Bacteria Lysinibacillus sp. for adsorbing gold and antimony

[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.

[0034] Example 2: Manganese oxidation removal curve of Lysinibacillus sp. M14

[0035] Prepare 3 bottles of sterilized liquid K medium (the formula is the same as above), each 100mL. Add 20mmol/L filtered and sterilized hydroxyethylpiperazine ethanesulfonic acid (HEPES), while adding 1mol/L sterile MnCl 2 The mother liquor makes the final concentration 0.3mmol/L. Then inoculate the log phase (OD 600 The value is about 0.7) M14 bacterial liquid, inoculated at a volume ratio of 1%. Take a sample of 10 mL immediately after mixing, and place this sample in a clean centrifuge tube as the first sample. Place each Erlenmeyer flask in a shaker at 28°C and 160r/min for shaking culture. After that, the sampling interval was 24h, and the experiment was set to repeat three times. After sampling, the samples are processed as follows:

[0036] 1) The 10mL sample obtained each time is centrifuged at 8000r/min for 6min at high speed, the supernatant is retained, and filtered with an aqueous membrane with a pore size of 0.22μm, and the filtrate is stored at -20°C. The applicant refers to the Mn(II) present in this partial solution as "remaining Mn(II)".

[0037] 2) The precipitate obtained by centrifugation is processed as follows: After repeated washing and centrifugation with deionized water 3 times, 4 mL of CuSO with a concentration of 0.1 mol/L is added 4 For the solution, use a pipette to disperse the bacteria and shake at a speed of 160r/min for 12h. Then take it out, dilute the bacterial suspension in the tube to 10 mL with deionized water, centrifuge at 8000r/min for 6min, retain the supernatant, filter it with an aqueous membrane with a pore size of 0.22μm, and store the filtrate at -20°C. The applicant calls the Mn(II) present in this partial solution "adsorbed Mn(II)".

[0038] 3) The remaining precipitate is treated as follows: After repeated washing with deionized water for 3 times, add 4 mL of hydroxylamine hydrochloride mother liquor (0.1 mol/L), use a pipette to break up the bacteria, and shake at a speed of 160r/min 12h. Then take it out, dilute the bacterial suspension in the tube to 10 mL with deionized water, centrifuge at 8000r/min for 5 minutes, save the supernatant, filter it with an aqueous membrane with a pore size of 0.22μm, and store the filtrate at -20°C. The applicant calls the Mn(II) present in this partial solution "oxidized Mn(II)".

[0039] The concentration of Mn(II) contained in the sample was measured with a flame atomic absorption spectrophotometer, and the measured Mn(II) concentration of the sample was used as the ordinate and time as the abscissa.

[0040] In the M14 bacteria's oxidation removal experiment on Mn(II), because the manganese oxidizing bacteria will produce a stress response to the concentration of Mn(II) and secrete protein substances, so in order to better understand this strain oxidation removal of Mn(II) ), the initial concentration of Mn(II) was selected to be 30mM. Figure 4 It is a graph of the oxidation removal of Mn(II) by M14 bacteria. It can be seen from the figure that the removal rate of manganese by the isolated M14 strain of the present invention has been increasing over time, especially in 3-7 days. The fastest, probably because the strain began to oxidize the Mn(II) in the solution during this period to produce manganese oxide. At 10d, the concentration of "remaining Mn(II)" reached the lowest, and the removal rate reached 92.62%. From the beginning of the reaction, Mn(II) began to be adsorbed on the surface of the bacteria or manganese oxide, and it increased continuously before 5d, but decreased after 5d, which may be part of the adsorbed Mn(II) After being oxidized, the concentration of "adsorbed Mn(II)" is about 0.04mM. The applicant found that the manganese oxidation curve has always been on a rising trend. At 3-7 days, the remaining Mn(II) in the solution is continuously decreasing, but the "oxidized Mn(II)" is continuously increasing, while the adsorbed Mn(II) It did not increase significantly, indicating that most of the Mn(II) was oxidized. At 10d, the oxidation efficiency of the isolated M14 bacteria of the present invention reached the highest, about 82.66% (see Figure 4 ).

[0041] Example 3: Determination of the ability of manganese oxide produced by Lysinibacillus sp.M14 to absorb gold in a gold-containing solution

[0042] The Lysinibacillus sp. M14 cells of the present invention are inoculated into 5mM Mn 2+ K medium (the formula is the same as above), cultured in a shaker at 28℃, 150r/min for 10 days, the total amount of medium is 3000mL, after the brown-black precipitate is formed in the medium, the culture is centrifuged at a high speed of 8000r/min After removing the supernatant, the precipitate was freeze-dried at -56°C, ground, and passed through a 100-mesh sieve. Add 1.87g of bio-manganese oxide to an Erlenmeyer flask containing 100mL of double-distilled water. The Erlenmeyer flask must be covered with black plastic before the experiment to prevent Au(III) from changing color when exposed to light. After shaking, add 200μL 100mmol/L After mixing, sample 3mL as the first sampling. Place each Erlenmeyer flask in a shaker at 28°C and 150r/min, take samples every 0.5h for the first 1h, and then take samples every 1h for centrifugal filtration. Sampling for 6 hours, the 3mL suspension is centrifuged at a high speed of 8000r/min for 5min each time, the supernatant is retained, and filtered with an aqueous membrane with a pore size of 0.22μm. The filtrate is placed in a clean 5ml centrifuge tube and stored at -20℃. Detection. After sampling, the remaining Au(III) concentration was measured with a flame atomic absorption spectrophotometer.

[0043] Using time as the abscissa and Au(III) concentration as the ordinate, draw the change curve of Au(III) remaining in the biomanganese oxide adsorption gold system (see Figure 5 ).

[0044] In this example, two reaction systems were set up, namely a blank control and a biological manganese oxide adsorption system. Each reaction system was tested according to the method described in Example 3. The manganese oxide produced by the M14 bacteria was collected, and the Au(III ) For the adsorption capacity see Figure 5.

[0045] In the blank experiment, no manganese oxide was added to the gold-containing solution. The initial concentration of Au(III) was 200μM. Within 6h, the As(III) concentration in the solution was always in a relatively stable state, but it was lower than the initial concentration. About 21.53μM Au(III), the analysis may be adsorbed by other means, such as the Erlenmeyer bottle.

[0046] The biomanganese oxide adsorption system for Au(III) is composed of 100ml of double distilled water, 200μM Au(III) and 1.87g of manganese oxide produced by M14 bacteria. In the first 0.5h, the adsorption amount of Au(III) increased rapidly. After 1h, the adsorption of Au(III) by biomanganese oxide reached equilibrium, and the adsorption efficiency was close to 100%.