CN112725240B - Acinetobacter livelii, and microbial inoculum and application thereof - Google Patents
Acinetobacter livelii, and microbial inoculum and application thereof Download PDFInfo
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- 229910052799 carbon Inorganic materials 0.000 claims abstract description 9
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- 241000894006 Bacteria Species 0.000 claims description 11
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Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
Abstract
The invention discloses an acinetobacter alive, a microbial inoculum and application thereof, belongs to the technical field of microorganisms, is named as acinetobacter alive KJ-1, and is preserved in China general microbiological culture Collection center (CGMCC) in 9-17 th of 2020, with the strain preservation number of CGMCC No. 20664. The strain and the microbial inoculum thereof can grow and reproduce by using n-dodecane as a unique carbon source, have a petroleum hydrocarbon degradation function, particularly have a very obvious degradation effect on n-alkane which is one of petroleum components, can be applied to treatment of petroleum hydrocarbon pollutants, and have wide application prospects.
Description
Technical Field
The invention belongs to the technical field of microorganisms and environmental protection, and particularly relates to acinetobacter livening, a microbial inoculum thereof and application thereof.
Background
With the rapid development of economy, petroleum is one of the most important energy sources for human beings, and the pollution problem is increasingly serious, thereby causing the attention of experts in various countries. At present, the technology for restoring the petroleum polluted soil mainly comprises physical, chemical and biological restoration technologies. The damage to the restored soil is large no matter in a physical or chemical method, and even if the content of petroleum hydrocarbon can reach the standard, the reutilization rate of the soil is questionable. In addition, the physical method has a large engineering quantity, sometimes requires ex-situ repair, and has large consumption of manpower, material resources and financial resources, while the chemical method also has the problems of secondary pollution and cost. Research shows that microbial remediation is a key technology for remediating petroleum-polluted soil due to the characteristics of low cost, no secondary pollution, good treatment effect and the like, and has remarkable social and environmental benefits. Therefore, obtaining the petroleum efficient degrading bacteria is an important link for improving the biodegradation efficiency of the petroleum efficient degrading bacteria.
Petroleum is a mixture consisting of 4 major substances such as alkane, aromatic hydrocarbon, asphaltene and resin with different molecular weights, wherein the proportion of the alkane with various chain lengths is between 50 and 70 percent, and the alkane is the main component of petroleum hydrocarbon pollution, and the biodegradation of the petroleum is always a research hotspot and a focus. At present, more than 90 genera and more than 200 kinds of petroleum hydrocarbon degrading bacteria are separated, including bacteria, yeast and fungi, and the bacteria are main participants of the metabolic degradation of the petroleum hydrocarbon. With regard to the research on alkane degrading bacteria, various national scholars have screened out a plurality of bacteria which can metabolize by taking alkane as a unique carbon source and energy source, including pseudomonas, bacillus, Acinetobacter, microbacterium, arthrobacter, micrococcus and the like, the microorganisms distribute in the environment by taking alkane as the unique carbon source and energy source, and provide new strain resources to a greater extent for the field of microbial remediation of petroleum hydrocarbon contaminated soil, and the Acinetobacter (Acinetobacter) is taken as an important alkane degrading microorganism genus which is generally distributed in the soil and can realize the digestion of alkane through the emulsification of the Acinetobacter.
Because alkane is the main component in petroleum and products thereof and is one of main pollutants in various petrochemical wastewater, the actual restoration application cannot be met only by degrading alkane by indigenous microorganisms in nature, so how to obtain high-efficiency alkane degrading bacteria has very important significance for improving the restoration effect of petroleum hydrocarbon polluted soil.
Disclosure of Invention
Aiming at the problem of poor alkane degradation capability of indigenous microorganisms in the prior art, the invention provides an acinetobacter calcoaceticus strain, a microbial inoculum thereof and application thereof.
The invention is realized by the following technical scheme:
acinetobacter livers strainAcinetobacter vivianii) The strain is named as Acinetobacter livelii KJ-1, is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms in 9 months and 17 months in 2020, and has the culture preservation number of CGMCC No. 20664.
The Acinetobacter livers microbial agent of the present invention comprises the active ingredient of KJ-1 of Acinetobacter livers described in claim 1.
