CN112646724A - Preparation method of compound bacterial liquid preparation - Google Patents
Preparation method of compound bacterial liquid preparation Download PDFInfo
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- CN112646724A CN112646724A CN202110027754.6A CN202110027754A CN112646724A CN 112646724 A CN112646724 A CN 112646724A CN 202110027754 A CN202110027754 A CN 202110027754A CN 112646724 A CN112646724 A CN 112646724A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 55
- 239000007788 liquid Substances 0.000 title claims abstract description 34
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 16
- 150000001875 compounds Chemical class 0.000 title claims abstract description 16
- 241000122973 Stenotrophomonas maltophilia Species 0.000 claims abstract description 39
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 claims abstract description 21
- 238000000855 fermentation Methods 0.000 claims abstract description 20
- 230000004151 fermentation Effects 0.000 claims abstract description 20
- 230000015556 catabolic process Effects 0.000 claims abstract description 17
- 238000006731 degradation reaction Methods 0.000 claims abstract description 17
- 230000004913 activation Effects 0.000 claims abstract description 16
- 235000015097 nutrients Nutrition 0.000 claims abstract description 14
- 238000012807 shake-flask culturing Methods 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims abstract description 7
- YNPNZTXNASCQKK-UHFFFAOYSA-N phenanthrene Chemical compound C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 239000001888 Peptone Substances 0.000 claims description 15
- 108010080698 Peptones Proteins 0.000 claims description 15
- 235000019319 peptone Nutrition 0.000 claims description 15
- 239000001963 growth medium Substances 0.000 claims description 14
- 238000011049 filling Methods 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 10
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 238000007710 freezing Methods 0.000 claims description 9
- 230000008014 freezing Effects 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 7
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 6
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 5
- QDGAVODICPCDMU-UHFFFAOYSA-N 2-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]propanoic acid Chemical compound OC(=O)C(N)CC1=CC=CC(N(CCCl)CCCl)=C1 QDGAVODICPCDMU-UHFFFAOYSA-N 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 5
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims description 5
- 239000007836 KH2PO4 Substances 0.000 claims description 5
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 claims description 5
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- 229960004889 salicylic acid Drugs 0.000 claims description 5
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 235000002906 tartaric acid Nutrition 0.000 claims description 5
- 239000011975 tartaric acid Substances 0.000 claims description 5
- 229960001367 tartaric acid Drugs 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- 241001052560 Thallis Species 0.000 claims description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims 1
- 239000002131 composite material Substances 0.000 abstract description 9
- 239000002068 microbial inoculum Substances 0.000 abstract description 5
- 239000002253 acid Substances 0.000 abstract 1
- 230000002035 prolonged effect Effects 0.000 abstract 1
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 239000000758 substrate Substances 0.000 abstract 1
- 239000012533 medium component Substances 0.000 description 9
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 230000000813 microbial effect Effects 0.000 description 5
- 244000005700 microbiome Species 0.000 description 4
- 239000003208 petroleum Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 241000193749 Bacillus coagulans Species 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 229940039407 aniline Drugs 0.000 description 3
- 229940054340 bacillus coagulans Drugs 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000000306 component Substances 0.000 description 3
- 230000000593 degrading effect Effects 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 150000001924 cycloalkanes Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003209 petroleum derivative Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
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- 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
- C12N1/04—Preserving or maintaining viable microorganisms
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- 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
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- Bioinformatics & Cheminformatics (AREA)
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- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention discloses a preparation method of a compound bacterial liquid preparation, which comprises the steps of firstly, obtaining stenotrophomonas maltophilia fermentation liquor through strain activation, seed liquid preparation and shake flask culture; then preparing a microecological preparation; and preparing inorganic nutrient solution, finally adding the stenotrophomonas maltophilia microecological preparation into the inorganic nutrient solution, and regulating the pH to 6.0-7.0 while stirring to obtain the compound bacterial liquid preparation. The composite microbial inoculum is added with small molecular acid and a co-metabolism substrate, so that the degradation period of strains is shortened, the polycyclic aromatic hydrocarbon degradation capability of the strains in unit time and the adaptability of the strains in an industrial environment are improved, the viable count of the composite microbial inoculum is kept at a higher level of 95-105 hundred million cfu/ml, the storage time is prolonged, and the polycyclic aromatic hydrocarbon degradation rate reaches 85%.
