CN108707561A - Complex microorganism preparations and its preparation method and application for rehabilitating soil - Google Patents
Complex microorganism preparations and its preparation method and application for rehabilitating soil Download PDFInfo
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- CN108707561A CN108707561A CN201810513636.4A CN201810513636A CN108707561A CN 108707561 A CN108707561 A CN 108707561A CN 201810513636 A CN201810513636 A CN 201810513636A CN 108707561 A CN108707561 A CN 108707561A
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Abstract
The invention discloses a kind of complex microorganism preparations for rehabilitating soil, the complex microorganism preparations include coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum, Corynebacterium glutamicum ATCC13032 microbial inoculums and denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums.The invention also discloses the preparation method and application of the complex microorganism preparations for rehabilitating soil.Inventor by above-mentioned three kinds of microorganism formulations by being used in combination discovery, effect in terms of the denitrogenation phosphorus and in terms of heavy metal adsorption be far superior to individually with effect that two kinds of microbial inoculums are used in combination two-by-two respectively, the removal rate that ammonia nitrogen removal frank has reached 95% or more, P reaches 95%, heavy metal Cu, the removal rate of Cd while also having reached 96% or more.
Description
Technical solution
The invention belongs to technical field of soil remediation, and in particular to be used for the complex microorganism preparations and its system of rehabilitating soil
Preparation Method and application.
Background technology
Soil be in air, water body and solid waste pollutant migrate in the environment, the target of direct current and deposition, be
Long-range circumstances pollution subjects, and as social economy and industrialization development accelerate, certain industrial wastewaters cause a soil huge sum of money
Belong to pollution, ammonia and nitrogen pollution, organic pollution etc..
For the recovery technique and method of soil pollution, foreign countries have begun working on from the beginning of the eighties at the end of the seventies, initial stage
The general reparation that contaminated soil is carried out using physics and chemical method, such as heat treating process and Chemical Leaching method, but somewhat expensive, no
Suitable for large-area applications.The end of the eighties and the initial stage nineties, foreign countries begin one's study based on physics, the microorganism of chemical process, plant
The biological renovation method of object catabolism soil pollution, developed in recent years it is very fast, not only compared with physics, chemical method economy,
It is not easy to produce secondary pollution simultaneously, is more suitable for the reparation of large area soil, and bioremediation technology has begun as soil
The main treatment technology of earth reparation, and more and more important role is played, but current recovery technique can have ammonia nitrogen
The problems such as removal rate is low, and microorganism is unstable, enzymatic treatment is easy inactivation, it would therefore be highly desirable to weight in comprehensively solve and rehabilitating soil
The problems such as metallic pollution, ammonia and nitrogen pollution and organic pollution.
Invention content
Goal of the invention：The object of the present invention is to provide a kind of complex microorganism preparations for soil remediation, this is compound micro-
Biological agent is mainly used for removing the pollution of ammonia nitrogen, phosphorus, heavy metal etc. in soil.
Another object of the present invention is to provide the preparation method of the above-mentioned complex microorganism preparations for soil remediation and
Using.
Technical solution：The present invention provides a kind of complex microorganism preparations for rehabilitating soil, the complex microorganism systems
Agent includes coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum, Corynebacterium glutamicum ATCC13032 microbial inoculums and denitrogenation
Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums.
Wherein, coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum, Corynebacterium glutamicum
The weight of ATCC13032 microbial inoculums and denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620
Than being 0.1~2:1~5:3~7.
Wherein, the viable bacteria content of coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum is 3 × 108~5
×1010The viable bacteria content of cfu/g, the Corynebacterium glutamicum ATCC13032 microbial inoculums are 2 × 109~5 × 1010Cfu/g, it is described
The viable bacteria content of denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 is 5 × 108~7
×1010cfu/g。
The content of present invention further includes a kind of method preparing the complex microorganism preparations for rehabilitating soil, the party
Method includes the following steps：By coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum, Corynebacterium glutamicum
ATCC13032 microbial inoculums and denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums are pressed
0.1~2:1~5:3~7 mass ratio is uniformly mixed to obtain the final product.