Further, the preparation method of the acinetobacter liverwort microbial inoculum comprises the following steps: mixing the Acinetobacter livening KJ-1 fermentation liquor with sterilized grass carbon according to the volume-mass ratio of 1:1.5-2.5, and naturally drying to obtain the Acinetobacter livening microbial inoculum.
Further, the preparation method of the acinetobacter liverwort KJ-1 fermentation liquor comprises the following steps:
(1) inoculating acinetobacter calcoaceticus KJ-1 into a nutrient agar culture medium, and culturing at 28-30 ℃ for 20-28 h to obtain an activated strain;
(2) inoculating the activated strain prepared in the step (1) into a nutrient broth liquid culture medium according to the inoculum size of 2-10%, and carrying out shake cultivation at 25-30 ℃ and 150-200 rpm for 20-28 h to prepare a fermentation liquid.
Further, each liter of the nutrient agar culture medium in the step (1) comprises 10g of peptone, 3g of beef powder, 5g of sodium chloride, 18.0 g of agar and the balance of water, and the pH value is 7.0-7.4; the nutrient broth liquid culture medium in the step (2) comprises 10g of peptone, 3g of beef powder, 5g of sodium chloride and the balance of water per liter of culture medium, and the pH value is 7.0-7.4.
In the invention, the acinetobacter alive is applied to the treatment of petroleum pollutants.
In the invention, the acinetobacter liverwort microbial inoculum is applied to the treatment of petroleum pollutants.
According to the invention, the acinetobacter livening microbial inoculum and petroleum polluted soil are uniformly mixed according to the weight ratio of 1 (10-20), soil turning is carried out once a week, the water content of the soil is maintained at 20-50 wt%, and the soil is repaired for 30-60 days.
The acinetobacter calcoaceticus is obtained by separating from polluted soil near a gas station, adding n-dodecyl alkane into an inorganic salt culture medium as a substrate through enrichment culture, and obtaining the acinetobacter calcoaceticus KJ-1 through separation and screening, wherein n-dodecyl alkane can be used as a unique carbon source for growth and propagation.
Weighing 5.0g of polluted soil sample, adding the polluted soil sample into a 50mL triangular flask containing an enrichment culture medium containing 3765mg/L n-dodecane, carrying out shaking culture at 30 ℃ and 150r/min for 24h, sucking 2mL of culture solution, transferring the culture solution into a fresh enrichment culture medium, continuously transferring and culturing for 3 times according to the culture conditions and time, carrying out strain separation by adopting a plate dilution coating method, respectively sucking 100 mu L of culture solution with different dilution gradients, coating the culture solution onto an inorganic salt plate containing 3765mg/L n-dodecane, and carrying out standing culture in a 30 ℃ constant temperature incubator for 2 d. Observing the growth condition of bacterial colony on the plate, selecting different bacterial strains according to the time, shape, size and color of bacterial colony, drawing lines on inorganic salt plate containing 3765mg/L n-dodecane, culturing for 2d in 30 deg.C constant temperature incubator, repeating the above operations for 3 times until obtaining single bacterial strain, separating to obtain Acinetobacter liverdna KJ-1, and detecting to obtain 16S rDNA sequence result belonging to Proteobacteria (DENTIABIA)Proteobacteria) Proteobacteria (A)Gammaproteobacteria) Pseudomonales (, (ii) and (iii)Pseudomonadales) Moraxeridae (Moraxeridae)Moraxellaceae) Acinetobacter (A), (B) and (C)Acinetobacter）。
Said strain Acinetobacter livers: (Acinetobacter vivianii) KJ-1 is streaked on nutrient agar medium (NB) for 48h,the bacterial colony is light yellow, the edge is thin, the middle is thick, the edge of a single bacterial colony is smooth and neat, the bacterial colony has a moist feeling and is facultative anaerobic, the bacterial colony is identified as gram-negative bacteria through gram staining, and the bacterial colony is in a ball-bar shape under a microscope.
According to Acinetobacter livers: (Acinetobacter vivianii) The 16S rRNA sequence of KJ-1 was compared with the 16S rRNA sequences of other registered bacterial strains by the BLAST program of the NCBI official website (https:// www.ncbi.nlm.nih.gov /), and the results showed that the strain had the highest similarity to the Acinetobacter vivianii strain NIPH 2168 and the homology was 99%.
