Method for recovering rare earth ions by adsorption of bacillus subtilis during culture

Abstract

The application provides a method for adsorbing and recovering rare earth ions in the culture process of bacillus subtilis, which comprises the following steps: 1) enlarged culture of the strain; 2) culture adsorption: inoculating the logarithmic phase seed liquid obtained in step 1) into LB liquid medium containing rare earth ions and carrying out shaker culture adsorption for 22-26 hours to obtain a bacterial body and spore suspension after adsorbing terbium; 3) elution and recovery: after centrifugal separation of the bacterial body and spore suspension obtained in step 2) at room temperature to remove the supernatant, adding excessive 2,6-pyridine dicarboxylic acid for elution, centrifuging again, collecting the supernatant, and realizing the recovery of the rare earth ions. The application belongs to the field of biological environmental protection technology, realizes the cooperation of culture and adsorption, effectively saves time and reduces cost, and improves the rare earth adsorption and recovery effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological environmental protection, and particularly relates to a method for adsorbing and recovering rare earth ions in the cultivation process of Bacillus subtilis. BACKGROUND

[0002] Rare earth is the general term of 17 metals of lanthanide series, scandium and yttrium. It is widely used in new energy, national defense and medical diagnosis due to its unique photoelectric and magnetic properties, and is called "industrial vitamin". As a non-renewable resource and important strategic resource, it is urgent to solve the waste and recycling problems of rare earth resource exploitation.

[0003] Traditional rare earth extraction methods, such as CN 108285979A and other prior art, have problems such as the need to use a large amount of acid, alkali, organic extractant, complex process and easy to cause secondary pollution. In contrast, microbial adsorption method has the advantages of simple process, less organic reagent and economic and environmental protection, and is widely used in rare earth adsorption research. CN 109402390A discloses a method for recovering rare earth ions by Bacillus subtilis spores, which utilizes the strong adsorption of Bacillus subtilis spores to rare earth ions formed under poor nutritional conditions, adsorbs rare earth ions, and then elutes and recovers; although the removal rate is high, the spores need to be enriched and the overall process time is long. CN110938565A discloses a method for recovering rare earth ions by Bacillus cereus DW019, which collects cell bacteria by culture, then incubates in a solution containing rare earth ions to collect cell bacteria loaded with rare earth ions, and then elutes with ethylenediaminetetraacetic acid solution and recovers rare earth ions by centrifugation; although the recovery rate of 17 kinds of rare earth ions is high, multiple collection operations are required.

[0004] Therefore, it is of great significance to provide a method for adsorbing and recovering rare earth ions in the cultivation process of Bacillus subtilis with simple operation. SUMMARY

[0005] To solve the problems in the prior art, the present application has unexpectedly found that by inoculating Bacillus subtilis into LB liquid medium containing terbium for cultivation, the cultivation and adsorption are carried out simultaneously, effectively saving time and reducing cost. In addition, compared with the existing technology of cultivating first and then adsorbing, the present application not only saves the step of microbial enrichment and purification, but also improves the rare earth adsorption and recovery effect. Under similar biomass and the same concentration of rare earth terbium, the adsorption rate is increased by 26.96% to 80.53%, the eluted bacteria can be reused, and the waste of resources and environmental pollution are effectively reduced. Based on the above findings, the present application is obtained.

[0006] The object of the present application will be further illustrated by the following detailed description.

[0007] The present application provides a method for recovering rare earth ions by adsorption during cultivation of Bacillus subtilis, comprising the following steps: 1) enlarged cultivation of the strain (referred to as expansion cultivation): inoculating activated Bacillus subtilis into LB liquid medium and shaking bed cultivation to obtain logarithmic phase seed liquid;

[0008] 2) cultivation and adsorption: inoculating the logarithmic phase seed liquid obtained in step 1) into LB liquid medium containing rare earth ions and cultivating and adsorbing for 22-26 h to obtain bacterial body and spore suspension after adsorption of terbium;

[0009] 3) elution and recovery: after centrifugal separation of the bacterial body and spore suspension obtained in step 2) at room temperature to remove supernatant, adding excess 2,6-pyridinedicarboxylic acid for elution, centrifuging again, collecting the supernatant, and realizing recovery of the rare earth ions.

[0010] The above technical solution realizes simultaneous cultivation and adsorption of Bacillus subtilis, effectively saves time and reduces cost, not only eliminates the step of microbial enrichment and purification, but also improves the effect of rare earth adsorption and recovery, and under similar biomass and the same concentration of terbium, the adsorption rate is increased by 26.96%-80.53%, the bacterial body after elution can be reused, and resource waste and environmental pollution are effectively reduced.

[0011] Preferably, the rare earth ions are terbium (Tb). The absorption spectrum of terbium fluorescence detection is 275 nm, and the emission spectrum is 545 nm.