Wherein, the preparation method of coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum includes：By plate-like
Logarithmic phase is arrived in spiral cyanobacteria (Spirulina platensis) culture, then presses the inoculum concentration of culture volume 3%~10%
The strain of culture to logarithmic phase is inoculated into culture medium, 30~35 DEG C, shaking table shakes 180~220rpm, and culture 40~48 is small
When, the zymotic fluid of coiled spiral cyanobacteria (Spirulina platensis) is obtained, adsorbent is added into the zymotic fluid, is done
It is dry to get coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum；The adsorbent is that bentonite or activated carbon are a kind of
Or it is several.
Wherein, the culture medium is 1.5g/L sodium acetates, 0.06g/L peptones, 0.8g/L sodium bicarbonates, 0.5g/L thio
Sodium sulphate, 0.3g/L sodium chloride, 0.2g/L magnesium sulfate and 0.07g/L potassium dihydrogen phosphates.
Wherein, the preparation method of the Corynebacterium glutamicum ATCC13032 microbial inoculums includes：By Corynebacterium glutamicum
ATCC13032 cultivated in LB culture mediums arrive logarithmic phase, then press culture volume 3%~6% inoculum concentration will culture to pair
The strain of number phase is inoculated into complete medium, and at 30~40 DEG C, shaking table shakes 120~180rpm, cultivates 24~48 hours, adds
Enter adsorbent, it is dry, Corynebacterium glutamicum ATCC13032 microbial inoculums are made；The adsorbent is one in bentonite or activated carbon
Kind is several.
Wherein, denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) the CGMCC No.10620 microbial inoculums
Preparation method include：Denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 are being planted
Logarithmic phase is arrived in culture in sub- culture medium, and logarithmic phase then is arrived in culture according to 5%~10% inoculum concentration of culture volume
Strain is inoculated into fermentation medium, 28~30 DEG C, and shaking table shakes 160~180rpm, is cultivated 24~48 hours, and absorption is added
Agent, it is dry, denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums are made；It is described
Adsorbent is one or more of bentonite or activated carbon.
Wherein, the seed culture medium includes：7g/L methanol, 3g/L ammonium sulfate, 2g/L potassium dihydrogen phosphates, 3g/L sulfuric acid
Magnesium, 2g/L disodium hydrogen phosphates；The fermentation medium includes：15g/L methanol, 2g/L ammonium sulfate, 3g/L potassium dihydrogen phosphates,
1.5g/L magnesium sulfate, 3g/L disodium hydrogen phosphates, 1.5g/L liquid microelements, the auxiliary liquid of 1.3g/L vitamins.
In fact, the liquid microelement includes that ferrous sulfate, zinc sulfate, manganese sulfate, calcium chloride and water are mixed to get,
A concentration of 100g/L of middle ferrous sulfate, a concentration of 20g/L of zinc sulfate, a concentration of 10g/L of manganese sulfate, calcium chloride concentration 200g/
L。
Wherein, the auxiliary liquid of the vitamin includes that folic acid, riboflavin, calcium pantothenate, biotin and water are mixed to get, the folic acid
A concentration of 3g/L, riboflavin concentration 300g/L, calcium pantothenate concentration 500g/L, biotin concentration 5g/L.
The content of present invention further includes that the complex microorganism preparations for rehabilitating soil are dirty in heavy metal, ammonia nitrogen, phosphorus
Contaminate the application in soil remediation.
Advantageous effect：The present invention is for the first time by coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum, glutamic acid rod
Bacillus ATCC13032 microbial inoculums and denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 are mixed
Microorganism formulation has been prepared in conjunction, and the microorganism formulation is by means of coiled spiral cyanobacteria (Spirulina platensis)
Microbial inoculum can effectively adsorb heavy metal in soil, while absorption ammonia nitrogen, phosphorus and realize that the self-purification function of soil, denitrogenation raw silk are micro-
Bacterium (Hyphomicrobium denitrificans) CGMCC No.10620 can be in same system by NH4 +- N is converted into harmless
Gas, while discovery is used in combination in above-mentioned three kinds of microorganism formulations by inventor, in terms of denitrogenation phosphorus and heavy metal adsorption
The effect of aspect is far superior to individually with effect that two kinds of microbial inoculums are used in combination two-by-two respectively, and ammonia nitrogen removal frank reaches
The removal rate of 95% or more, P reach 95%, heavy metal Cu, the removal rate of Cd while also having reached 96% or more.
