Immobilized particles for degrading crystal violet and preparation method thereof

Abstract

The invention discloses immobilized particles capable of efficiently and rapidly degrading crystal violet and a preparation method of the immobilized particles, and belongs to the technical field of biology, and the immobilized particles are mainly prepared from bacterial strains, sodium alginate, chitosan and biochar; the bacterial strain is Arthrobacter sp., and the preservation number of the bacterial strain is CICC (China General Microbiological Culture Collection Center) No. 23408. The method for obtaining the crystal violet degrading bacteria is simple, stable and good in operability, and the prepared immobilized particles can solve the problems that a traditional carrier material is poor in microbial activity, low in treatment efficiency, small in recycling frequency and the like. The obtained immobilized particles can rapidly decolorize and degrade crystal violet dye, the treatment cost is low, the efficiency is high, and compared with a chemical method, the immobilized particles are safer and free of secondary pollution.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to immobilized particles for degrading crystal violet and a preparation method thereof. Background Art

[0002] With the rapid development of society, the dye industry has become one of the world's three major chemical industries. At the same time, more and more printing and dyeing wastewater pollution problems have become increasingly prominent. Dye wastewater has attracted much attention due to its light and thermal stability, difficult biodegradability, and the toxicity and carcinogenicity of its degradation products, making it one of the most difficult environmental pollution problems to deal with.

[0003] Crystal violet is a triphenylmethane dye that is widely used as a biological dye in human and veterinary medicine, and can also be used as a fabric dye in the textile industry. Although crystal violet is widely used in cytology and bacteriology, it is notorious for causing environmental pollution because its discharge into water bodies will seriously affect the transmittance of water bodies, threaten the growth of plants and animals in water bodies, and once it enters the human system, it may interfere with DNA replication, cause mutations and carcinogenesis, and pose a huge threat to human health. However, since most triphenylmethane dyes have low production costs, strong stability and good use effects, it is difficult to find suitable substitutes, so this type of dye is still widely used, seriously damaging the ecological balance in the use area. Therefore, studying methods for efficiently and low-consumption degradation of wastewater containing crystal violet is of great significance to alleviating environmental pressure, protecting ecological balance, and maintaining the healthy and stable life of the people.

[0004] Immobilized microorganisms technology is a technology that uses physical or chemical methods to fix free cells or enzymes on carrier materials to reduce bacterial loss and improve microbial utilization. However, in previous microbial immobilization technologies, there are problems such as short service life of immobilized particles, low reuse rate and poor microbial activity, which greatly limit the application of decolorization strains. Summary of the invention

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an immobilized particle for efficiently and rapidly degrading crystal violet and a preparation method thereof, which solves the problems of short service life, low reuse rate and poor microbial activity existing in the prior art.

[0006] Technical solution: An immobilized particle for degrading crystal violet is prepared by cross-linking and fixing crystal violet degrading bacteria, sodium alginate, biochar, and chitosan; the crystal violet degrading bacteria is Arthrobacter sp. domesticated with a crystal violet solution, and the Arthrobacter sp. has a preservation number of CICC No. 23408.

[0007] Furthermore, the method for preparing the immobilized particles comprises the following steps:

[0008] Step 1, preparing sodium alginate solution, calcium chloride solution and chitosan solution;

[0009] Step 2: Sterilize the sodium alginate solution, calcium chloride solution and chitosan solution obtained in step 1 in an autoclave, then take them out and cool them to room temperature;

[0010] Step 3, adding biochar to the bacterial suspension of crystal violet degrading bacteria, mixing evenly, filtering to obtain an adsorption carrier, and adding all of it to the sodium alginate solution, mixing evenly, to obtain a mixed solution;

[0011] The crystal violet degrading bacteria are Arthrobacter sp. domesticated with crystal violet solution, and the Arthrobacter sp. is of the genus Arthrobacter sp., and the preservation number is CICC No. 23408;

[0012] Step 4: Add the mixed solution dropwise into the calcium chloride solution, stir until the gel balls harden, let stand for cross-linking and fixation, put into the chitosan solution and stir, filter out and rinse to obtain the immobilized particles.

[0013] Furthermore, in step 1: the sodium alginate solution is formed by mixing sodium alginate and ultrapure water, and the mass volume ratio of sodium alginate to ultrapure water is (1-3) g: 100 mL; the calcium chloride solution is formed by mixing calcium chloride and ultrapure water, and the mass volume ratio of calcium chloride to ultrapure water is (1-3) g: 100 mL; the chitosan solution is formed by mixing chitosan and ultrapure water, and the mass volume ratio of chitosan to ultrapure water is (1-3) g: 100 mL;

[0014] Furthermore, in step three, the biochar is corn straw biochar, and the mass volume ratio of the biochar to the bacterial suspension is (0.5-1.5) g:100 mL.

