[0023] Example 1: Isolation and identification of Lysinibacillus sp. M14 from manganese contaminated soil
[0024] (1) Sample collection: The manganese soil sample for this test was collected from the surface soil of the manganese raw material warehouse of the Malleable Iron & Steel Plant in Xiqing District, Tianjin, China in late June 2007.
[0025] (2) Sample enrichment: Take 100g soil sample and add sterile MnCl to the soil sample 2 Make the solution to a final concentration of 989.55mg/Kg, gently stir evenly and place it in a 28°C incubator for one week, pay attention to adding sterile water to ensure the humidity of the sample.
[0026] (3) Separation of manganese oxidizing bacteria: accurately weigh the MnCl 2 10g of the enriched soil sample is placed in an Erlenmeyer flask containing 90ml of sterile saline, shaken in a shaker at 28°C for half an hour, then 1ml is added to 9ml of sterile saline and gradually diluted to 10 -2 , 10 -3 , 10 -4 , Take 0.1ml coating containing 20mmol/L N’-a-hydroxythylpiperazine-N’-ethanesulfanic acid (HEPES for short, adjust the pH to 7.0) and 0.3mmol/L MnCl respectively 2 K solid medium plate (van Waasbergen et al., 1993), each dilution was coated with 3 plates, and placed in a 28°C incubator for one week. The brown-brown colonies were regarded as suspected manganese-oxidizing bacteria. Store the plate in a refrigerator at 4°C for later use. The above K solid medium formula is as follows: yeast extract 0.5g, peptone 2g, agar 15g, 1L artificial seawater (1L artificial seawater: NaCl 13.14g, KCl 0.56g, MgSO 4 ·7H 2 O 9.24g, CaCl 2 0.83g).
[0027] (4) Streak separation: pick different colonies of the suspected manganese oxidizing bacteria obtained in step (3) to streak to ensure that a single clone is obtained. Use K medium plate for streaking, and place in a 4℃ refrigerator after the bacteria grow. Medium for use and keep one part in a glycerin freezer in a refrigerator at -80°C.
[0028] (5) Identification of the manganese oxidation properties of the strain: transfer the single clone obtained in step (4) to containing 20mmol/L HEPES (pH 7.0) and 0.3mmol/LMnCl 2 K solid medium plate, place it in a 28℃ incubator, and when a brown colony appears on the plate, add Berbelin blue-I(LBB,N.N'-Dimethylamino-p,p'-triphenyhnethane-o"-sulphonicacid ) Drop it on the brown colony to detect its oxidizing property. If the color of the solution does not change, it means that the bacteria has no manganese oxidizing ability. If the color changes from light blue to dark blue, it means that the bacteria has manganese oxidizing ability (see image 3 , LBB is a redox colorant, Mn 3+/4+ +LBB(reduced)> Mn 2+ +LBB (oxidized), the oxidized LBB is dark blue). The preparation method of LBB is as follows: Dissolve 40 mg of LBB in 100ml of 40mmol/L acetic acid aqueous solution at 4°C overnight in the dark.
[0029] (6) Classification and identification of manganese-oxidizing bacteria: Amplify 16S rDNA using colony PCR method: use toothpicks to pick out monoclonal strains and mix them in 50μL sterile double-distilled water; heat shock at 100°C for 5 minutes, -20°C for 5 minutes, cold and hot After repeating 2~3 times, centrifuge at 12000r/min for 3min, and the supernatant obtained was directly used as a PCR amplification template. The amplification system was a 50μL system. Universal primers: 27 (5'AGAGTTTGATCMTGGCTCAG3') and 1492R (5'GGYTACCTTGTTACGACTT3) ′). The amplification conditions were: pre-denaturation, 95°C, 5min; denaturation, 94°C, 45sec; annealing, 49°C, 45sec; extension, 72°C, 1.5min denaturation-extension, 35 cycles; extension: 72°C, 10min. The nucleotide sequence shown in SEQID NO:1 in the sequence table is obtained, and the sequence length is 1395 bp. Use 1% agarose gel electrophoresis to detect PCR products. Purify the PCR product with the column DNA recovery kit produced by Shanghai Cyberway Gene Technology Co., Ltd., operate according to the instructions, and send it to Sanbo Yuanzhi Company for sequencing. Compare the obtained 16S rDNA gene sequence (see SEQ ID NO:1 for the sequence list) with the existing sequence in the GenBank library (see the comparison result figure 1 ).
[0030] Mycological characteristics of the manganese oxide producing bacteria (Lysinibacillus sp. M14) isolated in the present invention:
[0031] Short rod-shaped bacteria, gram-positive bacteria, suitable for growth temperature 28-30 ℃, suitable pH 7.0-7.5, without MnCl 2 On the K solid medium, the colonies are round, milky white, convex, moist on the surface, and irregular edges. With 0.3mmol/L MnCl 2 On K medium, the colony surface is brown (see figure 2 ); see the scanning electron micrograph of the bacteria image 3.
[0032] The preservation method of Lysinibacillus sp.M14 is as follows:
[0033] Lysinibacillus sp. M14 can be cultured at 28°C on K liquid or solid medium (the formula is the same as above), and can be stored at 4°C for short-term storage after cultivation. For long-term preservation, glycerin freezing tubes or freeze-dried tubes (Zhao Bin, He Shaojiang, 2002) can be used to preserve this strain.