Specific implementation mode
Coiled spiral cyanobacteria (Spirulina platensis) is presented for Institute of Microorganism, Academia Sinica in the present invention
It gives, Corynebacterium glutamicum ATCC13032 purchases are in U.S.'s ATCC cell banks, denitrogenation Hyphomicrobium (Hyphomicrobium
Denitrificans) CGMCC No.10620 purchases are in China General Microbiological bacterial strain preservation administrative center.Own in the present invention
Reagent be on the market purchase obtain.
Preparation of the embodiment 1 for the complex microorganism preparations of soil remediation
The preparation method of coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum includes：Coiled spiral is blue thin
Logarithmic phase is arrived in bacterium (Spirulina platensis) culture, and culture is arrived logarithmic phase by the inoculum concentration for then pressing culture volume 3%
Strain be inoculated into culture medium, 30 DEG C of shaking tables shake 220rpm, cultivate 48 hours, obtain coiled spiral cyanobacteria
Activated carbon is added into the zymotic fluid for the zymotic fluid of (Spirulina platensis), dry to get coiled spiral cyanobacteria
(Spirulina platensis) microbial inoculum.The culture medium is 1.5g/L sodium acetates, 0.06g/L peptones, 0.8g/L bicarbonates
Sodium, 0.5g/L sodium thiosulfate, 0.3g/L sodium chloride, 0.2g/L magnesium sulfate and 0.07g/L potassium dihydrogen phosphates.The coiled spiral is blue
The viable bacteria content of bacterium (Spirulina platensis) microbial inoculum is 3 × 108cfu/g。
The preparation method of Corynebacterium glutamicum ATCC13032 microbial inoculums includes：Corynebacterium glutamicum ATCC13032 is trained in LB
It supports culture in base and arrives logarithmic phase, be then inoculated into the strain cultivated to logarithmic phase completely by the inoculum concentration of culture volume 3%
In culture medium, at 30 DEG C, shaking table shakes 180rpm, cultivates 48 hours, and activated carbon is added, dry, and Corynebacterium glutamicum is made
ATCC13032 microbial inoculums.The viable bacteria content of the Corynebacterium glutamicum ATCC13032 microbial inoculums is 2 × 109cfu/g。
The preparation side of denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums
Method includes：By denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 in seed culture medium
Logarithmic phase is arrived in middle culture, and the strain cultivated to logarithmic phase is then inoculated into fermentation according to 5% inoculum concentration of culture volume
In culture medium, 28 DEG C, shaking table shakes 180rpm, cultivates 24 hours, and activated carbon is added, dry, and denitrogenation Hyphomicrobium is made
(Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums.Seed culture medium includes：7g/L methanol, 3g/L
Ammonium sulfate, 2g/L potassium dihydrogen phosphates, 3g/L magnesium sulfate, 2g/L disodium hydrogen phosphates；The fermentation medium includes：15g/L methanol,
2g/L ammonium sulfate, 3g/L potassium dihydrogen phosphates, 1.5g/L magnesium sulfate, 3g/L disodium hydrogen phosphates, 1.5g/L liquid microelements, 1.3g/L
The auxiliary liquid of vitamin.Liquid microelement includes that ferrous sulfate, zinc sulfate, manganese sulfate, calcium chloride and water are mixed to get, and wherein sulfuric acid is sub-
Concentration of iron is 100g/L, a concentration of 20g/L of zinc sulfate, a concentration of 10g/L of manganese sulfate, calcium chloride concentration 200g/L.Vitamin
Auxiliary liquid includes that folic acid, riboflavin, calcium pantothenate, biotin and water are mixed to get, and the folic acid concentration is 3g/L, and riboflavin concentration is
300g/L, calcium pantothenate concentration 500g/L, biotin concentration 5g/L.Denitrogenation Hyphomicrobium (the Hyphomicrobium
Denitrificans) viable bacteria content of CGMCC No.10620 is 5 × 108cfu/g。
The method of complex microorganism preparations for rehabilitating soil, this approach includes the following steps：By above-mentioned coiled spiral
Cyanobacteria (Spirulina platensis) microbial inoculum, Corynebacterium glutamicum ATCC13032 microbial inoculums and denitrogenation Hyphomicrobium
(Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums press 0.1:1:3 mass ratio is uniformly mixed i.e.
.