Description
Technical Field
The invention relates to a preparation method of a compound bacterial liquid preparation, in particular to a preparation method of a polycyclic aromatic hydrocarbon degrading bacterial preparation, which can be used for polycyclic aromatic hydrocarbon degrading treatment in polluted areas to guide biotechnology research and repair polluted areas or polycyclic aromatic hydrocarbon pollutant treatment, and belongs to the field of bioengineering.
Technical Field
Petroleum is an indispensable part of mineral resources and is widely used in the fields of manufacturing industry, agriculture, and medicine. With the increasing development speed of the petroleum industry, the application range of petrochemical products is continuously expanded, and the problem of petroleum pollution is increasingly serious. The petroleum hydrocarbons include alkanes, cycloalkanes, and aromatics, of which polycyclic aromatic hydrocarbons are the most toxic.
Compared with foreign oil fields, the development of oil fields in China is inseparable and staggered with urban construction, so that the influence of oil field polluted soil on ecological environment and human health is more sensitive and prominent. The removal of Polycyclic Aromatic Hydrocarbons (PAHs) comprises a biological treatment means besides common physical and chemical methods. The microbial degradation method gradually becomes a main research direction for petroleum hydrocarbon degradation because of the advantages of lower cost, convenient operation and the like compared with other degradation methods.
The configuration and optimization of the composite bacterial liquid are key technologies for industrial treatment of polycyclic aromatic hydrocarbon pollution. The existing biological method for treating polycyclic aromatic hydrocarbon pollution generally has various problems, a single strain is weak in adaptability and poor in stress resistance, and is always in a weak position in competition with indigenous microorganisms, so that the development of polycyclic aromatic hydrocarbon microbial degradation is severely restricted.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provide a preparation method of a compound bacterial liquid preparation for degrading polycyclic aromatic hydrocarbons.
The technical scheme of the invention is as follows: a preparation method of a compound bacteria liquid preparation comprises the following specific steps:
(1) the stenotrophomonas maltophilia fermentation broth is obtained by strain activation (the strain is commercially available stenotrophomonas maltophilia CGMCC 1.14975), preparation of seed solution and shake flask culture;
(2) preparing a microecological preparation: centrifuging the fermentation liquor, removing supernatant, collecting centrifuged thalli, uniformly mixing the stenotrophomonas maltophilia thalli, trehalose and skim milk according to the volume ratio of (1.5-2) to 1, pre-freezing a sample in an ultra-low temperature refrigerator, and putting the sample into a freeze dryer for vacuum freeze drying to obtain the stenotrophomonas maltophilia microecological preparation, wherein the degradation rate of polycyclic aromatic hydrocarbon of the target microecological preparation reaches 60-70%;
(3) preparing an inorganic nutrient solution: MgSO (MgSO)4Aniline, L-phenylalanine, tartaric acid, salicylic acid, KH2PO4And water according to the mass ratio (2-4): (2-4): (1-2): (1-2): (1-2): (1-2): 10000 of the raw materials are mixed, stirred evenly, fully dissolved, kept stand and cooled to room temperature to obtain a mixed solution;
(4) preparing a compound bacterial liquid preparation: adding the stenotrophomonas maltophilia microecological preparation obtained in the step (2) into the inorganic nutrient solution prepared in the step (3), wherein the volume ratio of the added mass of the stenotrophomonas maltophilia microecological preparation to the inorganic nutrient solution is 10-15 mg/mL; adjusting pH to 6.0-7.0 under stirring to obtain compound bacterial liquid preparation.
The strain is commercially available stenotrophomonas maltophilia, and the number of the stenotrophomonas maltophilia preserved in the China Committee for culture Collection of microorganisms is CGMCC 1.14975.
Preferably, the strain activation in the step (1) is as follows: transferring the stenotrophomonas maltophilia single colony to a seed slant culture medium, and culturing at the activation culture temperature of 28-30 ℃ for 24-30 h; wherein the seed slant culture medium comprises the following components: 100 portions of phenanthrene, 5 to 10g/L of peptone, 1 to 5g/L of sodium chloride and 10 to 25g/L of agar.
Preferably, the preparation of the seed solution in the step (1) is: inoculating activated stenotrophomonas maltophilia into a seed culture medium with the filling volume of 15-20%, controlling the pH at 6.0-7.0, the temperature at 28-3 ℃, and the rotating speed at 130-160r/min, and culturing for 16-20h to obtain a seed solution; wherein the seed culture medium comprises the following components: 100 portions of phenanthrene, 5 to 10 portions of peptone, 1 to 5 portions of yeast extract and 1 to 5 portions of sodium chloride.
