT2 is derived from local oily sludge of Liaohe oil fields, and the strain enters into a soil flora to occupy a certain ecological niche, so that the degradation of petroleum hydrocarbons is effectively promoted.
2. According to the invention, the Achromobacter freundii (Achromobacter piechaudii) XT2 single strain is used for degrading the thick oil sludge, so that the effect of greatly reducing the petroleum hydrocarbon content (oil content) of the sludge can be achieved. In the indoor test, the degradation rate of the total petroleum hydrocarbon content in the thick oil sludge inoculated with the Achromobacter pekinensis (Achromobacter piechaudii) strain for 21 days is 15.17%. In the outdoor test, the degradation rate of the total petroleum hydrocarbon content in the thick oil sludge inoculated with the Achromobacter freundii (Achromobacter piechaudii) strain for 52 days is 51.60%. The strain can occupy a certain ecological place in the indigenous strain, promote the degradation of petroleum hydrocarbon, further reduce the total petroleum hydrocarbon content in the sludge treated by the thick oil fine hot washing thick oil sludge chemical fine hot washing treatment technology, and has lower treatment cost, good application effect and obvious economic and social benefits.
3. According to the invention, the Achromobacter freundii (Achromobacter piechaudii) XT2 is prepared by using a single strain to realize excellent degradation of petroleum hydrocarbon in the oily sludge, so that complex steps of activating each strain respectively and mixing the strains in proportion in the preparation process of the mixed microbial inoculum are reduced, the optimal reaction condition of petroleum hydrocarbon degradation is easy to control, and the petroleum hydrocarbon substances in the oily sludge are easy to be further removed by combining other methods.
Drawings
FIG. 1 shows the form of Xylobacter cutanensis (Achromobacter piechaudii) XT2 obtained in example 1 of the present invention.
FIG. 2 is an electron micrograph of Achromobacter Pitussilago (Achromobacter piechaudii) XT2 in example 1 of the invention.
FIG. 3 is a graph showing the degradation profile of Achromobacter freundii (Achromobacter piechaudii) XT2 in accordance with example 3 of the invention for petroleum hydrocarbons in oily sludge at various pH values.
FIG. 4 shows the degradation profile of Achromobacter freundii (Achromobacter piechaudii) XT2 of example 4 of the invention on petroleum hydrocarbons in oily sludge at different temperatures.
The information on the preservation of Achromobacter freundii XT2 microorganisms according to the invention is as follows:
preservation date: 2019, 1 month and 11 days;
preservation unit name: china general microbiological culture Collection center (CGMCC);
deposit unit address: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no.1, no. 3, north Chen West Lu, the Korean region of Beijing;
preservation number: CGMCC No.17160;
classification naming: achromobacter freundii Achromobacter piechaudii.
Detailed Description
The following detailed description of the present invention will be presented in order to provide a clearer understanding of the technical features, objects and advantages of the present invention, but should not be construed as limiting the scope of the invention.
Example 1 screening and identification of Achromobacter Pisi-2
Material preparation
1 soil material
Taking petroleum polluted soil from a place of Liaohe oil field, carrying out scattering treatment, and storing at 4 ℃.
2 culture medium:
inorganic salt enrichment medium (1L): k (K) 2 HPO 4 ·3H 2 O 1g，KH 2 PO 4 1g，Mg 2 SO 4 ·7H 2 O 0.5g，NH 4 NO 3 1g，CaCl 2 0.02g，FeCl 3 Trace amount, diesel 3, pH 7.2-7.4.
Beef extract peptone medium (1L): 5g of beef extract; 10g of peptone; 5g of sodium chloride; 18-20 g of agar; 1000ml of distilled water; pH 7.0-7.4.
Degradation medium (1L): (NH 4) 2 SO 4 0.5g，NaNO 3 0.5g，CaCl 2 0.02g，MgSO 4 0.2g，KH 2 PO 4 1.0g，NaH 2 PO 4 ·H 2 O1.0 g, pH7, and 50g of crude oil containing silt.
Isolation, screening and domestication of strains (II)
Adding 1% (w/v) petroleum oil polluted soil or residual oil into an inorganic salt enrichment culture medium taking diesel oil as a unique carbon source, carrying out shake cultivation for 3d at 30 ℃, inoculating 1mL of enrichment liquid into the same fresh culture medium, and carrying out cultivation for 3d under the same condition, thus continuously carrying out enrichment cultivation for 4 times. After the second culture, a large amount of microorganisms were observed to be produced in the culture medium, and the culture medium was in a suspended state.