Preparation of the embodiment 2 for the complex microorganism preparations of soil remediation
The preparation method of coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum includes：Coiled spiral is blue thin
Logarithmic phase is arrived in bacterium (Spirulina platensis) culture, and culture is arrived logarithm by the inoculum concentration for then pressing culture volume 10%
The strain of phase is inoculated into culture medium, 35 DEG C, and shaking table shakes 180rpm, is cultivated 40 hours, is obtained coiled spiral cyanobacteria
Adsorbent is added into the zymotic fluid for the zymotic fluid of (Spirulina platensis), dry to get coiled spiral cyanobacteria
(Spirulina platensis) microbial inoculum；The adsorbent is that bentonite or activated carbon are one or more of.Culture medium is 1.5g/L
Sodium acetate, 0.06g/L peptones, 0.8g/L sodium bicarbonates, 0.5g/L sodium thiosulfate, 0.3g/L sodium chloride, 0.2g/L sulfuric acid
Magnesium and 0.07g/L potassium dihydrogen phosphates.The viable bacteria content of coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum is
5×1010cfu/g。
The preparation method of Corynebacterium glutamicum ATCC13032 microbial inoculums includes：Corynebacterium glutamicum ATCC13032 is trained in LB
It supports culture in base and arrives logarithmic phase, be then inoculated into the strain cultivated to logarithmic phase completely by the inoculum concentration of culture volume 6%
In culture medium, at 40 DEG C, shaking table shakes 120rpm, cultivates 48 hours, and bentonite is added, dry, and Corynebacterium glutamicum is made
ATCC13032 microbial inoculums.The viable bacteria content of the Corynebacterium glutamicum ATCC13032 microbial inoculums is 2 × 109cfu/g。
The preparation side of denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums
Method includes：By denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 in seed culture medium
Logarithmic phase is arrived in middle culture, and the strain cultivated to logarithmic phase is then inoculated into fermentation according to 10% inoculum concentration of culture volume
In culture medium, 30 DEG C, shaking table shakes 180rpm, cultivates 48 hours, and bentonite is added, dry, and denitrogenation Hyphomicrobium is made
(Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums.Seed culture medium includes：7g/L methanol, 3g/L
Ammonium sulfate, 2g/L potassium dihydrogen phosphates, 3g/L magnesium sulfate, 2g/L disodium hydrogen phosphates；The fermentation medium includes：15g/L methanol,
2g/L ammonium sulfate, 3g/L potassium dihydrogen phosphates, 1.5g/L magnesium sulfate, 3g/L disodium hydrogen phosphates, 1.5g/L liquid microelements, 1.3g/L
The auxiliary liquid of vitamin.Liquid microelement includes that ferrous sulfate, zinc sulfate, manganese sulfate, calcium chloride and water are mixed to get, and wherein sulfuric acid is sub-
Concentration of iron is 100g/L, a concentration of 20g/L of zinc sulfate, a concentration of 10g/L of manganese sulfate, calcium chloride concentration 200g/L.Vitamin
Auxiliary liquid includes that folic acid, riboflavin, calcium pantothenate, biotin and water are mixed to get, and the folic acid concentration is 3g/L, and riboflavin concentration is
300g/L, calcium pantothenate concentration 500g/L, biotin concentration 5g/L.Denitrogenation Hyphomicrobium (the Hyphomicrobium
Denitrificans) viable bacteria content of CGMCC No.10620 is 7 × 1010cfu/g。
The method of complex microorganism preparations for rehabilitating soil, this approach includes the following steps：By above-mentioned coiled spiral
Cyanobacteria (Spirulina platensis) microbial inoculum, Corynebacterium glutamicum ATCC13032 microbial inoculums and denitrogenation Hyphomicrobium
(Hyphomicrobium denitrificans) CGMCC No.10620 press 2:5:7 mass ratio is uniformly mixed to obtain the final product.
