CN114717152B - Composite functional microbial agent taking pseudomonas stutzeri as carrier for in-situ remediation of polluted soil and application thereof - Google Patents
Composite functional microbial agent taking pseudomonas stutzeri as carrier for in-situ remediation of polluted soil and application thereof Download PDFInfo
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- 239000002689 soil Substances 0.000 title claims abstract description 69
- 241000589614 Pseudomonas stutzeri Species 0.000 title claims abstract description 49
- 239000002131 composite material Substances 0.000 title claims abstract description 38
- 238000005067 remediation Methods 0.000 title claims abstract description 20
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 17
- 239000003795 chemical substances by application Substances 0.000 title claims description 36
- 230000000813 microbial effect Effects 0.000 title claims description 20
- 230000001580 bacterial effect Effects 0.000 claims abstract description 46
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 43
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 43
- UDCXNLDCQOXJNC-UHFFFAOYSA-N 2-n,4-n,1,3-tetraphenyl-1,3,2,4-diazadiphosphetidine-2,4-diamine Chemical compound C=1C=CC=CC=1NP(N1C=2C=CC=CC=2)N(C=2C=CC=CC=2)P1NC1=CC=CC=C1 UDCXNLDCQOXJNC-UHFFFAOYSA-N 0.000 claims abstract description 42
- 238000004321 preservation Methods 0.000 claims abstract description 16
- 238000000855 fermentation Methods 0.000 claims description 27
- 230000004151 fermentation Effects 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 24
- 238000001694 spray drying Methods 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 12
- 239000003209 petroleum derivative Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 241000209149 Zea Species 0.000 claims 2
- 238000011081 inoculation Methods 0.000 claims 2
- 229930195733 hydrocarbon Natural products 0.000 abstract description 24
- 150000002430 hydrocarbons Chemical class 0.000 abstract description 24
- 239000004215 Carbon black (E152) Substances 0.000 abstract description 23
- 241000894006 Bacteria Species 0.000 abstract description 19
- 230000015556 catabolic process Effects 0.000 abstract description 11
- 238000006731 degradation reaction Methods 0.000 abstract description 11
- 244000005700 microbiome Species 0.000 abstract description 6
- 230000004083 survival effect Effects 0.000 abstract description 4
- FCBUKWWQSZQDDI-UHFFFAOYSA-N rhamnolipid Chemical compound CCCCCCCC(CC(O)=O)OC(=O)CC(CCCCCCC)OC1OC(C)C(O)C(O)C1OC1C(O)C(O)C(O)C(C)O1 FCBUKWWQSZQDDI-UHFFFAOYSA-N 0.000 description 10
- 239000003225 biodiesel Substances 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- 239000000306 component Substances 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 239000004094 surface-active agent Substances 0.000 description 7
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- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000194107 Bacillus megaterium Species 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 4
- 230000000593 degrading effect Effects 0.000 description 4
- 238000009792 diffusion process Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 230000000644 propagated effect Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 241000589651 Zoogloea Species 0.000 description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 235000010344 sodium nitrate Nutrition 0.000 description 3
- 239000004317 sodium nitrate Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000003344 environmental pollutant Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 230000002906 microbiologic effect Effects 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 241000194103 Bacillus pumilus Species 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000004115 Sodium Silicate Substances 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005056 compaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 230000001094 effect on targets Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000008394 flocculating agent Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- -1 physical Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
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- 230000008439 repair process Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
- 229910052911 sodium silicate Inorganic materials 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Abstract
The invention discloses a composite functional microbial inoculum for in-situ remediation of polluted soil by taking pseudomonas stutzeri as a carrier and application thereof, wherein the composite functional microbial inoculum comprises pseudomonas stutzeri VTCQ-1 and pseudomonas aeruginosa VTS-1, the preservation number of the pseudomonas stutzeri VTCQ-1 is CGMCC24469, and the preservation number of the pseudomonas aeruginosa VTS-1 is CGMCC2200, wherein the pseudomonas stutzeri VTCQ-1 is taken as the carrier of the pseudomonas aeruginosa VTS-1. According to the invention, by utilizing the characteristics of large specific surface area of pseudomonas stutzeri VTCQ-1 colony and capability of providing a large and stable attachment place for other bacteria, the bacterial colony and pseudomonas aeruginosa VTS-1 are mixed and cultured, so that the survival rate of pseudomonas aeruginosa VTS-1 can be improved, the prepared composite functional microbial inoculum can improve the degradation efficiency of hydrocarbon in soil, greatly shorten the hydrocarbon polluted soil treatment period and maintain the diversity and durability of soil microorganisms.