And (3) carrying out plate culture on the beef extract peptone culture medium for 3 days after gradient dilution of the enrichment culture medium, wherein a large number of microbial colonies are generated on the surface of the culture medium. After the bacterial colony grows out of the flat plate, a proper amount of single bacterial colony with different colors and morphologies is picked by an inoculating loop, and repeated streaking, separation and purification are carried out until the single bacterial colony is obtained. Uniformly numbering, and preserving at 4 ℃ inclined plane.
The oil sludge sample for domestication test is the oil sludge sample to be treated, and the oil content is 20%. And (5) carrying out scattering treatment on the soil sample, and sealing at 4 ℃ for later use. The strain obtained by separation and purification was inoculated with an inoculating needle into a sterilized 500ml Erlenmeyer flask containing 200ml of a liquid medium for degradation, and cultured at 30℃for 14 days by shaking at 180 rpm. After the completion of the culture, 0.1ml of each culture was cultured in a plate medium, and the growth was observed. The strain domesticated and cultured by the high oil-containing culture medium has a large amount of strain growth on the beef extract peptone culture medium.
DNA identification and morphological identification were performed by the Qingdao West letter detection analysis Co., ltd. The colony morphology of the strain XT2 is as follows: as shown in FIG. 1, the bacteria were light white, round, translucent, smooth, moist, clean-edged, gram-negative. The bacterial morphology is shown in scanning electron microscope figure 2.
The sequence of the 16S ribosomal RNA gene of the strain is compared with a database by an analysis method for analysis, and the strain is found to belong to Achromobacter freundii (Achromobacter piechaudii) and the DNA sequence table is shown in the sequence table.
Example 2 degradation repair test of certain oily sludge in Liaohe oilfield
The thick oil sludge sample for degradation test is collected in the mud sand crude oil in the treatment process of a certain treatment plant in the period of 5.14.2018, and the soil is air-dried, scattered, ground and sealed for later use. The isolated and purified strain XT2 was inoculated with an inoculating needle into a sterilized 250ml triangular flask containing 100ml of a liquid medium for degradation, and a medium not inoculated with microorganisms was set as a control group (CK), and the culture was shake-cultured at 30℃and 180rpm for 21 days, and each treatment was repeated three times. And after drying, measuring the total petroleum hydrocarbon content in the oily sludge by using an infrared spectroscope. The bacterium has good degradation effect on thick oil sludge, after 21 days of culture, the total petroleum hydrocarbon content of CK group is 5.40+/-0.31%, the total petroleum hydrocarbon content of thick oil sludge inoculated with XT2 strain is 4.58+/-0.34%, and the degradation rate is 15.17% in 21 days.
EXAMPLE 3 Achromobacter Pitussilago (Achromobacter piechaudii) effect on the degradation of Petroleum hydrocarbons in oily sludge at different pH values
And collecting thick oil sludge in the treatment process of a certain treatment plant in 7.23.2018, air-drying, scattering and grinding the soil, and sealing the bag for later use. 100ml degradation culture medium is added into a 250ml conical flask, the initial pH value of the degradation culture medium for degrading liquid inorganic salt is adjusted to be 5.0, 6.0, 7.0, 8.0 and 9.0 respectively by NaOH or HCl, the bacterial strain XT2 obtained in the separation and purification in the example 1 is inoculated by an inoculating needle after sterilization, the bacterial strain XT2 is cultured for 14 days by shaking at a constant temperature of 30 ℃ and 180rpm, and the total petroleum hydrocarbon content in the oily sludge is measured by an infrared spectroscope after drying, so that the degradation rate is calculated. The effect of the strain Achromobacter freundii (Achromobacter piechaudii) on the degradation of petroleum hydrocarbons in oily sludge at different pH conditions is shown in FIG. 3.
The oil content of the control treated (CK) soil sample without inoculating microorganisms is 7.19+/-0.36%, the pH adaptability is strong, the oil degradation effect is good in the test range of pH 5-9, the degradation rate is 8.60% -18.69%, and the degradation effect is optimal under the condition of pH 7-8.