[0015] Furthermore, in step 3, the method for preparing the bacterial solution of crystal violet-degrading bacteria is as follows:

[0016] Step (1), inoculating untamed Arthrobacter on a plate medium containing crystal violet, culturing at 30° C. for 24 h, selecting Arthrobacter with good growth potential and inoculating it into a basic culture dish, and placing it under shaking culture at 30° C. and 150 r / min for 48 h;

[0017] Step (2), taking 1 mL of the enriched bacterial solution on the basic culture dish in step (1), transferring it to a basic culture medium containing 20 mg / L crystal violet solution, placing it under constant temperature shaking culture at 30° C. and 150 r / min for 48 h, and then sequentially increasing the crystal violet content in the basic culture medium containing 20 mg / L crystal violet solution, and increasing the mass concentration of crystal violet to 40, 60, 80, and 100 mg / L in sequence, for strain directional induction;

[0018] Step (3), applying the acclimated bacterial suspension capable of decolorizing 100 mg / L crystal violet solution on a solid dye plate containing 30 mg / L crystal violet, isolating crystal violet-degrading bacteria, and performing strain identification to obtain crystal violet-degrading bacteria;

[0019] Step (4), taking crystal violet degrading bacteria to a plate culture medium, placing the plate culture medium in a biochemical incubator at 30°C and culturing for 24 hours until a single colony grows, picking a colony with good growth and inoculating it into a liquid basal culture medium, culturing at 30°C and 150r / min under shaking conditions until the logarithmic growth phase, centrifuging the bacterial solution after the cultivation, discarding the supernatant, taking the wet bacterial cells at the bottom of the centrifuge tube, washing the bacterial cells with sterile water several times, and collecting the bacterial cells after centrifugation, adding physiological saline to the collected bacterial cells, and adjusting the OD 600 to 1.0 to obtain bacterial suspension, which was stored in a refrigerator at 4°C.

[0020] Furthermore, in step three, the volume ratio of the bacterial suspension to the sodium alginate solution is 1:1.

[0021] Furthermore, in step 4, the volume of the calcium chloride solution is at least 5 times that of the mixed solution in step 3.

[0022] Furthermore, step four is specifically as follows:

[0023] The mixture was added dropwise to the calcium chloride solution and stirred until the gel balls hardened. The cross-linking fixation time was 4 h, the small balls were filtered out, rinsed with sterile water for several times, and placed in the chitosan solution. After uniform stirring for 10 min, the small balls were filtered out and rinsed with sterile water for several times again to obtain the immobilized particles. The particles were sealed and stored at 4 °C for later use.

[0024] Furthermore, when the immobilized particles are used to degrade crystal violet, the feed ratio of the immobilized particles to the solution containing crystal violet is (2-16) g:100 mL, and the concentration of the crystal violet solution is 50-300 mg / L.

[0025] Beneficial effects:

[0026] 1) The present invention utilizes immobilized particles synthesized from efficient decolorizing strains, sodium alginate, chitosan and biochar to efficiently and quickly degrade wastewater containing crystal violet, opens up a new method and new idea for treating crystal violet wastewater, provides a direction for completely degrading crystal violet wastewater, and also provides a practical method for effectively preventing crystal violet wastewater pollution, which has wide application significance.

[0027] 2) The immobilized particles of the present invention are strains that are directional induced to obtain highly efficient decolorizing strains. The original strain is Arthrobacter sp., original number: WN3-1, which was deposited in the China Industrial Microbiological Culture Collection Management Center, Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing on October 31, 2008, with a deposit number of CICC No. 23408. The present invention prepares immobilized particles of the strains after directional induction of the original strains, thereby improving the problems of easy loss of the microorganisms themselves in practical applications, poor environmental adaptability, and long degradation period, so that the residual crystal violet in the environment can be removed or reduced more stably and efficiently, achieving the effect of environmental treatment.

[0028] 3) The preparation method of the immobilized particles of the present invention is simple, the production cost is low, the recovery is easy, the practical application is convenient and fast, there is no secondary pollution, and it is more environmentally friendly.

[0029] 4) In the preparation method of the immobilized particles provided by the present invention, the mechanical strength of the composite carrier is increased after biochar modification and chitosan coating, and it has the ability to resist hydraulic shock and adapt to acid-base changes, so that the mechanical strength and mechanical stability of the immobilized particles prepared by the present invention far exceed those of traditional embedding carriers, greatly improving the service life of the present invention and the recycling rate.