Preparation of the embodiment 3 for the complex microorganism preparations of soil remediation
The preparation method of coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum includes：Coiled spiral is blue thin
Logarithmic phase is arrived in bacterium (Spirulina platensis) culture, and culture is arrived logarithmic phase by the inoculum concentration for then pressing culture volume 5%
Strain be inoculated into culture medium, 33 DEG C, shaking table shake 200rpm, cultivate 44 hours, obtain coiled spiral cyanobacteria
Bentonite is added into the zymotic fluid for the zymotic fluid of (Spirulina platensis), dry to get coiled spiral cyanobacteria
(Spirulina platensis) microbial inoculum.Culture medium be 1.5g/L sodium acetates, 0.06g/L peptones, 0.8g/L sodium bicarbonates,
0.5g/L sodium thiosulfate, 0.3g/L sodium chloride, 0.2g/L magnesium sulfate and 0.07g/L potassium dihydrogen phosphates.The coiled spiral is blue
The viable bacteria content of bacterium (Spirulina platensis) microbial inoculum is 2.5 × 1010cfu/g。
The preparation method of Corynebacterium glutamicum ATCC13032 microbial inoculums includes：Corynebacterium glutamicum ATCC13032 is trained in LB
It supports culture in base and arrives logarithmic phase, be then inoculated into the strain cultivated to logarithmic phase completely by the inoculum concentration of culture volume 4%
In culture medium, at 35 DEG C, shaking table shakes 150rpm, cultivates 36 hours, and bentonite is added, dry, and Corynebacterium glutamicum is made
ATCC13032 microbial inoculums.The viable bacteria content of the Corynebacterium glutamicum ATCC13032 microbial inoculums is 2.5 × 1010cfu/g。
The preparation side of denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums
Method includes：By denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 in seed culture medium
Logarithmic phase is arrived in middle culture, and the strain cultivated to logarithmic phase is then inoculated into fermentation according to 7% inoculum concentration of culture volume
In culture medium, 29 DEG C, shaking table shakes 170rpm, cultivates 36 hours, and bentonite is added, dry, and denitrogenation Hyphomicrobium is made
(Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums.Seed culture medium includes：7g/L methanol, 3g/L
Ammonium sulfate, 2g/L potassium dihydrogen phosphates, 3g/L magnesium sulfate, 2g/L disodium hydrogen phosphates；The fermentation medium includes：15g/L methanol,
2g/L ammonium sulfate, 3g/L potassium dihydrogen phosphates, 1.5g/L magnesium sulfate, 3g/L disodium hydrogen phosphates, 1.5g/L liquid microelements, 1.3g/L
The auxiliary liquid of vitamin.Liquid microelement includes that ferrous sulfate, zinc sulfate, manganese sulfate, calcium chloride and water are mixed to get, and wherein sulfuric acid is sub-
Concentration of iron is 100g/L, a concentration of 20g/L of zinc sulfate, a concentration of 10g/L of manganese sulfate, calcium chloride concentration 200g/L.Vitamin
Auxiliary liquid includes that folic acid, riboflavin, calcium pantothenate, biotin and water are mixed to get, and the folic acid concentration is 3g/L, and riboflavin concentration is
300g/L, calcium pantothenate concentration 500g/L, biotin concentration 5g/L.Denitrogenation Hyphomicrobium (the Hyphomicrobium
Denitrificans) viable bacteria content of CGMCC No.10620 is 3.5 × 1010cfu/g。
The method of complex microorganism preparations for rehabilitating soil, this approach includes the following steps：By above-mentioned coiled spiral
Cyanobacteria (Spirulina platensis) microbial inoculum, Corynebacterium glutamicum ATCC13032 microbial inoculums and denitrogenation Hyphomicrobium
(Hyphomicrobium denitrificans) CGMCC No.10620 press 2.5：3：4 mass ratio is uniformly mixed to obtain the final product.
The complex microorganism preparations for the rehabilitating soil that 1 two kinds of microbial inoculums of comparative example are mixed to get
The preparation method of coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum includes：Coiled spiral is blue thin
Logarithmic phase is arrived in bacterium (Spirulina platensis) culture, and culture is arrived logarithmic phase by the inoculum concentration for then pressing culture volume 3%
Strain be inoculated into culture medium, 30 DEG C of shaking tables shake 220rpm, cultivate 48 hours, obtain coiled spiral cyanobacteria
Activated carbon is added into the zymotic fluid for the zymotic fluid of (Spirulina platensis), dry to get coiled spiral cyanobacteria
(Spirulina platensis) microbial inoculum.The culture medium is 1.5g/L sodium acetates, 0.06g/L peptones, 0.8g/L bicarbonates
Sodium, 0.5g/L sodium thiosulfate, 0.3g/L sodium chloride, 0.2g/L magnesium sulfate and 0.07g/L potassium dihydrogen phosphates.The coiled spiral is blue
The viable bacteria content of bacterium (Spirulina platensis) microbial inoculum is 3 × 108cfu/g。
The preparation method of Corynebacterium glutamicum ATCC13032 microbial inoculums includes：Corynebacterium glutamicum ATCC13032 is trained in LB
It supports culture in base and arrives logarithmic phase, be then inoculated into the strain cultivated to logarithmic phase completely by the inoculum concentration of culture volume 3%
In culture medium, at 30 DEG C, shaking table shakes 180rpm, cultivates 48 hours, and activated carbon is added, dry, and Corynebacterium glutamicum is made
ATCC13032 microbial inoculums.The viable bacteria content of the Corynebacterium glutamicum ATCC13032 microbial inoculums is 2 × 109cfu/g。
The method of complex microorganism preparations for rehabilitating soil, this approach includes the following steps：By above-mentioned coiled spiral
Cyanobacteria (Spirulina platensis) microbial inoculum, Corynebacterium glutamicum ATCC13032 microbial inoculums press 0.1:1 mass ratio mixing
Uniformly to obtain the final product.