EXAMPLE 4 Achromobacter Pitussilago (Achromobacter piechaudii) effect on the degradation of Petroleum hydrocarbons in oily sludge at different temperatures
And collecting thick oil sludge in the treatment process of a certain treatment plant in 7.23.2018, air-drying, scattering and grinding soil, and bagging and sealing for later use. Adding 100ml of degradation culture medium into a 250ml conical flask, regulating the initial pH value of the degradation culture medium for degrading liquid inorganic salt to 7.0 by using NaOH or HCl, inoculating the strain XT2 obtained in the separation and purification in the example 1 by using an inoculating needle after sterilization, respectively setting the temperature of a shaking table to 10 ℃, 20 ℃,30 ℃, 40 ℃ and 180rpm for shaking culture for 14 days, drying, measuring the total petroleum hydrocarbon content in the oily sludge by using an infrared spectroscope, and calculating the degradation rate. The degradation effect of the strain Achromobacter freundii (Achromobacter piechaudii) on petroleum hydrocarbons in the oily sludge under different temperature conditions is shown in figure 4. The oil content in the control treated (CK) soil sample without inoculating microorganism is 7.24+ -0.14%, the optimal culture temperature of XT2 is 20-30deg.C, and the degradation rate is 14.60-15.97%.
EXAMPLE 5 Achromobacter Pitussilago (Achromobacter piechaudii) remediation of actual oily sludge
Based on the above examples, achromobacter freundii (Achromobacter piechaudii) was used to repair the actual oily sludge, and the experimental steps were as follows:
(1) Test oily sludge: in 2018, 8 and 3 days, in a field for recycling crude oil containing silt in Liaohe oil field, placing a batch of treated thick oil sludge into the same plastic box with the volume of 40cm multiplied by 55cm multiplied by 6cm, adding microbial agent into the plastic box, and setting control treatment without microbial agent. The pH of the test sludge sample was 8.4 (soil: water=1:5).
(2) Preparation of Achromobacter freundii (Achromobacter piechaudii) bacterial suspension:
achromobacter suis (Achromobacter piechaudii) XT2 is selected and inoculated into beef extract peptone liquid culture medium (without agar) for culture, and shaking is carried out for 3 days at 30 ℃ and 180r/min for testing. 300ml of bacterial liquid is added in each treatment, and stirring is carried out. Repeated inoculations were performed after 2 weeks.
Stirring (with manual stirring) was performed every two weeks during the test to provide the oxygen required for microbial degradation of petroleum. In the experimental process, part of water is properly added according to the dry and wet conditions of the crude oil containing the silt, so that the crude oil containing the silt is in a wet state in the experimental process, and the upper cover of the plastic box is covered in the weather such as heavy rain. The total petroleum hydrocarbon content of the soil is measured once every half month in the first month of the test (the total petroleum hydrocarbon content of the soil is actually the oil content, because the collected samples are wet samples, the directly measured values are greatly influenced by the water content of the soil, the dry oil content needs to be calculated for comparison of the samples), and the water content is measured once a month from the second month.
When the first sampling is carried out in 8 months and 3 days, 3 groups of crude oil samples containing silt are taken, the oil content (dry) =total petroleum hydrocarbon content of soil/(1-water content) ×100%) of the crude oil samples is between 2.18 and 2.96%, but the error between the oil contents of the thick oil sludge on the surface layer and the thick oil sludge on the bottom layer is larger when the sampling is found in the test. Thus, the oil content of each group on 8 months and 17 days was used as the initial value of each group of strains, and the degradation rate after the addition of petroleum for 52 days (10 months and 8 days) was calculated, and the results are shown in Table 1:
TABLE 1
After 52 days, the oil content of the control group is reduced from 2.05% to 1.38%, and the degradation rate reaches 32.35%. The soil bacteria has a certain degradation effect. After Achromobacter freundii (Achromobacter piechaudii) XT2 is added, the thick oil sludge with the oil content of 3.98 percent is degraded to 1.93 percent, and the degradation rate reaches 51.60 percent. The degradation function bacteria are added to promote the degradation of the thick oil sludge. The pH of the sample of the thick oil sludge to be tested is 8.4 (soil: water=1:5), and the degradation speed is high at 20-30 ℃, which accords with the optimal degradation condition that the degradation temperature is 20-30 ℃ and the pH is 7-8.