[0030] In summary, the present invention provides a method for preparing immobilized particles that can efficiently and quickly degrade crystal violet, and has the advantages of fast degradation rate, good stability, long service life, simple process operation, low production cost, etc. It is an effective way to solve the problem of crystal violet pollution in wastewater and is conducive to industrial promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A visual representation of the immobilized particles.

[0032] Figure 2 The surface and cross-sectional images of the scanning electron microscope images of the immobilized particles, where (a) is the surface image and (b) is the cross-sectional image.

[0033] Figure 3 Comparison of mechanical strength of different types of gel balls.

[0034] Figure 4 The effect of the dosage of immobilized particles on the decolorization rate of crystal violet.

[0035] Figure 5 This is the effect of the initial concentration of crystal violet on the decolorization rate of crystal violet by immobilized particles. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is described in detail below through embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0037] Example 1

[0038] The original strain selected in this example was purchased from the China Industrial Microbiological Culture Collection Center, and is: Arthrobacter sp., original number: WN3-1, and was preserved in the China Industrial Microbiological Culture Collection Center on October 31, 2008, Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing, with a preservation number: CICC No. 23408.

[0039] The culture medium and reagents involved in this embodiment are:

[0040] Basic culture medium: 5 g peptone; 3 g beef extract; 5 g sodium chloride; 1 L deionized water; pH = 7 ± 0.2.

[0041] Acclimation culture medium: 5 g peptone; 3 g beef extract; 5 g sodium chloride; 0.02-0.1 g crystal violet; 1 L deionized water; pH = 7 ± 0.2.

[0042] Decolorization medium: After the crystal violet mother solution is filtered through a 0.22 μm filter membrane, it is added to the enrichment medium after high-pressure sterilization. The initial concentration of crystal violet in the medium is adjusted according to the amount of crystal violet added.

[0043] Plate culture medium: 3 g glucose; 0.3 g magnesium sulfate; 0.5 g ammonium sulfate; 0.3 g sodium chloride; 0.03 g ferrous sulfate heptahydrate; 2 g potassium hydrogen sulfate; 0.03 g manganese sulfate; 0.1 g crystal violet; 1 L deionized water; pH = 7 ± 0.2.

[0044] Solid dye culture medium: 3 g glucose; 0.3 g magnesium sulfate; 0.5 g ammonium sulfate; 0.3 g sodium chloride; 0.03 g ferrous sulfate heptahydrate; 2 g potassium hydrogen sulfate; 0.03 g manganese sulfate; 0.03 g crystal violet; 1 L deionized water; pH = 7 ± 0.2.

[0045] The steps for strain-directed induction of the strain and preparation of bacterial suspension are as follows:

[0046] Step (1), inoculating the untamed Arthrobacter purchased from the China Industrial Microbiological Culture Collection Administration Center. The untamed Arthrobacter is inoculated on the above-mentioned plate culture medium containing crystal violet, and cultured at 30° C. for 24 hours, then picking the Arthrobacter with better growth on the plate culture medium and inoculating it into a 50 ml basic culture dish, and placing it at 30° C. and 150 r / min for 48 hours of shaking culture;

[0047] Step (2), take 1 mL of the enriched bacterial solution on the basic culture dish in step (1), transfer it to 50 mL of the basic culture medium containing 20 mg / L crystal violet solution, place it under constant temperature shaking culture conditions of 30° C. and 150 r / min for 48 h, and observe the decolorization of the crystal violet. It can be observed that the crystal violet solution slowly changes from dark purple to light purple, and finally to light yellow, or even colorless.

[0048] In a basic culture medium containing 20 mg / L crystal violet solution, the crystal violet content was increased in sequence, and the mass concentration of crystal violet was increased to 40, 60, 80, and 100 mg / L in sequence, and the strain was induced in a directed manner, and the degradation and decolorization effects of different concentrations of crystal violet were measured;

[0049] Step (3), applying the acclimated bacterial suspension capable of decolorizing 100 mg / L crystal violet solution on a solid dye culture medium containing 30 mg / L crystal violet, isolating crystal violet-degrading bacteria, and performing strain identification to obtain crystal violet-degrading bacteria, i.e., a highly efficient decolorizing strain.

[0050] Step (4), take the above strain on a plate culture medium, place the plate culture medium in a biochemical incubator at 30°C and culture for 24 hours until a single colony grows, pick the colony with good growth and inoculate it into a liquid basal culture medium, shake and culture at 30°C and 150r / min until the logarithmic growth phase, centrifuge the bacterial solution at 8000r / min for 10 minutes after the culture, discard the supernatant, take the wet bacteria at the bottom of the centrifuge tube, rinse the bacteria with sterile water, centrifuge again, and repeat 3 times. Add an appropriate amount of physiological saline to the collected bacteria, adjust the OD 600 to 1.0 to obtain bacterial suspension, which was stored in a refrigerator at 4°C.