Information on strain preservation
Preservation time: 9 month 17 days 2020;
the preservation unit: china general microbiological culture Collection center;
the preservation number is: CGMCC No. 20664;
the address of the depository: the microbiological research institute of western road 1, 3, national academy of sciences, north-kyo, chaoyang, the postal code: 100101;
and (3) classification and naming: acinetobacter livelii: (Acinetobacter vivianii）。
Advantageous effects
The invention discovers for the first time that a strain KJ-1 of Acinetobacter livestocks (Acinetobacter viviani) can grow and reproduce by using n-dodecane as a unique carbon source, has a petroleum hydrocarbon degradation function, particularly has a very obvious degradation effect on n-alkane which is one of petroleum components, can be applied to treatment of petroleum hydrocarbon pollutants, and has a wide application prospect.
Drawings
FIG. 1 is a tree phylogenetic from Acinetobacter livelii KJ-1;
FIG. 2 is a graph showing the effect of incubation time on strain growth and n-dodecane degradation;
FIG. 3 is a graph of the effect of initial concentration on strain growth and n-dodecane degradation;
FIG. 4 is a graph showing the effect of inoculum size on strain growth and n-dodecane degradation;
FIG. 5 is a graph of the effect of temperature on strain growth and n-dodecane degradation;
FIG. 6 is a graph of the effect of initial pH on strain growth and n-dodecane degradation;
FIG. 7 is a gas phase spectrum of Acinetobacter livers KJ-1 before degradation of petroleum hydrocarbon wastewater;
FIG. 8 is a gas phase diagram of Acinetobacter livers KJ-1 after degradation of petroleum hydrocarbon wastewater.
Detailed Description
The technical solutions of the present invention are further described below with reference to the drawings and examples of the specification, but the present invention should not be construed as being limited thereto. It is within the scope of the present invention to make simple modifications or alterations to the methods, procedures or conditions of the present invention without departing from the spirit and substance of the invention; the technical means used in the examples are conventional means well known to those skilled in the art, unless otherwise specified.
Example 1
Isolation and characterization of strains
(1) The source of the strain
The strain is obtained by separating from polluted soil near a gas station.
(2) Reagent and culture medium formula
PBS buffer: NaCl 8.0g, KCl 0.2g, Na2HPO4 1.15g、KH2PO40.2g, distilled water to 1000ml, pH7.0, sterilizing at 121 deg.C for 20 min;
enrichment culture medium: NH (NH)4NO3 1.0g、K2HPO4 0.5g、KH2PO4 1.0g、MgSO4 0.2g、NaCl 5.0g、CaCl2 0.02g、FeCl30.002g, 12.6g of yeast powder, 5.8g of peptone and distilled water, wherein the volume is fixed to 1000ml, the pH value is 7.0-7.2, and the sterilization is carried out for 20min at 121 ℃;
inorganic salt culture medium: NaH2PO4 1g、（NH4）2SO4 0.5g、CaCl2 0.1g、NaNO3 0.5g、MgSO4·7H2O 0.2g、K2HPO41g of agar, 12g of agar and distilled water, wherein the volume is fixed to 1000mL, the pH value is 7.0, and the mixture is sterilized for 20min at 121 ℃;
NB medium: 10.0g of peptone, 3.0g of beef extract powder, 5.0g of NaCl, 1.5 percent (mass fraction) agar and distilled water to a constant volume of 1000mL, and the pH value is 7.2+Sterilizing at 121 deg.C for 20min at 0.2;
induction medium: NH (NH)4NO3 1.0g、K2HPO4 0.5g、KH2PO4 1.0g、MgSO4 O.2g、NaCl 5.0g、CaCl20.02g、FeCl30.002g, 12.6g of yeast powder, 5.8g of peptone and distilled water, wherein the volume is fixed to 1000ml, the pH value is 7.0-7.2, and the sterilization is carried out for 20min at 121 ℃;
nutrient agar medium: 10g of peptone, 3g of beef powder, 5g of sodium chloride, 18.0 g of agar, and distilled water with constant volume of 1000mL and pH value of 7.2;
nutrient broth liquid medium: 10g of peptone, 3g of beef powder, 5g of sodium chloride and distilled water to reach the constant volume of 1000mL, and the pH value is 7.2.