Sequence listing
<110> China Petroleum and Natural gas stock Co., ltd
<120> an oil-containing contaminant degrading strain XT2, microbial agent and use
<130> GAI20CN2949
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1399
<212> DNA
<213> Achromobacter pekoe (Achromobacter pechaudii)
<400> 1
atcgcccccc ttacggttag gctaactact tctggtaaaa cccactccca tggtgtgacg 60
ggcggtgtgt acaagacccg ggaacgtatt caccgcgaca tgctgatccg cgattactag 120
cgattccgac ttcacgcagt cgagttgcag actgcgatcc ggactacgat cgggtttctg 180
ggattggctc cccctcgcgg gttggcgacc ctctgtcccg accattgtat gacgtgtgaa 240
gccctaccca taagggccat gaggacttga cgtcatcccc accttcctcc ggtttgtcac 300
cggcagtctc attagagtgc cctttcgtag caactaatga caagggttgc gctcgttgcg 360
ggacttaacc caacatctca cgacacgagc tgacgacagc catgcagcac ctgtgttcca 420
gttctcttgc gagcacttcc aaatctcttc ggaattccag acatgtcaag ggtaggtaag 480
gtttttcgcg ttgcatcgaa ttaatccaca tcatccaccg cttgtgcggg tccccgtcaa 540
ttcctttgag ttttaatctt gcgaccgtac tccccaggcg gtcaacttca cgcgttagct 600
gcgctaccaa ggcccgaagg ccccaacagc tagttgacat cgtttagggc gtggactacc 660
agggtatcta atcctgtttg ctccccacgc tttcgtgcat gagcgtcagt gttatcccag 720
gaggctgcct tcgccatcgg tgttcctccg catatctacg catttcactg ctacacgcgg 780
aattccacct ccctctgaca cactctagct cggtagttaa aaatgcagtt ccaaagttaa 840
gctctgggat ttcacatctt tctttccgaa ccgcctgcgc acgctttacg cccagtaatt 900
ccgattaacg cttgcaccct acgtattacc gcggctgctg gcacgtagtt agccggtgct 960
tattctgcag gtaccgtcag tttcacgggg tattagccca tgacgtttct ttcctgccaa 1020
aagtgcttta caacccgaag gccttcatcg cacacgcggg atggctggat cagggtttcc 1080
cccattgtcc aaaattcccc actgctgcct cccgtaggag tctgggccgt gtctcagtcc 1140
cagtgtggct ggtcgtcctc tcaaaccagc tacggatcgt cgccttggtg agccgttacc 1200
ccaccaacta gctaatccga tatcggccgc tctaatagtg caaggtcttg cgatcccctg 1260
ctttcccccg tagggcgtat gcggtattag ctacgctttc gcgtagttat cccccgctac 1320
taggcacgtt ccgatacatt actcacccgt tcgccactcg ccaccagacc gaagtccgtg 1380
ctgccgttcg actgcatgt 1399
Claims (12)
1. Achromobacter pekinensis (L.) KuntzeAchromobacter piechaudii) XT2, characterized by the fact that it has the deposit number: CGMCC No.17160.
2. A microbial agent comprising achromobacter pekinensis XT2 as claimed in claim 1.
3. Use of achromobacter suis XT2 as claimed in claim 1 for degradation repair of oily contaminants.
4. Use according to claim 3, wherein the oleaginous contaminants comprise oleaginous sludge or bodies of water contaminated with crude oil.
5. The use according to claim 3, characterized by the following steps:
inoculating Achromobacter freundii XT2 into a culture medium for culture to prepare bacterial suspension;
and step two, mixing the bacterial suspension with the oil-containing pollutant, repeatedly inoculating after a proper time, and then preserving for a proper time to finish degradation and repair.
6. The use according to claim 5, wherein in step one, the medium is beef extract peptone liquid medium.
7. The use according to claim 5, wherein in step one, the achromobacter pisiformis XT2 is inoculated into a culture medium for culturing at 30 ℃ at 180r/min, and is shaken for 3 days.
8. The method according to claim 5, wherein in step two, the inoculation is repeated after mixing the bacterial suspension with the oily contaminants for 13-15 days.
9. The use according to claim 6, wherein the beef extract peptone liquid medium is prepared from the following raw materials: each 1000mL of water contains 5g of beef extract, 10g of peptone and 5g of sodium chloride, and the pH value is 7.0-7.4.