Preferably, the shake flask culture in step (1) is: according to the volume inoculation amount of the seed liquid and the fermentation liquid being 10-40%, the seed liquid is filled into a container with 15-20% of the volume filling amount of the fermentation liquid, the container is placed in a shaking table, shaking culture is carried out for 10-14h at the temperature of 28-30 ℃, and the rotating speed is 160 r/min; wherein the seed liquid fermentation medium comprises the following components: 100 portions of phenanthrene, 5 to 10 portions of peptone, 1 to 2 portions of monopotassium phosphate and 1 to 2 portions of dipotassium phosphate.
Preferably, the centrifugal speed in the step (2) is 4000-; the pre-freezing temperature is-60 ℃ to-80 ℃, and the pre-freezing time is 5-7 h; vacuum degree of vacuum freeze drying is 40-60 Pa.
Preferably, the number of the target compound bacterial liquid stenotrophomonas maltophilia viable bacteria in the compound bacterial liquid preparation prepared in the step (4) reaches 95-105 hundred million cfu/mL.
Has the advantages that:
the composite microbial inoculum has the protective agent, enhances the weak adaptability and poor stress resistance of the composite microbial inoculum, enhances the competitiveness of the composite microbial inoculum and indigenous microorganisms due to the existence of an exogenous additive, effectively reduces the degradation cost, and lays a solid foundation for the industrial application of the polycyclic aromatic hydrocarbon microorganism degradation.
Detailed Description
The present invention is further explained by the following examples, which are not intended to limit the present invention in any way.
Example 1:
activating strains: transferring the single colony of stenotrophomonas maltophilia CGMCC1.14975 to a seed slant culture medium, and culturing at the activation culture temperature of 28 ℃ for 24h to obtain a seed solution; wherein the solid activation medium component (g/L): 120mg/L phenanthrene, 5g/L peptone, 1g/L sodium chloride and 13g/L agar.
Preparing a seed solution: inoculating activated stenotrophomonas maltophilia into a seed culture medium with the filling volume of 15%, controlling the pH at 6.0, the temperature at 28 ℃, and the rotating speed at 150r/min, and culturing for 16h to obtain a seed solution; wherein the activation medium component (g/L): 110mg/L phenanthrene, 5g/L peptone, 2g/L yeast extract and 1g/L sodium chloride;
and (3) shake flask culture: taking 1mL of seed solution, filling the seed solution into a 50mL triangular flask with the fermentation liquid filling volume of 15%, placing the triangular flask in a shaking table, and carrying out shake culture at 28 ℃ for 11h at the rotating speed of 130 r/min; wherein the fermentation medium components (g/L): 110g/mg/L phenanthrene, 6g/L peptone, 1.2g/L potassium dihydrogen phosphate and 1.3g/L dipotassium hydrogen phosphate;
preparing a microecological preparation: centrifuging the fermentation liquor at 4000r/min for 15min, removing supernatant, collecting centrifuged thallus, uniformly mixing stenotrophomonas maltophilia, trehalose and skim milk according to the volume ratio of 1.5:1.5:1, pre-freezing the sample in an ultralow temperature refrigerator at-60 ℃ for 5h, putting the sample into a freeze dryer for vacuum freeze drying at the vacuum degree of 40Pa to obtain a bacillus coagulans microecological preparation, wherein the polycyclic aromatic hydrocarbon degradation rate of the target microecological preparation reaches 62%;
MgSO4, aniline, L-phenylalanine, tartaric acid, salicylic acid, KH2PO4 and water are mixed according to a mass ratio of 2: 4: 1: 1.5: 1.3: 1: 10000 of the raw materials are mixed, stirred evenly, fully dissolved, kept stand and cooled to room temperature to obtain a mixed solution;
and adding the stenotrophomonas maltophilia microecological preparation into the inorganic nutrient solution, wherein the ratio of the added mass of the stenotrophomonas maltophilia microecological preparation to the volume of the inorganic nutrient solution is 10mg/mL, the pH is adjusted to 6.3 while stirring, the number of viable bacteria of the target composite microbial agent stenotrophomonas maltophilia reaches 95cfu/mL, and the degradation rate of polycyclic aromatic hydrocarbon reaches 87%.