(3) Isolation of the Strain
Weighing 5.0g of polluted soil sample, adding the polluted soil sample into a 50mL triangular flask containing an enrichment culture medium containing 3765mg/L n-dodecane, carrying out shaking culture at 30 ℃ for 24h, sucking 2mL of culture solution, transferring the culture solution into a fresh enrichment culture medium, and continuously transferring and culturing for 3 times according to the culture conditions and time; separating strains by adopting a plate dilution coating method, respectively sucking 100 mu L of culture solution with different dilution gradients, coating the culture solution on an inorganic salt culture medium plate containing 3765mg/L n-dodecane, and performing static culture in a constant temperature incubator at 30 ℃ for 2d;
seven strains with degrading capability to the dodecane are finally screened out through enrichment culture, separation and purification, and are named as KJ-1-KJ-7 respectively.
(4) Bacterial strain rescreening
Transferring the bacterial liquid cultured in the step (3) to an induction culture medium, carrying out induction culture for 1d, centrifuging for 5min at 8000r/min, pouring out supernatant, taking a thallus precipitate, washing the thallus precipitate for 2-3 times by PBS (phosphate buffer solution), carrying out heavy suspension on thallus, carrying out ultrasonic crushing for 17min under the power condition of 195W, carrying out ultrasonic treatment for 2s, stopping centrifuging for 5min at 8000r/min, collecting supernatant, namely enzyme liquid, adding 3765mg/L n-dodecane into a prepared enzyme liquid system, carrying out enzymolysis for 8h, and carrying out enzymolysis according to an enzymolysis system: petroleum ether = 1:1, extracting for 2-3 times, collecting supernatant, dehydrating with anhydrous sodium sulfate, drying, and fixing volume. The residual amount of n-dodecane was measured by ultraviolet spectrophotometry, and the degradation rate of n-dodecane was calculated from a standard curve (y =0.1631 x-0.0576).
After re-screening, a strain Acinetobacter liverwhicus KJ-1 which takes n-dodecane as a unique carbon source and has a better growth condition is obtained, and the degradation rate of the n-dodecane is up to 91.99 percent.
(5) Identification of strains
According to the physiological and biochemical reactions of the strain and the identification work of the strain carried out by 16S rRNA, the colony of the strain acinetobacter calcoaceticus KJ-1 is round, smooth in edge, yellow-white, opaque, moist and glossy in surface, free of spores, negative in gram stain, positive in oxidase and positive in starch hydrolysis, and the strain is in a ball-bar shape under a microscope.
Inoculating the strain Acinetobacter livelii KJ-1 into a nutrient broth liquid culture medium, performing shake culture at 28 ℃ for 16h, centrifuging at 12000rpm to collect thalli, and extracting the genome of the strain KJ-1 by using an Ezup column type bacterial genome DNA extraction kit. PCR amplification was performed using primers 7F and 1540R.
The primer sequences are as follows:
7F：5’-CAGAGTTTGATCCTGGCT-3’
1540R：5’-AGGAGGTGATCCAGCCGCA-3’
and (3) purifying the PCR product, then sending the purified PCR product to Shanghai bioengineering company Limited for sequence determination, and performing homology comparison analysis on a sequencing result and a sequence in an NCBI website database. The result shows that the 16S rRNA sequence of the strain Acinetobacter livelii KJ-1 obtained by separation and purification has the highest similarity with Acinetobacter vivianii strain NIPH 2168 and the homology reaches 99 percent, and has higher homology with other strains to which the strain belongs, and the strain belongs to the proteobacteria according to the sequence of the Acinetobacter livelii KJ-116S rDNAProteobacteria) Proteobacteria (A)Gammaproteobacteria) Pseudomonales (1)Pseudomonadales) Moraxeridae (Moraxeridae)Moraxellaceae) Acinetobacter (A), (B) and (C)Acinetobacter）。
In conclusion, according to the physiological and biochemical characteristics of the strainsMolecular verification, the strain is named Acinetobacter liverwhile (Acinetobacter vivianii）KJ-1
The strain is preserved in China general microbiological culture Collection center (China academy of sciences, institute 3, Ministry of microbiology, west Lu 1, Beijing, Chaoyang, and North Cheng) within 17 days 9 and 17 months 2020, and the preservation number of the strain is CGMCC No. 20664.
