Pseudomonadaceae petroleum degrading bacterium L-1, microbial mixed agent of Pseudomonadaceae petroleum degrading bacterium L-1 and in-situ remediation method of petroleum-contaminated soil
A technology of Pseudomonas and mixed bacterial agents, which is applied in the fields of energy biotechnology and environmental biology, can solve the problems of secondary pollution, unsuitable use, and low degree of comprehensive utilization, so as to restore ecological functions and eliminate harm to the human body Effect
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Embodiment 1
[0033] Example 1 Isolation and Identification of Pseudomonas Pseudomonadaceae Strains
[0034] The present invention provides a Pseudomonas oleaginous bacteria L-1, which is isolated from the oily mud sand of Changqing Oilfield, and is initially identified as belonging to the Pseudomonas genus ( Pseudomonadaceae). The strain can use petroleum hydrocarbons as the sole carbon source for growth, and the simulation test under the conditions of temperature 25-35°C, pH between 7-8, and TDS between 0-800ppm shows that the Pseudomonas can significantly remove petroleum hydrocarbons , The removal rate can reach 77%.
[0035] The oil-contaminated soil suspension was inoculated in the nutrient solution NS-1 with crude oil (6g / L) as the only carbon source at an inoculation amount of 15% by mass, and cultured on a shaker at 150r / min at 28°C for 3-4d under aerobic conditions. , transfer once under the same conditions. Dilute and apply to a solid medium with crude oil as the only carbon s...
Embodiment 2
[0042] The preparation of embodiment 2 microbial mixed bacterial agents
[0043]1. Inoculation and activation of microbial strains
[0044] Under sterile conditions, mix Pseudomonas oleuropeaceae L-1 with indigenous bacteria, [indigenous bacteria are Bacillus encimensis strain encimensis, Alcaligenes faecalisstrain Nocardioid spp. (Nocardioides luteus) Acinetobacter sp) Penicillium oxalicum (Penicillium oxalicum)] according to the ratio of 1:1:1:1:1:1 were inoculated into the Erlenmeyer flask containing nutrient broth (after sterilization ), respectively put the above Erlenmeyer flasks into a shaker at 30°C and 200r / min to incubate for 48h, and set aside.
[0045] 2. Fermentation preparation of liquid mixed bacterial agent
[0046] 2.1 Preparation of bacterial seed solution
[0047] The above-mentioned Pseudomonas oleaginus L-1 and native bacteria cultured for 48 hours were respectively inserted into a bacterial fermentation tank according to the incorporation amount of 5%,...
Embodiment 3
[0050] Example 3 Microbial remediation treatment of oily sludge
[0051] The invention also provides an in-situ remediation method for petroleum-contaminated soil using a microbial mixed bacterial agent. The Pseudomonas oleobacterium L-1 of the present invention can be combined with the enriched and cultivated indigenous bacteria in oil-contaminated soil for in-situ restoration, and according to the crude oil content of the polluted soil, it generally accounts for 20 wt% of the bacterial agent content after mixing. In-situ remediation methods for oil-contaminated soil, including:
[0052] (1) Revive the required microbial strains first, then expand the culture, ferment and produce, and reserve; put them in a fermenter according to a certain ratio at 30°C, and ferment at 200r / min for standby;
[0053] (2) Add native bacteria and bacterial agent nutrient solution according to the corresponding proportion, so that C:N:P=100:10:3 in the soil; carry out ventilation and watering ev...
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