Description
Technical Field
The invention relates to the field of bioremediation of petroleum hydrocarbon contaminated soil, in particular to a composite functional microbial inoculum for in-situ remediation of contaminated soil by taking pseudomonas stutzeri as a carrier and application thereof.
Background
At present, a plurality of methods are adopted in the field of in-situ remediation of hydrocarbon contaminated soil, such as physical, chemical and microbial methods, wherein the microbial in-situ remediation of hydrocarbon contaminated soil is the most environmentally-friendly, economical, secondary pollution-free and sustainable and environmentally-friendly reliable treatment mode, and the method is widely focused and studied in various environmental protection fields in the world.
The key of the microbial remediation method is the screening of high-efficiency hydrocarbon degrading bacteria, the maintenance of activity of flora in soil environment, the density of microbial agents, the diffusion and propagation of microbial agents in soil environment, and the capture and aggregation of hydrocarbon pollutants. To meet the above requirements, methods such as increasing the input of the microbial inoculum, improving the injection of the nutrient components, adding a flocculating agent or a surfactant and the like are generally adopted to assist in improving the hydrocarbon treatment efficiency. Increasing the amount of the microbial inoculum increases the cost, and chemical components such as the flocculant, the surfactant and the like which are added are especially undegradable parts or cause secondary environmental pollution. In addition, the composite microbial agent carrier is generally prepared by mixing one or more of calcium carbonate, diatomite, sodium silicate, maltodextrin, sucrose, glucose, peptone and the like according to a certain proportion, but the carriers have the characteristic of quantification, once the microbial agent is dissolved or the microorganisms enter the soil environment to be completely released, the microorganisms are not subjected to carrier auxiliary diffusion, and the effect of gathering and degrading the hydrocarbons can not be completed by the microorganisms in a large range for a long time due to the limited addition of the carriers.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a composite functional microbial agent for in-situ remediation of polluted soil by taking pseudomonas stutzeri as a carrier and application thereof, so as to solve the problem that the carrier of the composite microbial agent cannot assist microorganisms to complete hydrocarbon aggregation and degradation for a long time in a large range, and further improve the degradation efficiency of petroleum hydrocarbon in soil.
The invention is realized in the following way:
in a first aspect, the invention provides a composite functional microbial agent, which comprises pseudomonas stutzeri VTCQ-1 and pseudomonas aeruginosa VTS-1;
the preservation number of the pseudomonas stutzeri VTCQ-1 is CGMCC24469;
the preservation number of the pseudomonas aeruginosa VTS-1 is CGMCC2200;
pseudomonas stutzeri VTCQ-1 is used as a carrier of pseudomonas aeruginosa VTS-1.
In a second aspect, the invention provides a preparation method of a composite functional microbial inoculum, which comprises the step of continuously mixed culturing pseudomonas stutzeri VTCQ-1 and pseudomonas aeruginosa VTS-1.