10. The use of claim 5, wherein the oleaginous contaminant is from a Liaohe field.
11. The use according to claim 5, wherein the pH of the oleaginous contaminant is from 8 to 9.
12. Use of the microbial agent of claim 2 for degrading and repairing oily contaminants.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010750313.4A CN114058527B (en) | 2020-07-30 | 2020-07-30 | Oil-containing pollutant degradation strain XT2, microbial agent and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010750313.4A CN114058527B (en) | 2020-07-30 | 2020-07-30 | Oil-containing pollutant degradation strain XT2, microbial agent and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114058527A CN114058527A (en) | 2022-02-18 |
| CN114058527B true CN114058527B (en) | 2023-05-26 |
Family
ID=80227136
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010750313.4A Active CN114058527B (en) | 2020-07-30 | 2020-07-30 | Oil-containing pollutant degradation strain XT2, microbial agent and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114058527B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745511A (en) * | 2015-03-30 | 2015-07-01 | 中国石油天然气集团公司 | Complex microbial agent for restoring coastline with heavy oil pollution, as well as preparation method and applications thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2472127C (en) * | 2002-01-07 | 2009-09-08 | Manas Ranjan Banerjee | Sulfur-oxidizing plant growth promoting rhizobacteria for enhanced canola performance |
| US7521236B2 (en) * | 2002-01-07 | 2009-04-21 | Manas Ranjan Banerjee | Sulfur-oxidizing plant growth promoting rhizobacteria for enhanced canola performance |
-
2020
- 2020-07-30 CN CN202010750313.4A patent/CN114058527B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745511A (en) * | 2015-03-30 | 2015-07-01 | 中国石油天然气集团公司 | Complex microbial agent for restoring coastline with heavy oil pollution, as well as preparation method and applications thereof |
Non-Patent Citations (2)
| Title |
|---|
| Chemical composition of the essential oils and extracts of Achillea species and their biological activities: A review;Majid Mohammadhosseini等;《J Ethnopharmacol》;20170306;第199卷;第257-315页 * |
| Isolation and characterization of alkane degrading bacteria from petroleum reservoir waste water in Iran (Kerman and Tehran provenances);Mehdi Hassanshahian等;《Mar Pollut Bull》;20130815;第73卷(第1期);第300-305页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114058527A (en) | 2022-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111748483B (en) | Bacillus for degrading petroleum hydrocarbon and application thereof | |
| CN108102979B (en) | Degradation strain JN5 for petroleum hydrocarbons in oily sludge and application thereof | |
| CN112980746B (en) | Petroleum degrading bacterium and application thereof | |
| CN110846257A (en) | Microbial bacterium for degrading long-chain alkane and application thereof | |
| CN114058528B (en) | Oil-containing pollutant degradation strain XT4, microbial agent and application | |
| CN108048374B (en) | Degradation strain JN4 for petroleum hydrocarbons in oily sludge and application thereof | |
| CN108034626B (en) | Degradation strain JN1 for petroleum hydrocarbons in oily sludge and application thereof | |
| CN108300674B (en) | Petroleum degrading bacteria, obtaining method thereof and application of petroleum degrading bacteria in crude oil degradation | |
| CN110438033B (en) | Grease degrading bacterium, application and grease degrading method | |
| CN110218689B (en) | Efficient marine oil degradation complex microbial inoculant as well as preparation method and application thereof | |
| CN108102978B (en) | Degradation strain JN8 for petroleum hydrocarbons in oily sludge and application thereof | |
| CN113234625B (en) | Acinetobacter pittericus and application thereof | |
| Hamzah et al. | Isolation and identification new bacterial strains isolated from different sources of Al-Rafidiyah oil field in Iraq | |
| CN114058529B (en) | Oily pollutant degradation strain ZT2, microbial agent and application | |
| CN115449489A (en) | Oil reducing bacteria and composite microbial inoculum thereof, preparation method and application | |
| CN112239731B (en) | Surface active bacterial strain for degrading petroleum hydrocarbon and application thereof | |
| CN114058527B (en) | Oil-containing pollutant degradation strain XT2, microbial agent and application | |
| CN108034625B (en) | Degradation strain JN7 for petroleum hydrocarbons in oily sludge and application thereof | |
| CN110157634B (en) | Sphingomonas paucimobilis and application thereof | |
| CN116640682A (en) | Microbial strain and microbial agent suitable for degrading petroleum and application thereof | |
| CN117264798A (en) | Screening and application of high-concentration shale gas oil-based drilling cuttings efficient degradation strain | |
| CN112011480B (en) | Aromatic hydrocarbon degrading bacterium and application thereof | |
| CN108048376B (en) | Degradation strain JN3 for petroleum hydrocarbons in oily sludge and application thereof | |
| CN108048375B (en) | Degradation strain JN6 for petroleum hydrocarbons in oily sludge and application thereof | |
| CN108102977B (en) | Degradation strain JN2 for petroleum hydrocarbons in oily sludge and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |