Microbes Bn-88, microbial agent and application in degradation of petroleum hydrocarbons
A microbial inoculum and microbacteria technology, applied in the field of microorganisms, can solve the problems of unstable bacterial function effect, difficult to ferment and expand cultivation, difficult to practical application and promotion, etc., and achieve the effects of short lag period, easy cultivation and strong degradation ability.
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Embodiment 1
[0045] The screening of embodiment 1 bacterial strain
[0046] Enrichment medium: nutrient broth medium, peptone 10g, sodium chloride 5g, beef extract 3g, distilled water 1000ml, if a solid medium is used, add 2% agar.
[0047] Degradation medium: NaCl 1.0g, (NH 4 ) 2 · SO 4 0.617g, KH 2 PO 4 0.50g, K 2 HPO 4 1.0g, MgSO 4 0.50g, CaCl 2 0.1g, KCl 0.10g, FeSO 4 ·7H 2 O 0.01g, 1000mg of crude oil, 1000ml of distilled water, adjust the pH to 7.0-7.2, if preparing a solid medium, add 2% agar, sterilize in an autoclave at 121°C for 20min, and set aside.
[0048] Take 10g of soil moderately polluted by petroleum hydrocarbons in a certain place in Dongying, add 90ml of distilled water, stir evenly, use a pipette gun to take 10ml of the mixed solution and inoculate it into 100mL of enrichment medium. The amount was transferred to a new enrichment medium and continuously enriched for 3 generations. The above-mentioned enrichment solution was serially diluted, spread and in...
Embodiment 2
[0049] The identification of embodiment 2 bacterial strains
[0050] The strains were identified by cell morphology, colony characteristics, physiological and biochemical characteristics and 16S rRNA sequence.
[0051] According to Dong Xiuzhu and Cai Miaoying's "Common Bacterial System Identification Manual", the morphological and physiological and biochemical characteristics of the strains were determined.
[0052] The cloning and sequence analysis methods of the strain 16S rRNA gene are as follows:
[0053] (1) The extraction of bacterial strain genomic DNA uses the bacterial genomic DNA extraction kit of Beijing Quanshijin Biotechnology Co., Ltd.;
[0054] (2) Using 16S rRNA gene universal primers
[0055] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1),
[0056] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.2),
[0057] PCR amplification was performed using genomic DNA as a template.
[0058] The amplification conditions were: pre-denaturation at 95°C for 2 min, followed...
Embodiment 3
[0065] The growth curve of embodiment 3 strain Bn-88
[0066] Strain growth curve drawing: Pick a ring of bacterial lawn, inoculate it into nutrient broth medium, culture at 30°C, 150r / min for 20h, and prepare seed solution. Pack several culture tubes, each tube is filled with 5mL of nutrient broth medium, each culture tube is inoculated with seed solution at 5% inoculum, put in 30°C, 150r / min for cultivation, at 2h, 4h, 6h, 8h , 10h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, and 48h, samples were taken respectively, and the bacterial concentration was determined by turbidimetric method. Three parallel samples were taken each time, and the average value of OD600 was measured to represent the bacterial concentration of the culture solution at the sampling time point. Measure the absorbance value of different culture time, draw the strain growth curve, the result is as follows: image 3 shown.
[0067] It can be known from the results that the growth and reproduction of the strain c...
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