[0012] Preferably, the concentration of terbium is 0-900 mg / L.

[0013] More preferably, the concentration of terbium is >0 and ≤300 mg / L.

[0014] When the concentration of terbium is 50 mg / L and 300 mg / L, respectively, the adsorption and recovery rate of terbium by the strain in the cultivation environment is close to 100% in 24 h, and when the concentration of terbium increases to 900 mg / L, the recovery rate of terbium is only 87%. By cultivating and adsorbing for an appropriate time (22-26 h), a higher biomass can be obtained, which is beneficial to the adsorption and recovery of terbium in the cultivation liquid. Compared with high concentration of terbium, the growth of the strain is promoted at low concentration, the maximum biomass is reached faster, and the total biomass in 12 h is slightly higher. Although increasing the concentration of terbium ions can increase the recovery amount of terbium, too high concentration of terbium significantly inhibits the growth of the bacterial body, greatly prolongs the time to reach the maximum biomass, and reduces the adsorption and recovery efficiency.

[0015] In addition, the present application adopts excess DPA to elute the precipitate after adsorption, and the time required for complete elution is different under different concentrations of terbium; under low concentration (≤300 mg / L) of terbium, the excess DPA elution requires 10-22 min; when the concentration of terbium increases to 900 mg / L, the time required for complete elution of terbium is only 1.5-3.5 min.

[0016] Preferably, the conditions of the shaking culture in step 1) are 36-38℃, 220-300 rpm, and 5-6 h.

[0017] Preferably, the preparation method of the LB liquid medium comprises the following steps: adding NaCl 10 g, proteose peptone 10 g, and yeast extract powder 5 g into 970 mL of deionized water, adjusting the pH to 7.0 with 1 mol / L NaOH, and constant volume to 1 L, and then high-pressure sterilization.

[0018] Preferably, the Bacillus subtilis is Bacillus subtilis 168 with the accession number of ATCC NO:27370, which has strong stress resistance and wide application range.

[0019] Preferably, the activation in step 3) comprises the following steps: streaking the preserved Bacillus subtilis strain onto LB solid medium and culturing overnight at 36-38℃.

[0020] Preferably, the centrifugal speed in step 3) is 8000-12000 rpm, and the time is 1-3 min.

[0021] Compared with the prior art, the present application has the following beneficial effects: (1) The present application realizes the simultaneous culture and adsorption by inoculating Bacillus subtilis into LB liquid medium containing terbium, saves the step of microbial enrichment and purification, avoids the problem that the adsorption performance of the microorganism is damaged in the step of microbial enrichment and purification, effectively saves time and reduces cost. (2) Compared with the existing technical route of culturing first and then adsorbing (referred to as post-culture adsorption), the simultaneous culture and adsorption (referred to as in-culture adsorption) adopted by the present application can significantly improve the adsorption recovery effect of rare earth, and under similar biomass and the same concentration of rare earth terbium, the adsorption rate is increased by 26.96%-80.53%, the eluted bacterial cells can be reused, and resource waste and environmental pollution are effectively reduced. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The post-culture adsorption of the prior art and the in-culture adsorption of the present application are schematically shown in the following figure.

[0023] Figure 2 The adsorption results of terbium in Bacillus subtilis culture and post-culture.

[0024] Figure 3 Growth of Bacillus subtilis under different terbium concentrations.

[0025] Figure 4 Terbium elution in the culture after 24h cultivation.

[0026] Figure 5 Effect of DPA elution on spore germination. DETAILED DESCRIPTION

[0027] The application will be further described in detail below with reference to the accompanying drawings and examples.

[0028] In the application, LB liquid medium and the like are all conventional commercially available products, or can be obtained by conventional technical means in the art. Bacillus subtilis is Bacillus subtilis 168, with the preservation number of ATCC NO:27370.

[0029] Example 1 Method for adsorbing and recovering rare earth ions by Bacillus subtilis during cultivation

[0030] The method for adsorbing and recovering rare earth ions by Bacillus subtilis during cultivation comprises the following steps:

[0031] 1) Expansion culture of the strain: inoculate the activated Bacillus subtilis into LB liquid medium and shake bed culture to obtain logarithmic phase seed liquid; inoculate the preserved Bacillus subtilis strain Bacillus subtilis 168 into LB solid medium and cultivate at 37°C overnight to obtain the activated Bacillus subtilis; the conditions for shake bed culture are 37°C, 250 rpm and 5h;

[0032] 2) Cultivation and adsorption: inoculate the logarithmic phase seed liquid obtained in step 1) into LB liquid medium containing 0, 50, 300 and 900 mg / L terbium respectively at a 2‰ volume ratio of inoculation amount, and shake bed cultivate and adsorb at 37°C and 200 rpm for 24h to obtain the bacterial body and spore suspension after adsorbing terbium; according to the plate experiment, the biomass adsorbed in the culture is OD 600 =4.5;

[0033] 3) Elution and recovery: after centrifugal separation of the bacterial body and spore suspension obtained in step 2) to remove the supernatant at room temperature, add excess 2,6-pyridine dicarboxylic acid for elution, centrifuge again, collect the supernatant, and realize the recovery of rare earth ions; the centrifugal speed is 10000 rpm and the time is 2 min. Take the supernatant and determine the content of terbium therein, so as to calculate the adsorption rate of terbium.