[0051] OD 600 Determination method: Use a spectrophotometer to measure the absorbance of bacterial culture solution at 600nm, that is, OD 600 .

[0052] Example 2

[0053] In this embodiment, sodium alginate, chitosan and biochar are all purchased from Sinopharm Chemical Reagent Co., Ltd. Sodium alginate and chitosan are both purchased chemically pure. The mesh number of biochar is 120 mesh, and the biochar can be corn straw biochar.

[0054] The preparation steps for the preparation of immobilized particles for the degradation of crystal violet are as follows:

[0055] Step 1: Solution preparation:

[0056] Preparation of sodium alginate solution: Mix 2 g of sodium alginate and 100 ml of ultrapure water, heat to 55° C. in a water bath and stir until the solution is completely dissolved to obtain a sodium alginate solution with a concentration of 2%. The pH value of the sodium alginate solution is preferably 7±0.02.

[0057] Preparation of calcium chloride solution: 15g CaCl2 and 500ml ultrapure water are mixed and stirred until completely dissolved to obtain a CaCl2 solution with a concentration of 3%. The pH value of the CaCl2 solution is preferably 7±0.02.

[0058] Preparation of chitosan solution: 1 g of chitosan and 100 ml of ultrapure water were mixed and stirred until completely dissolved to obtain a chitosan solution with a concentration of 1%. The pH value of the chitosan solution was preferably 7±0.02.

[0059] Step 2: Place the sodium alginate solution, CaCl2 solution and chitosan solution obtained in step 1 into a high pressure sterilizer respectively, sterilize at 121°C for 20 min, take out and cool to room temperature.

[0060] Step 3: Take 100 ml of the bacterial suspension prepared in Example 1, add 1 g of sterilized biochar, the concentration of biochar in the bacterial suspension is 1%, and shake at a constant temperature of 30°C and a rotation speed of 150 r / min for 2 hours, and filter to obtain an adsorption carrier. Add all the adsorption carriers to the sodium alginate solution (100 ml), mix evenly, and obtain a mixed solution;

[0061] Step 4: Use a syringe, preferably a 10 ml syringe, on a workbench to absorb the mixed solution (100 ml) in step 3 and drip it dropwise into an excess of CaCl2 solution (500 ml in this embodiment), stir until the gel hardens, put it in a 4°C refrigerator, let it stand for 4 hours for cross-linking and fixation, and then filter out the gel balls; put the gel balls into a 1% chitosan solution (100 ml, chitosan is 1 g) and stir at a uniform speed for 10 minutes, then filter out; finally, wash the gel balls with deionized water 3 times to obtain composite immobilized Arthrobacter particles, referred to as immobilized particles. The CaCl2 solution is at least 5 times the mixed solution in step 3. The above workbench is preferably a sterilized ultra-clean workbench.

[0062] The gel spheres prepared according to the above steps were recorded as SA / BC / CTS.

[0063] Gel spheres were prepared according to the above steps, except that the gel spheres filtered out after static cross-linking and fixation for 4 hours in step 4 were not placed in the chitosan solution for stirring. Other operations were the same, and the obtained gel spheres were recorded as SA / BC.

[0064] Gel spheres were prepared according to the above steps, except that in step 3, no biochar was added to the bacterial suspension. Other operations were the same, and the obtained gel spheres were recorded as SA / CTS.

[0065] Gel spheres were prepared according to the above steps, except that no biochar was added to the bacterial suspension in step 3, and no chitosan solution was added for stirring in step 4. The obtained gel spheres were recorded as SA.

[0066] Immobilized particles (SA / BC / CTS) such as Figure 1 As shown, the internal structural characteristics of the immobilized particles prepared in this example were observed using SEM technology, and the SEM scanning image is shown in Figure 2 The appearance of the immobilized particles is black spherical, without adhesion or tailing, and the particle size is uniform, with a diameter of 2 to 3 mm. The appearance is maintained for a long time and the performance is stable. The biochar is evenly distributed and embedded inside, which has a skeleton support effect, making the interior present a three-dimensional grid structure, enhancing the stability and mechanical strength of the immobilized particles, so that the internal microorganisms can effectively resist the influence of water turbulence and hydraulic shear force in the reactor.

[0067] The adaptability and tolerance of gel balls to changes in environmental factors are important factors reflecting their performance. The applicability of gel balls is evaluated by measuring their mechanical properties.