In a third aspect, the invention provides a method for in-situ remediation of contaminated soil, which comprises the step of remediating soil to be remediated by adopting the composite functional microbial inoculum.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a composite functional microbial inoculum for in-situ remediation of polluted soil by taking pseudomonas stutzeri as a carrier, which utilizes the characteristics that the specific surface area of a pseudomonas stutzeri VTCQ-1 colony is large, and a large and stable attachment place can be provided for other bacteria, and the microbial inoculum is mixed and cultured with pseudomonas aeruginosa VTS-1, so that the pseudomonas stutzeri VTCQ-1 colony can wrap pseudomonas aeruginosa VTS-1, and the survival rate of the VTS-1 is improved in the process of preparing the microbial inoculum by spray drying. The pseudomonas stutzeri VTCQ-1 is obtained by screening and domesticating the soil environment, has good environmental adaptability, can be rapidly propagated in the underground deep soil environment, has a large diffusion range in the soil environment, provides more attachment sites for other composite functional bacterial agents which are propagated simultaneously, and is easy to form zoogloea with a large range and a large number; meanwhile, the pseudomonas stutzeri VTCQ-1 can be adsorbed on the surface of hydrocarbon-polluted soil to fix functional bacteria and adsorb hydrocarbon on the surface of soil particles, so that the effect of gathering hydrocarbon substances for other composite functional bacterial agents with higher degradation efficiency in the soil can be achieved, the composite functional bacterial agents can be likened to a flowing restaurant in which the soil remediation functional bacterial agents degrade the hydrocarbon in the soil, and the operating range of the flowing restaurant can be infinitely expanded. The composite functional microbial agent provided by the invention can improve the degradation efficiency of hydrocarbon in soil, greatly shorten the treatment period of hydrocarbon polluted soil and maintain the diversity and durability of soil microorganisms.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. FIG. 1 shows the results of detection of rhamnolipid surfactant component in fermentation broth according to example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Some embodiments of the present invention provide a complex functional microbial agent comprising Pseudomonas stutzeri VTCQ-1 and Pseudomonas aeruginosa VTS-1; pseudomonas stutzeri VTCQ-1 is used as a carrier of pseudomonas aeruginosa VTS-1.
The preservation information of the Pseudomonas stutzeri VTCQ-1 is as follows: the preservation number is CGMCC24469; the classification was designated pseudomonas stutzeri Pseudomonas stutzeri; the preservation unit is China general microbiological culture Collection center; the preservation address is the microbiological institute of China academy of sciences of national institute of sciences No.1, 3 of north chen west way, the morning of Beijing city; the preservation time is 2022, 03 and 07.
The preservation information of the pseudomonas aeruginosa VTS-1 is as follows: the preservation number is CGMCC2200; the classification was named pseudomonas aeruginosa Pseudomonas aeruginosa; the preservation unit is China general microbiological culture Collection center; the preservation address is the microbiological institute of China academy of sciences of Datunlu in the Yang ward area of Beijing city; the preservation time was 10 months and 17 days of 2007, which is disclosed in patent document CN101177696B, and is an existing preserved strain.
The pseudomonas stutzeri VTCQ-1 is obtained by repeatedly domesticating and separating a water layer of deep polluted soil of a certain gas station, and the morphology characteristics of the pseudomonas stutzeri VTCQ-1 are as follows: the bacterial colony is round, the edge is irregular, the surface corrugation specific surface area is large, the bacterial colony is white and waxy, the bacterial colony is easy to attach to the surface of a common LB solid culture medium or the surface of other solids, and the bacterial colony is easy to strip in a piece; observing the form of the thallus under a microscope oil microscope to form a short rod shape with the length of 0.7-3.6 mu m and the width of 0.35-42 mu m; gram negative bacteria. The pure strain obtained after screening and separation is subjected to 16sRNA sequencing, and the 16sRNA gene sequence of the strain is shown in a sequence table SEQ ID No. 1:
AGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGAGTGGAGCTTGCTCCATGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCGGGGGACCTTCGGACCTCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTATGGCAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGGCTAATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCATTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTAAGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC。
the cell was identified as belonging to Pseudomonas stutzeri (Pseudomonas stutzeri) by comparison and was designated as Pseudomonas stutzeri VTCQ-1.
The morphology of the pseudomonas aeruginosa VTS-1 is characterized as follows: the colony is round, white, matt in surface and neat in edge. Microscopic examination shows that the Bacillus pumilus has a length of 0.66-1.31 μm and a width of 0.4-0.51 μm, and is gram-negative.
Through a series of creative labor, the inventor of the invention discovers that pseudomonas stutzeri VTCQ-1 easily grows surface-wrinkled colonies in a culture medium in liquid and solid, and the colony morphological characteristics lead the colonies to have the characteristic of larger specific surface area, so that larger and stable attachment sites can be provided for other bacteria; after the pseudomonas stutzeri VTCQ-1 colony is mixed with two bacterial bodies of pseudomonas aeruginosa VTS-1, the pseudomonas stutzeri VTCQ-1 colony wraps the pseudomonas aeruginosa VTS-1, so that the survival rate of the VTS-1 can be improved in the process of preparing the microbial inoculum by spray drying; pseudomonas stutzeri is screened and domesticated from soil environment, has good environmental adaptability, can be rapidly propagated in underground deep soil environment, has a large diffusion range in soil environment, provides more attachment sites for other composite functional bacterial agents propagated simultaneously, and is easy to form zoogloea with a large range and a large number; the adhesion of the pseudomonas stutzeri VTCQ-1 colony can adsorb functional bacteria to form zoogloea, and can adsorb functional bacteria on the surface of hydrocarbon-polluted soil, adsorb hydrocarbon on the surface of soil particles, play a role in gathering hydrocarbon substances for other composite functional bacterial agents with higher degradation efficiency in soil, can be compared with the bacterial agents into a flowing restaurant for degrading hydrocarbon in soil by the soil remediation functional bacterial agents, and can infinitely expand the operating range of the flowing restaurant.
Preferably, the density of the pseudomonas aeruginosa VTS-1 bacterial cells in the composite bacterial agent is 8.0x10 11 -9.45×10 11 Each/g.
In addition, the VTS-1 fermentation process can generate a surfactant, so that the surface tension of liquid can be reduced, the release of petroleum hydrocarbon on the surface of soil particles is facilitated, and the degradation of the hydrocarbon by thalli is promoted. In some embodiments, the VTS-1 fermentation process produces rhamnolipid surfactant ingredients, mainly including rhamnolipids of mono-C10, C12 and disaccharide C10, C12 structures.
Some embodiments of the invention provide a preparation method of a composite functional microbial inoculum, which comprises the step of continuously mixed culturing pseudomonas stutzeri VTCQ-1 and pseudomonas aeruginosa VTS-1.
Preferably, the medium for continuous mixed culture described above comprises: 30-50g/L of glucose, 12-17g/L of sodium nitrate, 6-7g/L of potassium dihydrogen phosphate, 4-5g/L of disodium hydrogen phosphate and 1.5-2.5g/L of peptone.
Preferably, the pH of the medium is 7.0-7.2.
Preferably, 15-20ml/L biodiesel is also added into the culture medium.
The invention uses the biodiesel as a part of the carbon source of the pseudomonas aeruginosa VTS-1 fermentation medium to carry out thallus fermentation culture, which not only saves cost, but also adapts to the degradation of petroleum hydrocarbon by hydrocarbon degrading bacteria in the fermentation stage, and the degradation effect on target pollutants in soil is more obvious after the biodiesel is applied to hydrocarbon polluted soil.
Preferably, the continuous mixed culture comprises two stages, wherein the first stage is to firstly access pseudomonas aeruginosa for culture, and the second stage is to access pseudomonas stutzeri for mixed culture after the first stage culture.
Preferably, the inoculum size of the pseudomonas aeruginosa VTS-1 is 2% -5%.
Preferably, pseudomonas stutzeri VTCQ-1 is inoculated in an amount of 2% -5%.
