Preparation method of petroleum degrading bacteria solid microbial inoculum and method for repairing petroleum-polluted soil by using solid microbial inoculum prepared by preparation method

Preparation method of petroleum degrading bacteria solid microbial inoculum and method for repairing petroleum-polluted soil by using solid microbial inoculum prepared by preparation method

  • CN 104,450,597 B
  • Filed: 12/10/2014
  • Issued: 07/28/2020
  • Est. Priority Date: 12/10/2014
  • Status: Active Grant
First Claim
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1. A preparation method of a solid microbial inoculum of petroleum degrading bacteria is characterized by comprising the following steps:

  • the method comprises the following specific steps;

    A. screening and domesticating petroleum degrading bacteria1) Collection of bacterial sourcesTaking the soil polluted by petroleum throughout the year by an oil refinery as a petroleum degradation indigenous bacteria source;

    2) primary screen for oil degradation indigenous bacteria(1) Taking a soil sample as a petroleum degrading indigenous bacteria source, adding the soil sample into a conical flask filled with sterile water, and placing the conical flask in a shaking incubator to shake for 0.5-2 h at room temperature, wherein the shaking frequency is 200 rpm;

    (2) taking the supernatant from the conical flask in the step (1), inoculating the supernatant into an enrichment medium, and carrying out shake culture on a shaking table at the temperature of 30 ℃ and

    the rotating speed of 175rpm for 48 h;

    coating the cultured enrichment solution on a screening culture medium flat plate, and culturing at constant temperature of 30 ℃

    for 48 h;

    3) domesticationInoculating and domesticating a plurality of single strains obtained by primary screening by using an inoculating loop in a domestication culture medium, and specifically comprises the steps of inoculating the plurality of single strains obtained by primary screening by using the inoculating loop into a 100m L domestication culture medium A respectively, culturing for a period, then taking a 10m L culture solution from the domestication culture medium A, adding the 10m L culture solution into a 90m L domestication culture medium B, culturing for a period, taking a 10m L culture solution from the domestication culture medium B, adding the 10m L culture solution into a 90m L domestication culture medium C, culturing for a period, taking a 10m L culture solution from the domestication culture medium C, adding the 10m L culture solution into a 90m L domestication culture medium D, culturing for a period, adding the 10m L culture solution into the 90m L domestication culture medium E from the domestication culture medium D, culturing from the domestication culture medium A to the domestication culture medium E, and gradually increasing the petroleum concentration gradient;

    the one cycle is as follows;

    culturing at 30 deg.C and rotation speed of 175rpm under constant temperature shaking for 7 d;

    4) purification of bacterial species(1) Streaking the domesticated culture solution on an oil-containing plate, and culturing at 30 deg.C for 7 d;

    (2) picking a single colony from an oil-containing flat plate, transferring the single colony to an L B inclined plane, and culturing for 24h at 30 ℃

    ;

    (3) re-inoculating the oil-containing plate from the L B slant;

    (4) repeating the steps until a purified colony with a single form is obtained;

    (5) inoculating the purified bacterial colony on a strain preservation culture medium slant, culturing at 30 ℃

    for 24h, and storing in a refrigerator at 4 ℃

    for later use;

    B. preparation of seed culture solutionRespectively and independently inoculating the petroleum degrading strains obtained by purification into a seed culture medium, and gradually performing amplification culture on the strains through a first-stage seed, a second-stage seed and a seed tank to obtain liquid seeds;

    the thallus density of the liquid seed reaches 1 x 107cfu;

    wherein;

    the first-level seeds are cultured by adopting a solid culture medium slant under the culture condition of 30 ℃

    for 24 hours;

    the secondary seeds are cultured in a liquid culture medium for 10-12 hours by shaking table under the conditions of 30 ℃ and

    175rpm of rotation speed;

    the seeding tank culture condition is liquid culture medium stirring culture, the culture condition is constant temperature of 30 ℃

    , the rotating speed is 80rpm, and the ventilation volume is 1;

    1;

    C. fermentation of solid microbial inoculumCrushing or grinding the waste biomass raw material dried by air into particles of 1-5 mm;

    the biomass particles and other ingredients are put into a high-temperature resistant plastic bag and sterilized for 1 hour at 110 ℃

    for standby;

    spraying a seed culture solution in a seed tank to the surface of the compost according to a solid-liquid ratio of 10;

    1 by weight, and uniformly mixing a fermentation seed culture solution with a solid fermentation culture medium;

    putting the inoculated material on a solid fermentation bed, and carrying out enrichment culture at the temperature of 28-30 ℃

    for 3-5 days to obtain a microorganism immobilization material;

    b, inoculating the primary seed solution in the step B into the treated immobilized carrier according to the proportion of 1;

    10(v/w), keeping the water content at 50-55%, and adsorbing, fixing and proliferating for 3-5 days at the temperature of 28-30 ℃

    ;

    proliferating for 3 times to obtain immobilized bacteria agent;

    D. drying, pulverizing, metering, and packaging the piled productDrying the immobilized microbial inoculum at the temperature of 40-60 ℃

    until the water content is 6-10%, crushing the microbial inoculum, and sieving by a 5mm sieve;

    the culture medium used in the steps A and B contains solubilization liquid of petroleum and polysorbate-80, the mass concentration of the petroleum in the solubilization liquid of the petroleum and polysorbate-80 is 2%, the mass concentration of the polysorbate-80 is 4%, and the balance is water.

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