[0068] Three gel balls of each of the four types of SA / BC / CTS, SA / CTS, SA / BC, and SA were randomly selected and placed under a glass sheet. A 50 g weight was placed on top of the glass sheet. The time was set for 60 seconds, and pressure was slowly applied (usually at a constant speed, such as 0.1-0.5 mm / s) until the gel ball broke or reached a predetermined deformation degree. The radius of the gel ball before and after pressure was measured, and the maximum pressure that the front of the gel ball could withstand was calculated using the following formula, which represented the average mechanical strength P (g / mm 2 ), and three parallel samples were set up in each group.

[0069]

[0070] Wherein: d is the front radius of the gel ball, mm; R is the radius of the gel ball before pressure, mm; r1, r2, r3 are the radius heights of the gel ball after pressure, mm.

[0071] The mechanical strength of the four groups of gel balls was measured by the pressing method. Figure 3As shown in the figure, compared with the other three gel balls, the SA ball has a lower density of internal cross-linked structure and the worst mechanical properties, with a mechanical strength of 8.44 g / mm 2 The surface of the coated SA / CTS ball has a polyelectrolyte film, which makes its surface structure more compact, so the mechanical strength is slightly increased. The mechanical strength of the SA / BC ball is 10.38g / mm 2 , proving that the skeleton support of biochar can improve the mechanical properties of immobilized particles. The mechanical strength of SA / BC / CTS pellets is 18.20 g / mm 2 , compared with the other three types of gel balls, it has obvious advantages. Therefore, in actual application, the SA / BC / CTS balls have a longer service life and are capable of resisting hydraulic shock.

[0072] The present invention provides a physical barrier for degradation bacteria through chitosan coating and sodium alginate matrix, avoids direct damage to strains by adverse external environment, significantly prolongs the service life of strains, and forms a suitable microenvironment inside the immobilized carrier, reduces the risk of strains exposed to adverse conditions, and ensures its continuous and efficient metabolic activity. Secondly, the composite carrier formed by sodium alginate and biochar has high mechanical strength, and the chitosan coating further enhances the toughness and wear resistance of the particles. The carrier can maintain structural integrity during multiple uses, is not easy to break, and thus achieves long-term reuse, and biochar has a high specific surface area and abundant surface functional groups, can effectively adsorb crystal violet, reduce the degradation pressure of strains, and the synergistic effect of adsorption and degradation improves the decolorization efficiency, so that the particles maintain high performance during multiple uses. Finally, the immobilized particles can be directly recovered by a simple separation method (such as filtration, sedimentation) after use, avoiding the cumbersome operation of repeated inoculation in suspended bacterial culture. After each use of the immobilized particles, the active strains are still retained in the carrier, without frequent supplementation or replacement, thereby reducing costs and improving utilization.

[0073] The immobilized particles prepared by the present invention improve the shortcomings of the microorganisms themselves in practical applications, such as easy loss, poor environmental adaptability, and long degradation period. The immobilized particles prepared by the present invention have a fast degradation rate, good stability and mechanical strength, and the microorganisms inside them can effectively resist the influence of water turbulence and hydraulic shear force in the reactor. At the same time, the problems of short service life, low reuse rate, and poor activity of microorganisms are also solved.

[0074] Example 3

[0075] In order to determine the optimal ratio of each raw material in the prepared immobilized particles, based on the results of the single factor preliminary test, four factors, namely, sodium alginate concentration, biochar content, CaCl2 concentration, and chitosan concentration, were selected as the investigation objects, respectively. The decolorization rate of crystal violet by immobilized particles was used as the evaluation index, and L9(34 ) orthogonal table was arranged to produce 9 kinds of sodium alginate / biochar / chitosan beads for orthogonal experiments.

[0076] The preparation method of immobilized particles and the decolorization experimental steps are as follows:

[0077] 1) Add sterilized biochar to 100 ml of bacterial suspension, oscillate at a constant temperature of 30° C. and a rotation speed of 150 r / min for 2 h, and filter to obtain the adsorption carrier.

[0078] Add sodium alginate to 100 ml of deionized water, heat in a water bath at 55°C until completely dissolved by stirring to obtain a sodium alginate solution, add all the adsorption carriers to the sodium alginate solution, mix well to obtain a mixed solution;

[0079] 2) Use a 10mL syringe to drop 100mL of the mixture into 500mL of calcium chloride solution while stirring. Continue stirring for 30min after the addition is complete until the gel balls harden. Place in a 4°C refrigerator and allow to crosslink for 4h. Filter out the balls and rinse with deionized water three times.

[0080] 3) Put it into 100 ml of chitosan solution, stir it at a constant speed for 10 minutes, filter out the small balls, rinse it three times with deionized water to obtain composite immobilized Arthrobacter particles (referred to as immobilized particles), and seal it and store it at 4°C for use.