Example 2
Inoculating acinetobacter calcoaceticus KJ-1 into 50mL of inorganic salt liquid culture medium containing 3765mg/L n-dodecane and having pH of 7.0 according to the inoculation amount of 5%, carrying out shake cultivation at 30 ℃ and 150r/min, taking an inorganic salt culture medium which is not inoculated under the same cultivation condition as a control group, and respectively investigating the influence of different cultivation time such as 4d, 6d, 8d, 10d and 12d on the degradation of the n-dodecane by the KJ-1; as a result, as shown in FIG. 2, Acinetobacter livelii KJ-1 was always in the growth phase and OD was observed during the whole culture period, as compared with the control group under the same conditions600The degradation rate shows a trend of steadily increasing and then decreasing. At 6d, the environment was still sufficient to sustain the vital activity of Acinetobacter liveweight KJ-1, which is metabolized by nutrients, OD600At 0.257, the degradation rate of n-dodecane increased to 82.07%. According to the growth curve of the strain KJ-1 and the degradation rate of the n-dodecane, the culture time suitable for the strain of the acinetobacter calcoaceticus KJ-1 to degrade the n-dodecane is 6 days.
Example 3
Initial concentration plays different roles in the growth and development process of microorganisms, Acinetobacter liverwort KJ-1 is inoculated into 50mL of inorganic salt liquid culture medium with pH7.0 according to the inoculation amount of 5 percent, shaking culture is carried out at 30 ℃ and 150r/min, the influence of different initial concentrations such as 3765mg/L, 7530mg/L, 11295mg/L, 15060mg/L, 18825mg/L on the degradation of n-dodecane by the Acinetobacter liverwort KJ-1 is respectively examined, and the results are shown in figure 3, when the n-dodecane concentration is 3765mg/L, the growth amount of the strain is higher, and the OD is6000.598, the utilization of the n-dodecane by the microorganism is maximized, and the degradation rate is maximized to 77.48%; in the 3765 mg/L-11295 mg/L concentration range, the growth of the microorganism shows a rising trend and a falling trend, and the OD is obtained in the period600A minimum value appears0.558, the degradation rate of n-dodecane also shows a tendency to decrease, and microorganisms may produce substances adverse to their growth during the metabolic activity of this process, thus affecting the degradation of n-dodecane by microorganisms; acinetobacter liverwort KJ-1 enters logarithmic growth phase when n-dodecane is higher than 11295mg/L and OD is 18825mg/L600When the maximum value is reached to 0.734, the degradation rate of n-dodecane shows a tendency of decreasing fluctuation, and the degradation rate is 20.68% when 18825mg/L, so that the optimal degradation concentration of the strain acinetobacter liverwortus KJ-1 is 3765 mg/L.
Example 4
The effect of the inoculation amount on the growth of microorganisms is important, in order to investigate the optimal inoculation amount of the strain Acinetobacter livelii KJ-1, the Acinetobacter livelii KJ-1 is inoculated into 50mL of inorganic salt liquid culture medium containing 3765mg/L of n-dodecane and having pH of 7.0, shaking culture is carried out at 30 ℃ and 150r/min, and the effect of different inoculation amounts of 2.5%, 5%, 7.5%, 10%, 15%, 20% and the like on the degradation of the n-dodecane by the Acinetobacter livelii KJ-1 is respectively investigated. As a result, as shown in FIG. 4, the OD was adjusted to 2.5 to 10%600And the degradation rate of n-dodecane is positively correlated with the inoculation amount, and when the inoculation amount is 10 percent, the OD is6000.572, the degradation rate reaches a maximum of 81.43%; the inoculation amount is within the range of 10-15%, OD600Shows a decreasing trend from 0.572 to 0.527, the degradation rate of n-dodecane also starts to decrease; OD when the inoculation amount is 15-20%600Again from 0.527 to 0.719, but the degradation rate continued to decrease, with 20% inoculum the degradation rate was as low as 34.51%. The result shows that the proper increase of the inoculation amount of the acinetobacter calcoaceticus KJ-1 can enhance the utilization of the microorganism on the n-dodecane and improve the degradation rate of the n-dodecane, but the continuous decrease of the degradation rate of the n-dodecane can be caused when the inoculation amount is too large, because the consumption of nutrients in the environment where the microorganism grows is accelerated due to the excessive inoculation amount, meanwhile, the metabolic activity of the microorganism causes the accumulation of metabolites, the content of oxygen is relatively insufficient, the toxicity of the whole growth environment is enhanced, and therefore, the optimal inoculation amount suitable for the growth of the strain KJ-1 is 10%.