The complex microorganism preparations for the rehabilitating soil that 2 two kinds of microbial inoculums of comparative example are mixed to get
The preparation method of coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum includes：Coiled spiral is blue thin
Logarithmic phase is arrived in bacterium (Spirulina platensis) culture, and culture is arrived logarithm by the inoculum concentration for then pressing culture volume 10%
The strain of phase is inoculated into culture medium, 35 DEG C, and shaking table shakes 180rpm, is cultivated 40 hours, is obtained coiled spiral cyanobacteria
Adsorbent is added into the zymotic fluid for the zymotic fluid of (Spirulina platensis), dry to get coiled spiral cyanobacteria
(Spirulina platensis) microbial inoculum；The adsorbent is that bentonite or activated carbon are one or more of.Culture medium is 1.5g/L
Sodium acetate, 0.06g/L peptones, 0.8g/L sodium bicarbonates, 0.5g/L sodium thiosulfate, 0.3g/L sodium chloride, 0.2g/L sulfuric acid
Magnesium and 0.07g/L potassium dihydrogen phosphates.The viable bacteria content of coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum is
5×1010cfu/g。
The preparation side of denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums
Method includes：By denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 in seed culture medium
Logarithmic phase is arrived in middle culture, and the strain cultivated to logarithmic phase is then inoculated into fermentation according to 10% inoculum concentration of culture volume
In culture medium, 30 DEG C, shaking table shakes 180rpm, cultivates 48 hours, and bentonite is added, dry, and denitrogenation Hyphomicrobium is made
(Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums.Seed culture medium includes：7g/L methanol, 3g/L
Ammonium sulfate, 2g/L potassium dihydrogen phosphates, 3g/L magnesium sulfate, 2g/L disodium hydrogen phosphates；The fermentation medium includes：15g/L methanol,
2g/L ammonium sulfate, 3g/L potassium dihydrogen phosphates, 1.5g/L magnesium sulfate, 3g/L disodium hydrogen phosphates, 1.5g/L liquid microelements, 1.3g/L
The auxiliary liquid of vitamin.Liquid microelement includes that ferrous sulfate, zinc sulfate, manganese sulfate, calcium chloride and water are mixed to get, and wherein sulfuric acid is sub-
Concentration of iron is 100g/L, a concentration of 20g/L of zinc sulfate, a concentration of 10g/L of manganese sulfate, calcium chloride concentration 200g/L.Vitamin
Auxiliary liquid includes that folic acid, riboflavin, calcium pantothenate, biotin and water are mixed to get, and the folic acid concentration is 3g/L, and riboflavin concentration is
300g/L, calcium pantothenate concentration 500g/L, biotin concentration 5g/L.Denitrogenation Hyphomicrobium (the Hyphomicrobium
Denitrificans) viable bacteria content of CGMCC No.10620 is 7 × 1010cfu/g。
The method of complex microorganism preparations for rehabilitating soil, this approach includes the following steps：By above-mentioned coiled spiral
Cyanobacteria (Spirulina platensis) microbial inoculum and denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans)
CGMCC No.10620 press 2:7 mass ratio is uniformly mixed to obtain the final product.