[0081] 4) 10 g of different immobilized particles were placed in 100 mL of decolorization medium with a pH value of 7.00±0.20 and a crystal violet concentration of 100 mg / L. After constant temperature oscillation at 30° C. for 120 min, the crystal violet decolorization rate was measured.

[0082] The level ranges of each factor are shown in Table 1.

[0083] Table 1. Table of factors and their levels in orthogonal experiment

[0084]

[0085] The results of the orthogonal test are shown in Table 2. The group with the highest crystal violet decolorization rate is Group 5 A2B2C3D1, and the decolorization rate reaches 90.20%. According to the principle that the larger the mean K is, the better the level is, the data in the table are analyzed, KA2>KA3>KA1, KB2>KB3>KB1, KC3>KC2>KC1, KD1>KD2>KD3, so A2B2C3D1 is also the theoretical optimal condition (experiment number 5), that is, the sodium alginate concentration is 2%, the biochar content is 1%, the calcium chloride concentration is 3%, and the chitosan concentration is 1%, which is the optimal level combination of each factor. Because the larger the range R is, the greater the influence of the factor on the index, as shown in Table 2, R2>R4>R1>R3, that is, the influence of the level changes of the four factors on the decolorization rate is: biochar>chitosan>sodium alginate>calcium chloride.

[0086] Table 2L9(3 4 )Orthogonal experimental design and results

[0087] Test No. A B C D Decolorization rate 1 1 0.5 1 1 69.07 2 1 1 2 2 73.07 3 1 1.5 3 2 69.62 4 2 0.5 2 3 70.41 5 2 1 3 1 90.20 6 2 1.5 1 2 80.75 7 3 0.5 3 2 68.25 8 3 1 1 3 73.53 9 3 1.5 2 1 81.92 <![CDATA[Mean K1]]> <![CDATA[KA 1 =0.6303]]> <![CDATA[KB 1 =0.6129]]> <![CDATA[KC 1 =0.6656]]> <![CDATA[KD 1 =0.7562]]> <![CDATA[Mean K2]]> <![CDATA[KA 2 =0.7338]]> <![CDATA[KB 2 =0.7534]]> <![CDATA[KC 2 =0.6998]]> <![CDATA[KD 2 =0.6981]]> <![CDATA[Mean K3]]> <![CDATA[KA 3 =0.7281]]> <![CDATA[KB 3 =0.7260]]> <![CDATA[KC 3 =0.7268]]> <![CDATA[KD 3 =0.6379]]> Extreme R <![CDATA[R 1 =0.1035]]> <![CDATA[R 2 =0.1405]]> <![CDATA[R 3 =0.0612]]> <![CDATA[R 4 =0.1183]]>

[0088] Example 4

[0089] In order to determine the effects of different influencing factors in the prepared immobilized particles on the decolorization of crystal violet, two influencing factors were produced to conduct exploratory experiments.

[0090] The preparation method of immobilized particles and the decolorization experimental steps are as follows:

[0091] 1) Add 1g of sterilized biochar to 100ml of bacterial suspension, oscillate at a constant temperature of 30°C and a speed of 150r / min for 2h, and filter to obtain an adsorption carrier. Add 2g of sodium alginate to 100ml of deionized water, heat in a 55°C water bath until completely dissolved by stirring to obtain a sodium alginate solution, add all the adsorption carriers to the sodium alginate solution, and mix well to obtain a mixed solution;

[0092] 2) Use a 10mL syringe to drop 100mL of the mixed solution into 500mL of calcium chloride solution (containing 15g of calcium chloride) while stirring. Continue stirring for 30min after the addition is complete until the gel balls harden. Place in a 4℃ refrigerator, let stand for 4h to crosslink, filter out the balls, and rinse with deionized water three times;

[0093] 3) Put it into 100 ml chitosan solution (1 g chitosan), stir it at a constant speed for 10 min, filter out the small balls, rinse them three times with deionized water to obtain composite immobilized Arthrobacter particles (referred to as immobilized particles), and seal them and store them at 4°C for use.

[0094] 4) The prepared immobilized particles of different masses were placed in a decolorization medium with a pH value of 7.00±0.20 and different crystal violet concentrations. After constant temperature oscillation at 30° C. for 120 min, the decolorization rate of crystal violet was measured.