Example 5
Inoculating Acinetobacter livelii KJ-1 strain at an inoculation amount of 5% to a strain containing 3765mg/L n-dodecane was cultured in 50mL of an inorganic salt liquid medium (pH7.0) at a shaking rate of 150r/min for 6d, and the influence of different temperatures, such as 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃, on the degradation of n-dodecane by Acinetobacter livelii KJ-1 was examined. As shown in FIG. 5, the growth of Acinetobacter lively KJ-1 was better at 20-30 ℃ compared to the control group under the same conditions, the environmental nutrients were sufficient to ensure the metabolic activity of Acinetobacter lively KJ-1, the normal vital activity could be performed, the growth rate of the microorganism was faster, and the OD was OD600From 0.261 to 0.299, at which time the OD is increased600The value is the maximum, the utilization of the n-dodecane is gradually increased, and the degradation rate of the n-dodecane reaches 70.57% at the temperature of 30 ℃; the growth of the microorganisms has already progressed into the decline phase, OD, at 30-40 deg.C600The drastic reduction to 0.124 is accompanied by the restriction of microbial growth, KJ-1 also affects the utilization of n-dodecane, and the degradation rate correspondingly decreases to 62.45%. From the results, the optimum degradation temperature of the strain KJ-1 was 30 ℃.
Example 6
Acinetobacter livelii KJ-1 is inoculated into 50mL of inorganic salt liquid culture medium containing 3765mg/L of n-dodecane according to the inoculation amount of 5%, shaking culture is carried out at the temperature of 30 ℃ and at the speed of 150r/min, and the influence of different pH values of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and the like on the degradation of the n-dodecane by the KJ-1 is examined. As a result, as shown in FIG. 6, the OD was determined when the initial pH of the medium was 5.0 to 5.5600The degradation rates of both n-dodecane and n-dodecane showed a tendency to increase continuously, and when the initial pH of the medium was 5.5, the OD was6000.325, the degradation rate of n-dodecane reaches 64.58 percent; OD when initial pH of the medium is 5.5-6.0600And degradation rate are reduced, and OD is 6.0600The small amplitude is reduced to 0.278, and the degradation rate is reduced to 58.26%, at which the degradation rate of the n-dodecane is at a minimum; when the initial pH of the culture medium is 6.0-6.5, the strain is still in the growth stage, OD600A maximum value of 0.423 appeared during growth, showing a sum OD of n-dodecane degradation600Synchronous trend, the degradation rate reaches 67.82%; OD when initial pH of the medium is 6.5-7.5600Continuously reduced from 0.423 to 0.220, but the utilization of n-dodecane by the microorganismsBut did not continue to decrease due to the decrease in the amount of microbial growth, but instead showed an opposite degradation profile, with the degradation rate instead reaching a maximum of 75.56% at an initial pH of 7.5 of the medium. From the results, it was found that the initial pH of the medium was controlled at a value of 6.0 or 7.5.
Example 7
Inoculating acinetobacter liverwort CGMCC number 20664 (acinetobacter liverwort KJ-1) stored at-80 ℃ to a nutrient agar culture medium, culturing for 24h at 28 ℃, transferring a grown single colony into a nutrient broth culture medium, and performing shake culture at 30 ℃ and 150rpm for 24h to finally obtain a bacterium solution of the acinetobacter liverwort KJ-1;
inoculating the bacterial liquid into an inorganic salt culture medium containing 5 g/L petroleum according to the inoculation amount of 5% of the volume ratio, detecting the degradation effect of the bacterial strain on the petroleum hydrocarbon by taking the oil-containing inorganic salt without inoculation as a contrast, wherein the gas phase maps of the petroleum hydrocarbon wastewater before and after degradation are respectively shown in figures 7 and 8, which shows that the bacterial strain has a certain degradation effect on the petroleum hydrocarbon, and compared with the contrast, the bacterial strain can degrade 51.7% of the total petroleum hydrocarbon, which shows that the bacterial strain has a certain degradation effect on n-dodecane and also has a degradation effect on other petroleum hydrocarbons.