The complex microorganism preparations for the rehabilitating soil that 3 two kinds of microbial inoculums of comparative example are mixed to get
The preparation method of Corynebacterium glutamicum ATCC13032 microbial inoculums includes：Corynebacterium glutamicum ATCC13032 is trained in LB
It supports culture in base and arrives logarithmic phase, be then inoculated into the strain cultivated to logarithmic phase completely by the inoculum concentration of culture volume 4%
In culture medium, at 35 DEG C, shaking table shakes 150rpm, cultivates 36 hours, and bentonite is added, dry, and Corynebacterium glutamicum is made
ATCC13032 microbial inoculums.The viable bacteria content of the Corynebacterium glutamicum ATCC13032 microbial inoculums is 2.5 × 1010cfu/g。
The preparation side of denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums
Method includes：By denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 in seed culture medium
Logarithmic phase is arrived in middle culture, and the strain cultivated to logarithmic phase is then inoculated into fermentation according to 7% inoculum concentration of culture volume
In culture medium, 29 DEG C, shaking table shakes 170rpm, cultivates 36 hours, and bentonite is added, dry, and denitrogenation Hyphomicrobium is made
(Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums.Seed culture medium includes：7g/L methanol, 3g/L
Ammonium sulfate, 2g/L potassium dihydrogen phosphates, 3g/L magnesium sulfate, 2g/L disodium hydrogen phosphates；The fermentation medium includes：15g/L methanol,
2g/L ammonium sulfate, 3g/L potassium dihydrogen phosphates, 1.5g/L magnesium sulfate, 3g/L disodium hydrogen phosphates, 1.5g/L liquid microelements, 1.3g/L
The auxiliary liquid of vitamin.Liquid microelement includes that ferrous sulfate, zinc sulfate, manganese sulfate, calcium chloride and water are mixed to get, and wherein sulfuric acid is sub-
Concentration of iron is 100g/L, a concentration of 20g/L of zinc sulfate, a concentration of 10g/L of manganese sulfate, calcium chloride concentration 200g/L.Vitamin
Auxiliary liquid includes that folic acid, riboflavin, calcium pantothenate, biotin and water are mixed to get, and the folic acid concentration is 3g/L, and riboflavin concentration is
300g/L, calcium pantothenate concentration 500g/L, biotin concentration 5g/L.Denitrogenation Hyphomicrobium (the Hyphomicrobium
Denitrificans) viable bacteria content of CGMCC No.10620 is 3.5 × 1010cfu/g。
The method of complex microorganism preparations for rehabilitating soil, this approach includes the following steps：By Corynebacterium glutamicum
ATCC13032 microbial inoculums and denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 press 3：4
Mass ratio be uniformly mixed to obtain the final product.
Embodiment 4
Ammonia nitrogen, heavy metal Cd, Cu, PO are measured according to current conventional test method4 3+Degradation capability：
1) it chooses by ammonia nitrogen, heavy metal Cd, Cu, PO4 3+Etc. contaminated soils, be divided into 18 parts, after being respectively placed in disinfection
Container in, by 18 parts sterilizing after soil be divided into 6 groups, every group 3 parts.
2) Example 1~3 and the complex microorganism preparations obtained for being used for rehabilitating soil of comparative example 1~3, it is each to implement
Example/comparative example complex microorganism preparations the correspondence obtained for rehabilitating soil is added thereto in one group of soil, is uniformly mixed,
The quality for the complex microorganism preparations for rehabilitating soil being wherein added in soil accounts for the 0.5% of soil quality.
3) ammonia nitrogen, heavy metal Cd, Cu, PO in soil after processing are detected after two months4 3+Deng content, every group 3 parts take
Average value, the results are shown in Table 1.
Table 1
Claims (10)
1. a kind of complex microorganism preparations for rehabilitating soil, which is characterized in that the complex microorganism preparations include plate-like spiral shell
Rotation cyanobacteria (Spirulina platensis) microbial inoculum, Corynebacterium glutamicum ATCC13032 microbial inoculums and denitrogenation Hyphomicrobium
(Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums.
2. the complex microorganism preparations according to claim 1 for rehabilitating soil, which is characterized in that the coiled spiral
Cyanobacteria (Spirulina platensis) microbial inoculum, Corynebacterium glutamicum ATCC13032 microbial inoculums and denitrogenation Hyphomicrobium
(Hyphomicrobium denitrificans) CGMCC No.10620 weight ratio be 0.1 ~ 2:1~5:3~7.
