Composite bacterial agent for repairing heavy oil polluted coastline and its preparation method and application
A compound inoculant, coastline technology, applied in microorganism-based methods, restoration of polluted soil, biochemical equipment and methods, etc., can solve the problems of late start, no large-scale practice, etc. The effect of outstanding advantages and efficient repair effect
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Embodiment 1
[0059] Enrichment and Screening of Example 1 Petroleum Degrading Strains
[0060] The bacterial strain involved in the present invention was isolated from the coastline of the Yellow Sea, which was severely polluted by oil spills during the explosion accident of the Dalian Xingang oil pipeline in 2010.
[0061]The specific separation operation is carried out as follows: Add 2 g of coastline and beach samples and a small amount of trace element solution to 100 mL of sterilized broad-spectrum culture solution, and enrich and cultivate on a shaking table at 25 ° C ~ 30 ° C, 140 rpm, and avoid light. 5d; After two consecutive enrichment treatments, transfer 1mL of the enrichment solution into the ASM medium containing 1g / L Venezuelan heavy oil, add a small amount of trace element solution, and culture for 7d at 25°C-30°C, 140rpm, and dark conditions , and then the culture medium was continuously transferred and cultured three times in the same fresh ASM medium containing Venezuela...
Embodiment 2
[0072] The degradability of embodiment 2 single bacterium and mixed flora to Venezuelan heavy oil
[0073] Taking Venezuelan heavy oil as pollutant, the oil degradation performance of the above single and mixed strains was investigated under simulated seawater salinity. Follow the steps below: (1) Pipette 200uL of cryopreserved strains into sterilized liquid LB culture medium with a pipette gun, vibrate and activate at 25°C-30°C, 140rpm for 48h, and repeat transfer to fresh LB culture Base 1 time. At this point, take the turbid solution in the logarithmic phase of growth and centrifuge at room temperature at 5000rpm for 10min, collect the thalli and wash with sterile phosphate buffered saline, after repeating this at least 3 times, dilute to a specific strain density with phosphate buffered saline ( OD 600 ), prepared as a single strain suspension. The above phosphate buffer contains 21.759g K per liter 2 HPO 4 ·H 2 O, 33.48 g Na 2 HPO 4 12H 2 O, 8.79gKH 2 PO 4 , 5....
Embodiment 3
[0075]Embodiment 3 relates to the salt tolerance of strain
[0076] Affected by seawater tides and evaporation, the salinity of the oil spilled coastline environment is relatively high and presents complex changes, which is an important factor affecting the growth, metabolism and competition of microorganisms. The present embodiment is carried out according to the following steps: take the suspensions of Sphingobacterium mizutaii XG-3, Achromobacter xylosoxidans XG-14, and Achromobacter piechaudii XG-11 of equal strain concentration by volume The ratio of 5% was inoculated in LB culture solution with NaCl mass concentration gradients of 0, 1%, 3%, 5%, 7%, and 10%, and cultured for 24 hours under shaking at 28°C, 140rpm, and dark conditions, Measure the OD of each mixture 600 , according to the test results to evaluate the saline-alkali tolerance performance of each dominant strain. If the OD of the bacterial solution 600 >0.25, it is considered that the strain grows well in...
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