Example 2:
activating strains: transferring the single colony of stenotrophomonas maltophilia CGMCC1.14975 to a seed slant culture medium, and culturing at the activation culture temperature of 29 ℃ for 26h to obtain a seed solution; wherein the solid activation medium component (g/L): 130mg/L phenanthrene, 7g/L peptone, 3g/L sodium chloride and 18g/L agar.
Preparing a seed solution: inoculating activated stenotrophomonas maltophilia into a seed culture medium with the filling volume of 17%, controlling the pH at 6.5, the temperature at 29 ℃, and the rotating speed at 140r/min, and culturing for 18h to obtain a seed solution; wherein the activation medium component (g/L): 130mg/L phenanthrene, 7g/L peptone, 3g/L yeast extract and 3g/L sodium chloride;
and (3) shake flask culture: taking 2mL of seed liquid, filling the seed liquid into a 50mL triangular flask with 17% of fermentation liquid filling volume, placing the triangular flask in a shaking table, and carrying out shake culture at 29 ℃ for 12h at the rotating speed of 140 r/min; wherein the fermentation medium components (g/L): 130g/mg/L phenanthrene, 7g/L peptone, 1.5g/L potassium dihydrogen phosphate and 1.6g/L dipotassium hydrogen phosphate;
preparing a microecological preparation: centrifuging the fermentation liquor for 13min at 5000r/min, discarding the supernatant, collecting the centrifuged thallus, uniformly mixing stenotrophomonas maltophilia, trehalose and skim milk according to the volume ratio of 1.7:1.7:1, pre-freezing the sample in an ultra-low temperature refrigerator at-70 ℃ for 6h, putting the sample into a freeze dryer for vacuum freeze drying at the vacuum degree of 50Pa to obtain the bacillus coagulans microecological preparation, wherein the polycyclic aromatic hydrocarbon degradation rate of the target microecological preparation reaches 66%;
MgSO4, aniline, L-phenylalanine, tartaric acid, salicylic acid, KH2PO4 and water are mixed according to the mass ratio of 3: 3: 1.2: 1.5: 1.6: 1.3: 10000 of the raw materials are mixed, stirred evenly, fully dissolved, kept stand and cooled to room temperature to obtain a mixed solution;
and adding the stenotrophomonas maltophilia microecological preparation into the inorganic nutrient solution, wherein the ratio of the added mass of the stenotrophomonas maltophilia microecological preparation to the volume of the inorganic nutrient solution is 15mg/mL, the pH is adjusted to 6.7 while stirring, the number of viable bacteria of the target composite microbial agent stenotrophomonas maltophilia reaches 98cfu/mL, and the degradation rate of the polycyclic aromatic hydrocarbon reaches 89%.
Example 3:
(1) activating strains: transferring the single colony of stenotrophomonas maltophilia CGMCC1.14975 to a seed slant culture medium, and culturing at the activation culture temperature of 30 ℃ for 29h to obtain a seed solution; wherein the solid activation medium component (g/L): phenanthrene 150mg/L, peptone 9g/L, sodium chloride 4g/L, agar 24 g/L.
Preparing a seed solution: inoculating activated stenotrophomonas maltophilia into a seed culture medium with a filling volume of 19%, controlling the pH at 7.0, the temperature at 30 ℃ and the rotating speed at 160r/min, and culturing for 20h to obtain a seed solution; wherein the activation medium component (g/L): 150mg/L phenanthrene, 9g/L peptone, 4g/L yeast extract and 5g/L sodium chloride;
and (3) shake flask culture: taking 3mL of seed solution, filling the seed solution into a 50mL triangular flask with the fermentation liquid filling volume of 20%, placing the triangular flask in a shaking table, and carrying out shake culture at 30 ℃ for 13h at the rotating speed of 160 r/min; wherein the fermentation medium components (g/L): 150g/mg/L phenanthrene, 9g/L peptone, 1.8g/L potassium dihydrogen phosphate and 1.9g/L dipotassium hydrogen phosphate;
preparing a microecological preparation: centrifuging the fermentation liquor at 6000r/min for 10min, removing supernatant, collecting centrifuged thallus, uniformly mixing stenotrophomonas maltophilia, trehalose and skim milk according to the volume ratio of 1.9:1.9:1, pre-freezing a sample in an ultra-low temperature refrigerator at-80 ℃ for 7h, putting the sample into a freeze dryer for vacuum freeze drying at the vacuum degree of 60Pa to obtain a bacillus coagulans microecological preparation, wherein the degradation rate of polycyclic aromatic hydrocarbon of the target microecological preparation reaches 69%;
MgSO4, aniline, L-phenylalanine, tartaric acid, salicylic acid, KH2PO4 and water are mixed according to a mass ratio of 4: 2: 2: 1.5: 1.8: 2: 10000 of the raw materials are mixed, stirred evenly, fully dissolved, kept stand and cooled to room temperature to obtain a mixed solution;
and adding the stenotrophomonas maltophilia microecological preparation into the inorganic nutrient solution, wherein the ratio of the added mass of the stenotrophomonas maltophilia microecological preparation to the volume of the inorganic nutrient solution is 13mg/mL, the pH is adjusted to 7.0 while stirring, the number of viable bacteria of the target composite microbial agent stenotrophomonas maltophilia reaches 102cfu/mL, and the degradation rate of the polycyclic aromatic hydrocarbon reaches 93%.