3. the complex microorganism preparations according to claim 1 for rehabilitating soil, which is characterized in that the coiled spiral
Cyanobacteria (Spirulina platensis) microbial inoculum viable bacteria content be 3 × 108~5×1010 Cfu/g, the glutamic acid rod
The viable bacteria content of bacterium ATCC13032 microbial inoculums is 2 × 109~5×1010 Cfu/g, the denitrogenation Hyphomicrobium
(Hyphomicrobium denitrificans) CGMCC No.10620 viable bacteria content be 5 × 108~7×1010 cfu/g。
4. a kind of method preparing the complex microorganism preparations for rehabilitating soil described in any one of claim 1 ~ 3,
It is characterized in that, this approach includes the following steps：By coiled spiral cyanobacteria (Spirulina platensis) microbial inoculum, paddy ammonia
Sour bar bacterium ATCC13032 microbial inoculums and denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC
No.10620 microbial inoculums press 0.1 ~ 2:1~5:3 ~ 7 mass ratio is uniformly mixed to obtain the final product.
5. the method according to claim 4 for preparing the complex microorganism preparations for rehabilitating soil, which is characterized in that institute
State coiled spiral cyanobacteria (Spirulina platensis) preparation method of microbial inoculum includes：By coiled spiral cyanobacteria
(Spirulina platensis) to logarithmic phase, culture is arrived logarithm by the inoculum concentration for then pressing culture volume 3% ~ 10% for culture
The strain of phase is inoculated into culture medium, 30 ~ 35 DEG C, and shaking table shakes 180 ~ 220rpm, is cultivated 40 ~ 48 hours, is obtained coiled spiral
Cyanobacteria (Spirulina platensis) zymotic fluid, be added adsorbent into the zymotic fluid, it is dry to get coiled spiral
Cyanobacteria (Spirulina platensis) microbial inoculum；The adsorbent is that bentonite or activated carbon are one or more of.
6. the method according to claim 5 for preparing the complex microorganism preparations for rehabilitating soil, which is characterized in that institute
It is 1.5g/L sodium acetates, 0.06g/L peptones, 0.8g/L sodium bicarbonates, 0.5g/L sodium thiosulfate, 0.3g/L chlorine to state culture medium
Change sodium, 0.2g/L magnesium sulfate and 0.07g/L potassium dihydrogen phosphates.
7. the method for complex microorganism preparations of the preparation according to claim 5 for rehabilitating soil, feature
It is, the preparation method of the Corynebacterium glutamicum ATCC13032 microbial inoculums includes：By Corynebacterium glutamicum ATCC13032 in LB
Logarithmic phase is arrived in culture in culture medium, is then inoculated into the strain cultivated to logarithmic phase by the inoculum concentration of culture volume 3% ~ 6%
In complete medium, at 30 ~ 40 DEG C, shaking table shakes 120 ~ 180 rpm, cultivates 24 ~ 48 hours, and adsorbent is added, dry, is made
Corynebacterium glutamicum ATCC13032 microbial inoculums；The adsorbent is one or more of bentonite or activated carbon.
8. the method according to claim 5 for preparing the complex microorganism preparations for rehabilitating soil, which is characterized in that institute
State denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums preparation method packet
It includes：By denitrogenation Hyphomicrobium (Hyphomicrobium denitrificans) CGMCC No.10620 are in seed culture medium
Logarithmic phase is cultivated, the strain cultivated to logarithmic phase is then inoculated into fermentation according to 5% ~ 10% inoculum concentration of culture volume
In culture medium, 28 ~ 30 DEG C, shaking table shakes 160 ~ 180 rpm, cultivates 24 ~ 48 hours, and adsorbent is added, dry, and denitrogenation life is made
Silk germ (Hyphomicrobium denitrificans) CGMCC No.10620 microbial inoculums；The adsorbent be bentonite or
One or more of activated carbon.
9. the method according to claim 8 for preparing the complex microorganism preparations for rehabilitating soil, which is characterized in that institute
Stating seed culture medium includes：7g/L methanol, 3g/L ammonium sulfate, 2g/L potassium dihydrogen phosphates, 3g/L magnesium sulfate, 2g/L phosphoric acid hydrogen two
Sodium；The fermentation medium includes：15g/L methanol, 2g/L ammonium sulfate, 3g/L potassium dihydrogen phosphates, 1.5g/L magnesium sulfate, 3g/L phosphorus
Sour disodium hydrogen, 1.5g/L liquid microelements, the auxiliary liquid of 1.3g/L vitamins.
10. complex microorganism preparations for rehabilitating soil described in any one of claim 1 ~ 5 heavy metal, ammonia nitrogen,
Application in phosphor polluted soil reparation.
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