Preferably, the culture temperature in the first stage is: constant temperature 37 ℃, stirring speed 150-200rpm and air quantity 0.2-0.4m 3 And/min, the tank pressure is 0.03-0.05MPa, and the fermentation is continued for 51-56h.
Preferably, the culture temperature of the second stage is: constant temperature of 35 ℃, stirring speed of 180-200rpm and air quantity of 0.6-0.7m 3 And/min, the tank pressure is 0.03-0.05MPa, and the fermentation is continued for 22-24h.
In the process of culturing the two bacteria, the oil and gas removal filter is arranged at the exhaust port of the fermentation tank, so that the volatilized biodiesel before degradation can be recovered, and the atmospheric pollution caused by volatilized gas in the biodiesel can be avoided.
Preferably, the preparation method further comprises adding corn steep liquor dry powder into the mixed bacterial liquid, and then spray drying.
Preferably, the corn steep liquor dry powder is added in an amount of 170-190g/L.
Preferably, the spray drying conditions are an inlet temperature of 110-120℃and an outlet temperature of 50-60 ℃.
The invention also provides a method for in-situ remediation of the polluted soil, which comprises the step of remediating the soil to be remediated by adopting the composite functional microbial inoculum.
When the composite functional microbial inoculum is used for restoration, the composite microbial inoculum is firstly added into water according to the addition concentration of 0.5-0.8 per mill by weight to prepare microbial inoculum use liquid, and then the microbial inoculum use liquid is used for soil restoration.
Preferably, the use amount of the microbial inoculum is 25-30% of the volume of the polluted soil.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a preparation method of a composite functional microbial agent, which comprises the following steps:
s1, preparing a fermentation medium
40g of glucose, 15g of sodium nitrate, 6.5g of potassium dihydrogen phosphate, 6g of disodium hydrogen phosphate, 2g of peptone and 340ml of biodiesel are respectively added into a 30L pilot fermentation tank, and water is added to fix the volume to 17L.
In this example, an oil removal filter was installed at the exhaust port of the fermenter, and after conventional sterilization of the fermenter, the pH of the above medium was adjusted to 7.0 using sodium hydroxide with a mass concentration of 20%.
S2, culturing pseudomonas stutzeri VTCQ-1 and pseudomonas aeruginosa VTS-1
Firstly, 425ml of pseudomonas aeruginosa VTS-1 is inoculated, and the culture conditions are as follows: constant temperature 37 ℃, stirring at 190rpm and air quantity of 0.3m 3 And/min, the tank pressure is 0.04MPa, and the fermentation is continued for 54h;
after 54h of fermentation, 425ml of Pseudomonas stutzeri VTCQ-1 was inoculated into the fermenter, and the culture conditions were: constant temperature 35 ℃, stirring speed 180rpm and ventilation rate 0.65m 3 And/min, the tank pressure is 0.03MPa, fermentation culture is continued for 23 hours, and then fermentation is finished, so that mixed bacterial liquid is obtained.
When the mixed bacterial liquid is placed in the tank, the surface tension of the mixed bacterial liquid is detected by a surface tension meter, the bacterial liquid has reduced surface tension, the bacterial liquid contains surface active substances, after the bacterial liquid is centrifugally concentrated, the fermentation liquid is detected by a further liquid phase-mass spectrometer, and the fermentation liquid contains rhamnolipid surfactant components mainly comprising R1, R3 and R4 rhamnolipid. As a result, the relative ratio between the respective rhamnolipid configurations in the sample was about R1: r3 type: r4 type = 27:31:15, the proportion of R1 type rhamnolipid is calculated to be 4.2-4.6%, the proportion of R3 type rhamnolipid is calculated to be 4.8-5.2%, and the proportion of R4 type rhamnolipid is calculated to be 1.2-1.6%. The rhamnolipid accounts for 10.3-10.7% of the total amount of the sample. As shown in fig. 1 below.