Claims (6)
1. A preparation method of a compound bacteria liquid preparation comprises the following specific steps:
(1) obtaining stenotrophomonas maltophilia fermentation broth through strain activation, preparation of seed solution and shake culture;
(2) preparing a microecological preparation: centrifuging the fermentation liquor, removing supernatant, collecting centrifuged thalli, uniformly mixing the stenotrophomonas maltophilia thalli, trehalose and skim milk according to the volume ratio of (1.5-2) to 1, pre-freezing a sample in an ultra-low temperature refrigerator, and putting the sample into a freeze dryer for vacuum freeze drying to obtain the stenotrophomonas maltophilia microecological preparation, wherein the degradation rate of polycyclic aromatic hydrocarbon of the target microecological preparation reaches 60-70%;
(3) preparing an inorganic nutrient solution: MgSO (MgSO)4Aniline, L-phenylalanine, tartaric acid, salicylic acid, KH2PO4And water according to the mass ratio (2-4): (2-4): (1-2): (1-2): (1-2): (1-2): 10000 of mixing, stirring uniformly, fully dissolving, standing and cooling to obtain a mixed solution;
(4) preparing a compound bacterial liquid preparation: adding the stenotrophomonas maltophilia microecological preparation obtained in the step (2) into the inorganic nutrient solution prepared in the step (3), wherein the volume ratio of the added mass of the stenotrophomonas maltophilia microecological preparation to the inorganic nutrient solution is 10-15 mg/mL; adjusting pH to 6.0-7.0 under stirring to obtain compound bacterial liquid preparation.
2. The method of claim 1, wherein: the strain activation in the step (1) is as follows: transferring the stenotrophomonas maltophilia single colony to a seed slant culture medium, and culturing at the activation culture temperature of 28-30 ℃ for 24-30 h; wherein the seed slant culture medium comprises the following components: 100 portions of phenanthrene, 5 to 10g/L of peptone, 1 to 5g/L of sodium chloride and 10 to 25g/L of agar.
3. The method of claim 1A, wherein: the preparation of the seed liquid in the step (1) comprises the following steps: inoculating activated stenotrophomonas maltophilia into a seed culture medium with the filling volume of 15-20%, controlling the pH at 6.0-7.0, the temperature at 28-3 ℃, and the rotating speed at 130-160r/min, and culturing for 16-20h to obtain a seed solution; wherein the seed culture medium comprises the following components: 100 portions of phenanthrene, 5 to 10 portions of peptone, 1 to 5 portions of yeast extract and 1 to 5 portions of sodium chloride.
4. The method of claim 1A, wherein: the shake flask culture in the step (1) comprises the following steps: according to the volume inoculation amount of the seed liquid and the fermentation liquid being 10-40%, the seed liquid is filled into a container with 15-20% of the volume filling amount of the fermentation liquid, the container is placed in a shaking table, shaking culture is carried out for 10-14h at the temperature of 28-30 ℃, and the rotating speed is 160 r/min; wherein the seed liquid fermentation medium comprises the following components: 100 portions of phenanthrene, 5 to 10 portions of peptone, 1 to 2 portions of monopotassium phosphate and 1 to 2 portions of dipotassium phosphate.
5. The method of claim 1, wherein: the centrifugal rotating speed in the step (2) is 4000-; the pre-freezing temperature is-60 ℃ to-80 ℃, and the pre-freezing time is 5-7 h; vacuum degree of vacuum freeze drying is 40-60 Pa.
6. The method of claim 1, wherein: the number of the target compound bacterial liquid stenotrophomonas maltophilia viable bacteria in the compound bacterial liquid preparation prepared in the step (4) reaches 95-105 hundred million cfu/mL.
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