[0095] The results are as follows:

[0096] 1) The dosage of immobilized particles has a great influence on the decolorization effect of crystal violet solution;

[0097] 100 mL of decolorization medium with an initial concentration of 100 mg / L of crystal violet was prepared, and the pH was adjusted to 7.00 ± 0.20. The dosage of immobilized particles was set to 2%, 4%, 6%, 8%, 10%, 12%, 14%, and 16% in order of solid-liquid ratio, that is, 2 g, 4 g, 6 g, 8 g, 10 g, 12 g, 14 g, and 16 g of immobilized particles were added to each 100 mL of decolorization medium with an initial concentration of 100 mg / L of crystal violet, and the culture was placed under shaking conditions at 30°C and 150 r / min. After 96 hours, samples were taken to determine the decolorization rate of crystal violet.

[0098] The decolorization effect of different dosages of immobilized particles on the culture medium with a crystal violet concentration of 100 mg / L is shown in Figure 2. Figure 4 As shown in the figure, the dosage of immobilized particles is proportional to the decolorization rate of crystal violet. When the dosage of immobilized particles increases from 2% to 10%, the decolorization rate of crystal violet increases from 33.03% to 90.20%, with a significant growth trend. This may be because the abundant oxygen-containing functional groups on the surface of the immobilized particles are combined with the crystal violet molecules by hydrogen bonds. The crystal violet is degraded after contacting the strains on the immobilized particles, which promotes more dye molecules to be adsorbed on the immobilized particles and continue to be used by the strains. A micro-reactor of adsorption-degradation is formed inside the immobilized particles. With the increase in the dosage of immobilized particles, the dye molecules in the solution obtain more active adsorption sites, and the total amount of microorganisms participating in the reaction gradually increases, which promotes the migration-enrichment of dye molecules in the system, so the decolorization effect of the crystal violet solution changes significantly. When the dosage of immobilized particles exceeds 10%, the growth trend of the decolorization rate weakens. This is because the addition of excessive immobilized particles causes a large number of active adsorption sites on the composite carrier to be in an unsaturated state, and the growth space, nutrients and dye molecules that can be provided to the strain are limited. Excessive immobilized particles cannot be fully utilized, so the decolorization rate change trend is no longer obvious. In order to use resources more efficiently and reasonably, the addition amount of immobilized particles is determined to be 10%, and the decolorization rate can reach a maximum of 90.20%.

[0099] 2) The initial concentration of crystal violet has a great influence on the decolorization effect of the crystal violet solution by the immobilized particles;

[0100] Prepare 100 mL of decolorization medium, set the initial concentration of crystal violet to 50, 100, 150, 200, 250, 300 mg / L, and adjust the pH to 7.00 ± 0.20. The dosage of immobilized particles is 10%, and the medium is placed in a shaking culture at 30°C and 150 r / min. Samples are taken at 6, 12, 24, 48, 72, and 96 hours to determine the decolorization rate of crystal violet.

[0101] The decolorization effect of immobilized particles on crystal violet solution under different initial crystal violet concentrations is as follows Figure 5 As shown in the figure, when the initial concentration of crystal violet is 50 mg / L, the decolorization rate of the immobilized particles is 90.2% after 96 hours of decolorization. This is because biochar has an adsorption effect on crystal violet. Even if the concentration of crystal violet in the culture medium is low, it can be enriched near the immobilized particles, increasing the chance of contact between the strain and crystal violet, which is conducive to enhancing the decolorization effect of the immobilized particles. When the initial concentration of crystal violet is 300 mg / L, the decolorization time is from 6h to 48h, and the decolorization rate of the immobilized particles increases from 16.64% to 64.90%, an increase of about 48.26%. The results show that under the condition of high initial concentration of crystal violet, the decolorization rate of the immobilized particles in the early stage of the reaction changes more significantly. It may be because the crystal violet adsorbed inside the pores of the immobilized particles has a concentration gradient in the process of being transferred to the strain, and the concentration is partially reduced. The strain is less affected by the toxicity of crystal violet, the environmental adaptation period is shortened, and it enters the logarithmic growth period quickly, producing a highly efficient decolorization effect. As the initial concentration of crystal violet increased from 200 mg / L to 300 mg / L, after 48 hours of decolorization, the decolorization rate of crystal violet by immobilized particles decreased from 84.24% to 64.90%, a decrease of about 19.34%. The results show that the concentration limit that immobilized particles can tolerate for crystal violet is relatively low. In order to better compare the decolorization effect, the initial crystal violet concentration was determined to be 100 mg / L, and the decolorization rate could reach up to 90.20%.

[0102] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention.

Claims

1. An immobilized particle for degrading crystal violet, characterized in that: The immobilized particles are prepared by cross-linking and fixing crystal violet degrading bacteria, sodium alginate, biochar and chitosan; The crystal violet degrading bacteria are Arthrobacter sp. domesticated with a crystal violet solution, and the Arthrobacter sp. is of the genus Arthrobacter sp., and the preservation number is CICC No. 23408.