S3, spray drying bacteria preparation
Adding 3.2kg of corn steep liquor dry powder into the mixed bacterial liquid in the step S2, dissolving and mixing uniformly, and performing high-temperature spray drying under the spray drying conditions: the inlet temperature is 115 ℃, the outlet temperature is 55 ℃, the brown yellow powdery microbial inoculum product is obtained after spray drying, and the cell density of the diluted plate count pseudomonas aeruginosa VTS-1 is 9.45 multiplied by 10 11 About one/g.
Example 2
The embodiment provides a preparation method of a composite functional microbial agent, which comprises the following steps:
s1, preparing a fermentation medium
To 30m 3 Adding 40kg of glucose, 15kg of sodium nitrate, 6.5kg of potassium dihydrogen phosphate, 6kg of disodium hydrogen phosphate, 2kg of peptone and 360L of biodiesel into a pilot fermentation tank, and adding water to constant volume to 18m 3 。
In this example, an oil removal filter was installed at the exhaust port of the fermenter, and after conventional sterilization of the fermenter, the pH of the above medium was adjusted to 7.0 using sodium hydroxide with a mass concentration of 20%.
S2, culturing pseudomonas stutzeri VTCQ-1 and pseudomonas aeruginosa VTS-1
First access 0.4m 3 Pseudomonas aeruginosa VTS-1 under the following culture conditions: constant temperature 37 ℃, stirring at 200rpm and air quantity of 0.3m 3 And/min, the tank pressure is 0.04MPa, and the fermentation is continued for 54h;
fermenting for 54h, and then introducing 0.4m into a fermentation tank 3 Pseudomonas stutzeri VTCQ-1, the culture conditions at this time are: constant temperature 35 ℃, stirring speed 180rpm and ventilation rate 0.65m 3 And/min, the tank pressure is 0.03MPa, fermentation culture is continued for 23 hours, and then fermentation is finished, so that mixed bacterial liquid is obtained.
S3, spray drying bacteria preparation
Adding 3.42 tons of corn steep liquor dry powder into the mixed bacterial liquid in the step S2, dissolving and mixing uniformly, and performing high-temperature spray drying under the spray drying conditions: the inlet temperature is 115 ℃, the outlet temperature is 55 ℃, the brown yellow powdery microbial inoculum product is obtained after spray drying, and the cell density of the diluted plate count pseudomonas aeruginosa VTS-1 is 9.45 multiplied by 10 11 About one/g.
Example 3
The embodiment provides a method for in-situ remediation of contaminated soil, which comprises the following steps:
adding the microbial inoculum product obtained in the example 1 into water according to the dosage of 0.5 per mill, and uniformly stirring to obtain microbial inoculum use solution;
the contaminated soil (average petroleum hydrocarbon content at three-dimensional sampling points within the contaminated range 1328 mg/m) retrieved from the underground of a gas station 3 ) Placing into a glass column with the diameter of 0.25m and the height of 1.0m, and compacting to be close to the original existing state of the soil. The metal pipe with holes on the pipe wall is used for punching holes from the middle and inserting into the soil center in the device, and 40L of microbial inoculum use liquid is injected. And periodically detecting the cell density and the petroleum hydrocarbon content of the multi-site soil sample.
After the microbial inoculum is added into soil, two types of bacteria are dominant bacterial groups, and the bacterial density is from the initial 10 5 Up to 10 per gram 8 The cell density is maintained at 6.8X10 after 1-12 months after only 7 days per gram 7 -7.5×10 8 The two bacteria are always taken as dominant bacteria; the hydrocarbon treatment rate is 91.36% after 12 months, and the treatment rate of the pseudomonas aeruginosa agent is 86.36% when being singly used, which is improved by about 5% compared with the hydrocarbon degradation bacteria when being singly used.