[0034] Comparative Example 1

[0035] The method for adsorbing terbium by Bacillus subtilis after cultivation comprises the following steps:

[0036] 1) the expansion culture of the strain: the activated Bacillus subtilis was inoculated into LB liquid medium and cultured in a shaker to obtain logarithmic phase seed liquid; the preserved Bacillus subtilis strain Bacillus subtilis 168 was streaked onto LB solid medium and cultured at 37°C overnight to obtain the activated Bacillus subtilis; the shaker culture conditions were 37°C, 250 rpm, and 5 h;

[0037] 2) spore enrichment: the logarithmic phase seed liquid obtained in step 1) was inoculated into LB liquid medium at a volume ratio of 2‰ to culture for 72 h; the bacterial liquid was centrifuged at 4°C and 8000 rpm for 10 min to obtain a bacterial mixture;

[0038] 3) spore purification: the bacterial mixture obtained in step 2) was washed with sterile water for 3 times, and further purified by a cell disrupter to obtain a spore mother liquor;

[0039] 4) adsorption detection: according to the biomass OD 600 = 4.5 in Example 1, a spore suspension with similar biomass was prepared, and the adsorption equilibrium after 50, 300, and 900 mg / L terbium was determined, with 0 mg / L as a control.

[0040] The existing technology culture adsorption and the culture adsorption process of the present application are shown in Figure 1 The adsorption results of Example 1 and Comparative Example 1 on terbium are compared, and the results are shown in Figure 2 Under similar biomass and the same terbium concentration, the adsorption rate of Bacillus subtilis in culture is significantly different from that after culture, and compared with the spore adsorption after culture, the adsorption rate of Bacillus subtilis in culture under 50, 300, and 900 mg / L terbium is increased by 30.40%, 26.96%, and 80.53%, respectively.

[0041] Example Two: Growth of Bacillus subtilis under Different Concentrations of Terbium Stress

[0042] 1) the expansion culture of the strain: the activated Bacillus subtilis was inoculated into LB liquid medium and cultured in a shaker to obtain logarithmic phase seed liquid; the preserved Bacillus subtilis strain Bacillus subtilis 168 was streaked onto LB solid medium and cultured at 37°C overnight to obtain the activated Bacillus subtilis; the shaker culture conditions were 37°C, 250 rpm, and 5 h;

[0043] 2) culture determination: the seed liquid was inoculated into LB liquid medium containing 0, 50, 300, 600, 900, and 1200 mg / L terbium at a volume ratio of 2% and cultured in a shaker at 37°C and 200 rpm; samples were taken every 1 h to determine the OD 600 of the culture liquid by an enzyme marker until it was relatively stable.

[0044] The growth of Bacillus subtilis under different concentrations of terbium is shown in Figure 3As shown, the addition of high concentrations of terbium severely inhibited the growth of Bacillus subtilis. Compared with the control group, the addition of 900 mg / L terbium significantly prolonged the growth retardation period of the strain, and the OD value at 14 h was also significantly lower. 600 The lag time was significantly lower, and as the terbium concentration increased to 1200 mg / L, the OD at steady state (14 h) increased. 600 The effect is even more pronounced.

[0045] Example 3: Elution and Recovery

[0046] 1) Centrifugation: The bacterial cultures that had been cultured and adsorbed for 24 hours in LB liquid medium containing 0, 50, 300 and 900 mg / L terbium were centrifuged at 10,000 rpm and room temperature for 2 min to obtain a precipitate containing bacterial cells.

[0047] 2) Elution and Recovery: The precipitate was eluted with DPA (concentration 2.5 times the terbium concentration in the culture medium) for different times, and the fluorescence intensity (excitation wavelength 275 nm, emission wavelength 545 nm) was measured until equilibrium was reached. The eluted terbium concentration and the total terbium concentration in the precipitate were calculated based on the fluorescence intensity, and the elution rate was determined. This allowed for the determination of the time required for complete terbium elution, enabling the separation of the supernatant and precipitate after centrifugation at 10,000 rpm at room temperature, thus achieving terbium recovery.