2. A method for preparing the immobilized particles according to claim 1, characterized in that: The steps include: Step 1, preparing sodium alginate solution, calcium chloride solution and chitosan solution; Step 2: Sterilize the sodium alginate solution, calcium chloride solution and chitosan solution obtained in step 1 in an autoclave, then take them out and cool them to room temperature; Step 3, adding biochar to the bacterial suspension of crystal violet degrading bacteria, mixing evenly, filtering to obtain an adsorption carrier, and adding all of it to the sodium alginate solution, mixing evenly, to obtain a mixed solution; The crystal violet degrading bacteria are Arthrobacter sp. domesticated with crystal violet solution, and the Arthrobacter sp. is of the genus Arthrobacter sp., and the preservation number is CICC No. 23408; Step 4: Add the mixed solution dropwise into the calcium chloride solution, stir until the gel balls harden, let stand for cross-linking and fixation, put into the chitosan solution and stir, filter out and rinse to obtain the immobilized particles.

3. The preparation method according to claim 2, characterized in that: In step one: The sodium alginate solution is prepared by mixing sodium alginate and ultrapure water, and the mass volume ratio of sodium alginate to ultrapure water is (1-3) g:100 mL; The calcium chloride solution is prepared by mixing calcium chloride and ultrapure water, and the mass volume ratio of calcium chloride to ultrapure water is (1-3) g:100 mL; The chitosan solution is prepared by mixing chitosan and ultrapure water, and the mass volume ratio of chitosan to ultrapure water is (1-3) g:100 mL; In step three: the biochar is corn straw biochar, and the mass volume ratio of the biochar to the bacterial suspension is (0.5-1.5) g:100 mL.

4. The preparation method according to claim 2, characterized in that: In step 3, the method for preparing the bacterial solution of crystal violet-degrading bacteria is as follows: Step (1), inoculating untamed Arthrobacter on a plate medium containing crystal violet, culturing at 30° C. for 24 h, selecting Arthrobacter with good growth potential and inoculating it into a basic culture dish, and placing it under shaking culture at 30° C. and 150 r / min for 48 h; Step (2), taking 1 mL of the enriched bacterial solution on the basic culture dish in step (1), transferring it to a basic culture medium containing 20 mg / L crystal violet solution, placing it under constant temperature shaking culture at 30° C. and 150 r / min for 48 h, and then sequentially increasing the crystal violet content in the basic culture medium containing 20 mg / L crystal violet solution, and increasing the mass concentration of crystal violet to 40, 60, 80, and 100 mg / L in sequence, for strain directional induction; Step (3), applying the acclimated bacterial suspension capable of decolorizing 100 mg / L crystal violet solution on a solid dye plate containing 30 mg / L crystal violet, isolating crystal violet-degrading bacteria, and performing strain identification to obtain crystal violet-degrading bacteria; Step (4), taking crystal violet degrading bacteria to a plate culture medium, placing the plate culture medium in a biochemical incubator at 30°C and culturing for 24 hours until a single colony grows, picking a colony with good growth and inoculating it into a liquid basal culture medium, culturing at 30°C and 150r / min under shaking conditions until the logarithmic growth phase, centrifuging the bacterial solution after the cultivation, discarding the supernatant, taking the wet bacterial cells at the bottom of the centrifuge tube, washing the bacterial cells with sterile water several times, and collecting the bacterial cells after centrifugation, adding physiological saline to the collected bacterial cells, and adjusting the OD 600 to 1.0 to obtain a bacterial suspension, which was stored in a refrigerator at 4°C.

5. The preparation method according to claim 4, characterized in that: In step 3, the volume ratio of the bacterial suspension to the sodium alginate solution is 1:

1.

6. The preparation method according to claim 4, characterized in that: In step 4, the volume of the calcium chloride solution is at least 5 times that of the mixed solution in step 3.

7. The preparation method according to claim 4, characterized in that: Step 4 is as follows: The mixture was added dropwise to the calcium chloride solution and stirred until the gel balls hardened. The cross-linking fixation time was 4 h, the small balls were filtered out, rinsed with sterile water for several times, and placed in the chitosan solution. After uniform stirring for 10 min, the small balls were filtered out and rinsed with sterile water for several times again to obtain the immobilized particles. The particles were sealed and stored at 4 °C for later use.

8. The preparation method according to claim 2, characterized in that: When the immobilized particles are used to degrade crystal violet, the feed ratio of the immobilized particles to the solution containing crystal violet is (2-16) g:100 mL, and the concentration of the crystal violet solution is 50-300 mg / L.

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