Example 4
The embodiment provides a method for in-situ remediation of contaminated soil, which comprises the following steps:
adding the microbial inoculum product obtained in the example 2 into water according to the dosage of 0.4 per mill, and uniformly stirring to obtain microbial inoculum use solution;
the contaminated soil (average petroleum hydrocarbon content of 1418mg/m at three-dimensional sampling points in the contaminated area) retrieved from the underground of a gas station was then recovered 3 ) Put into a reactor with the diameter of 0.25m and the height of 1.0m, compaction is close to the original existing state of the soil. A metal pipe with a plurality of holes on the pipe wall is inserted into the soil center in the device from the middle hole, 27% of microbial inoculum use liquid is injected according to the calculated pollution range volume, and the multi-site soil sample is periodically detected for the cell density and the petroleum hydrocarbon content.
Cell density in soil after 7 days of injection of microbial inoculum is 2.5X10 5 The number per gram is increased to 8.9X10 8 The density of the soil bacterial cells per gram at 12 months is 8.25 multiplied by 10 7 The hydrocarbon treatment rate per gram and 12 months is 92.3 percent.
Comparative example 1
Pseudomonas aeruginosa VTS-1 is singly inoculated into a culture medium for culture, and the obtained bacterial liquid is subjected to spray drying to prepare a bacterial agent after tank placing, and the components of the culture medium, the culture conditions and the spray drying strips are the same as those of example 1. The bacterial density of the pseudomonas aeruginosa VTS-1 in the prepared bacterial agent is 9.10X10 11 About one/g.
Comparative example 2
The Pseudomonas stutzeri VTCQ-1 and Pseudomonas aeruginosa VTS-1 are simultaneously inoculated into a culture medium for culture, the obtained bacterial liquid is subjected to spray drying to prepare a bacterial agent after tank placing, the culture medium components and the spray drying conditions are the same as those of example 1, the culture conditions are constant at 37 ℃, the stirring speed is 200rpm, and the air quantity is 0.3m 3 And/min, the tank pressure is 0.04MPa, and the fermentation is continued for 48 hours. Bacterial density of pseudomonas aeruginosa VTS-1 in bacterial agent prepared under culture conditions is 7.24 multiplied by 10 11 About one gram per gram, the content of the surface active substances in the bacterial liquid is low.
Comparative example 3
Mixing and culturing pseudomonas stutzeri VTCQ-1 and bacillus megatherium, wherein 17L of culture medium comprises the following components: 40g of glucose, 15g of ammonium chloride, 6.5g of monopotassium phosphate, 6g of dipotassium phosphate, 2g of peptone and 2g of yeast extract powder, wherein the culture conditions are as follows: constant temperature 35 ℃, stirring at 200rpm and air quantity of 0.4m 3 The culture period is 18-22 hours, the spray drying condition is the same as example 1, and the culture effect is that the density of bacillus megatherium thallus in the microbial inoculum is 4.25X10 11 The number per gram is 11 percent higher than that of single culture of bacillus megatherium, and the single culture of bacillus megatherium has the cell density of 3.78X10 11 About one/g.
Comparative example 4
Contaminated soil was repaired in situ using the microbial inoculum prepared in comparative example 1, and the repair method was the same as in example 3. The treatment rate of the microbial inoculum on hydrocarbon is 86.36% 12 months after the microbial inoculum is added into soil.