[0048] from Figure 4 It can be seen that, under excess DPA, it takes about 2 minutes to completely elute the terbium adsorbed in the precipitate obtained in a medium containing 900 mg / L terbium. However, the elution of terbium from precipitates with lower terbium concentrations is much slower, taking about 18 minutes.

[0049] Example 4: Effect of DPA elution on spore germination

[0050] 1) Centrifugal purification: After the cultured bacterial solution was adsorbed, it was centrifuged at 8000 rpm and 4℃ for 10 min to obtain a mixture containing bacterial cells. This mixture was washed with sterile water, and centrifuged after each wash, for a total of 3 times. The washed mixture was then subjected to a cell disruptor at 50% power for 6 min, and centrifuged to remove impurities around the precipitate to purify the spores. The purity of the spores was determined by microscopic examination to determine whether the purification steps of disruption and centrifugation needed to be repeated until the spore purity was >99%.

[0051] 2) Germination determination: The OD of the mother liquor was measured using a UV spectrophotometer. 600 And based on the initial OD of the obtained mother liquor 600 Preparation of OD 600 The spore suspension was 2.0 μL. After elution with 10 mM DPA, the suspension was heat-shocked at 60°C for 30 min and then incubated on ice for 15 min before being added to an ELISA plate to achieve the final OD value. 600was 1.0. Then 10 mmol·l -1 of valine (L-Val) was added to the above enzyme plate, and the OD 600 change was measured every 4 min with an enzyme plate reader to determine the spore germination (spore germination would cause the OD 600 of the spore population to decrease due to the outflow of the contents, and when the germination is complete, it would decrease by about 40% to 60%, and the OD 600 change can be used to determine the germination of the spore population). The spore germination of the non-eluted spores removes the DPA elution process, and the heat shock and germination determination steps are the same.

[0052] As shown in Figure 5 , the inhibition or promotion effect of spore germination after DPA elution is obviously removed, which indicates that the DPA elution process does not affect the germination of the spores and does not affect the reuse of the spores.

[0053] The above is a further detailed description of the present application in combination with specific preferred embodiments, and cannot be considered as limiting the specific implementation of the present application to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, a number of simple deductions or substitutions can be made without departing from the concept of the present application, and all of them should be considered as falling within the protection scope of the present application.

Claims

1. A method for adsorbing and recovering rare earth ions during the cultivation of Bacillus subtilis, characterized in that: Includes the following steps: 1) Large-scale culture of the strain: The activated Bacillus subtilis was inoculated into LB liquid medium and cultured on a shaker to obtain the logarithmic seed culture; 2) Culture and adsorption: The logarithmic seed culture obtained in step 1) was inoculated into LB liquid medium containing rare earth ions and cultured on a shaker for 22-26 h to obtain the bacterial cells and spore suspension after terbium adsorption. 3) Elution and recovery: After centrifuging the bacterial cells and spore suspension obtained in step 2) at room temperature to remove the supernatant, excess 2,6-pyridinedicarboxylic acid is added for elution, followed by centrifugation again to collect the supernatant, thereby recovering rare earth ions; the rare earth ions are terbium.

2. The method for adsorbing and recovering rare earth ions during the cultivation of Bacillus subtilis according to claim 1, characterized in that: The concentration of terbium is 0-900 mg / L.

3. The method for adsorbing and recovering rare earth ions during the cultivation of Bacillus subtilis according to claim 2, characterized in that: The concentration of terbium is >0 and ≤300mg / L.

4. The method for adsorbing and recovering rare earth ions during the cultivation of Bacillus subtilis according to any one of claims 1 to 3, characterized in that: The conditions for shaking culture in step 1) are 36–38℃, 220–300 rpm, and 5–6 h.

5. The method for adsorbing and recovering rare earth ions during the cultivation of Bacillus subtilis according to any one of claims 1 to 3, characterized in that: The preparation method of the LB liquid culture medium includes the following steps: add 10g of NaCl, 10g of peptone, and 5g of yeast extract to 970mL of deionized water, adjust the pH to 7.0 with 1mol / L NaOH and bring the volume to 1L, and then autoclave to obtain the medium.

6. The method for adsorbing and recovering rare earth ions during the cultivation of Bacillus subtilis according to any one of claims 1 to 3, characterized in that: The Bacillus subtilis strain mentioned is Bacillus subtilis 168, with accession number ATCC NO:27370.

7. The method for adsorbing and recovering rare earth ions during the cultivation of Bacillus subtilis according to any one of claims 1 to 3, characterized in that: The activation in step 1) includes the following steps: streaking the preserved Bacillus subtilis strain onto LB solid medium and culturing it overnight at 36-38°C.

8. The method for adsorbing and recovering rare earth ions during the cultivation of Bacillus subtilis according to any one of claims 1 to 3, characterized in that: In step 3), the centrifugation speed is 8000-12000 rpm and the time is 1-3 min.

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