Example 8
(1) Inoculating acinetobacter liverwort CGMCC number 20664 (acinetobacter liverwort KJ-1) stored at-80 ℃ to a nutrient agar culture medium, culturing for 24h at 28 ℃, transferring a grown single colony into a nutrient broth culture medium, and performing shake culture at 30 ℃ and 150rpm for 24h to finally obtain a fermentation broth of the acinetobacter liverwort KJ-1;
(2) and (2) sterilizing grass peat under high pressure, sterilizing at 121 ℃ for 2h, uniformly mixing the fermentation liquor obtained in the step (1) and the grass peat according to a ratio of 1:2 (V: W), and naturally drying to obtain the acinetobacter livelii KJ-1 microbial inoculum.
And (3) carrying out a petroleum-polluted soil test by adopting the acinetobacter liverwort KJ-1 microbial inoculum prepared in the step (2).
The preparation method of the soil polluted by petroleum comprises the following steps: collecting a soil sample, naturally drying, crushing, adding petroleum according to a volume/weight ratio, and simultaneously adding 6.5g of ammonium nitrate and 2.0g of potassium dihydrogen phosphate into per kilogram of petroleum soil.
Three groups of soil samples are respectively prepared from the soil polluted by the petroleum for comparison experiment:
a group of soil samples: weighing 1.0kg of petroleum-polluted soil, and adding 50g of bioremediation agent with acinetobacter liverwort CGMCC number 20664 as a main component;
two groups of soil samples: weighing 1.0kg of petroleum-polluted soil, and adding 100g of bioremediation agent with acinetobacter liverwort CGMCC number 20664 as a main component;
three groups of soil samples: 1.0kg of petroleum-contaminated soil was weighed.
The experimental procedure was as follows:
and (3) respectively putting the three groups of soil samples into flowerpots, adjusting the water content of the soil to 20-50 wt%, loosening the soil, standing at room temperature for one month, and ploughing and replenishing water every 7D, and keeping a certain humidity. After 21 times of remediation, the removal rate of petroleum hydrocarbon of one group of soil samples is 41.01%, and the removal rate of petroleum hydrocarbon of the two groups of soil samples is 49.08%.
Claims (6)
1. Acinetobacter livers strainAcinetobacter vivianii) The culture medium is characterized in that the culture medium is named as Acinetobacter livestocks KJ-1, is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms in 9 months and 17 days in 2020, and has the culture preservation number of CGMCC No. 20664.
2. The Acinetobacter vivacis inoculant according to claim 1, wherein the Acinetobacter vivacis inoculant is prepared by a method comprising: mixing the Acinetobacter livening KJ-1 fermentation liquor with sterilized grass carbon according to the volume-mass ratio of 1:1.5-2.5, and naturally drying to obtain the Acinetobacter livening microbial inoculum.
3. The Acinetobacter livers microbial inoculum according to claim 2, wherein the preparation method of the Acinetobacter livers KJ-1 fermentation broth comprises the following steps:
(1) inoculating acinetobacter calcoaceticus KJ-1 into a nutrient agar culture medium, and culturing at 28-30 ℃ for 20-28 h to obtain an activated strain;
(2) inoculating the activated strain prepared in the step (1) into a nutrient broth liquid culture medium according to the inoculum size of 2-10%, and carrying out shake cultivation at 25-30 ℃ and 150-200 rpm for 20-28 h to prepare a fermentation liquid.
4. The acinetobacter livetoo bacteria agent of claim 3, wherein the nutrient agar culture medium in step (1) comprises 10g of peptone, 3g of beef powder, 5g of sodium chloride, 18.0 g of agar, and the balance of water, and the pH value is 7.0-7.4; the nutrient broth liquid culture medium in the step (2) comprises 10g of peptone, 3g of beef powder, 5g of sodium chloride and the balance of water per liter of culture medium, and the pH value is 7.0-7.4.
5. Use of acinetobacter calcoaceticus according to claim 1 for treating petroleum pollutants.
6. Use of an Acinetobacter vivarium inoculant according to any one of claims 2 to 4 in the treatment of petroleum pollutants.
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