In conclusion, the invention realizes the multiplication of the pseudomonas aeruginosa VTS-1 by taking biodiesel as a carbon source in the fermentation culture process and can generate the rhamnolipid surfactant, and the VTS-1 pseudomonas aeruginosa is spray-dried by taking the VTCQ-1 as a carrier, the bacterial density of the bacterial agent is 3 percent higher than the survival rate of the single spray-dried pseudomonas aeruginosa, and the total bacterial density of two strains after spray drying is 9.45 multiplied by 10 11 Individual/g; the microbial inoculum is added into petroleum hydrocarbon soil, and the density of the microbial inoculum in the soil is from the initial 10 5 Up to 10 per gram 8 The cell density is maintained at 6.8X10 at 1-12 months after 7 days per g 7 -8.25×10 8 Two bacteria are always taken as dominant bacteria per gram; the treatment rate of 12 moons is 91.36%, which is at least 5% higher than that of VTS-1 used alone and higher than that of other bacteria agents reported at present.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Beijing Wo Taisi environmental protection technology development Co., ltd
<120> composite functional microbial inoculum for in-situ remediation of contaminated soil by taking pseudomonas stutzeri as carrier and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1383
<212> DNA
<213> Pseudomonas stutzeri
<400> 1
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gacactgagg tgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc 780
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aagtcgaccg cctggggagt acggccgcaa ggttaaaact caaatgaatt gacgggggcc 900
cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggcct 960
tgacatgcag agaactttcc agagatggat tggtgccttc gggaactctg acacaggtgc 1020
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acc 1383
Claims (12)
1. A composite functional microbial agent is characterized by comprising pseudomonas stutzeri VTCQ-1 and pseudomonas aeruginosa VTS-1;
the preservation number of the pseudomonas stutzeri VTCQ-1 is CGMCC24469;
the preservation number of the pseudomonas aeruginosa VTS-1 is CGMCC2200;
the Pseudomonas stutzeri VTCQ-1 is used as a carrier of the Pseudomonas aeruginosa VTS-1.
2. A preparation method of a composite functional microbial inoculum is characterized by comprising the step of continuously mixing and culturing pseudomonas stutzeri VTCQ-1 and pseudomonas aeruginosa VTS-1 according to claim 1.
3. The method for preparing the composite functional bacterial agent according to claim 2, wherein the mixed bacterial liquid is obtained through the continuous mixed culture, the continuous mixed culture comprises two stages, the first stage is to firstly insert pseudomonas aeruginosa for culture, and the second stage is to insert pseudomonas stutzeri for mixed culture on the basis of the first stage culture.
4. The method for preparing the composite functional microbial inoculum according to claim 3 wherein the inoculation amount of pseudomonas aeruginosa is 2% -5%.
5. The method for preparing the composite functional microbial agent according to claim 4, wherein the inoculation amount of the pseudomonas stutzeri is 2% -5%.
6. The method for preparing a composite functional bacterial agent according to claim 5, wherein the culture temperature in the first stage is: constant temperature 3At 7deg.C, stirring speed of 150-200rpm and air volume of 0.2-0.4m 3 The pressure of the tank is 0.03-0.05MPa, and the fermentation is continued for 51-56 h;
the culture temperature of the second stage is as follows: constant temperature of 35 ℃, stirring speed of 180-200rpm and air quantity of 0.6-0.7m 3 And/min, the tank pressure is 0.03-0.05MPa, and the fermentation is continued for 22-24h.
7. The method for preparing a composite functional bacterial agent according to claim 5, further comprising adding corn steep liquor dry powder to the mixed bacterial liquid and then spray drying.
8. The method for preparing the composite functional microbial agent of claim 7, wherein the addition amount of the corn steep liquor dry powder is 170-190g/L.
9. The method for preparing the composite functional microbial agent according to claim 8, wherein the spray drying condition is that the inlet temperature is 110-120 ℃ and the outlet temperature is 50-60 ℃.
10. A method for in-situ remediation of contaminated soil, comprising remediating soil to be remediated using the composite functional microbial inoculum of claim 1, wherein the soil is petroleum hydrocarbon contaminated soil.
11. The method for in-situ remediation of contaminated soil according to claim 10, comprising adding the composite microbial inoculum to water at an addition concentration of 0.5-0.8%o by weight to prepare a microbial inoculum use solution, and using the microbial inoculum use solution for soil remediation.
12. The method of in situ remediation of contaminated soil of claim 11 wherein the microbial inoculum is used in an amount of 25-30% by volume of contaminated soil.
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