Biological mediated aerobic decolorization method of dye and its application
By using Shewanella onedida MR-1 under aerobic conditions with a specific decolorizing medium to degrade dyes, the problems of strict environmental requirements and low efficiency in anaerobic biological decolorization methods have been solved, achieving a highly efficient and environmentally friendly dye decolorization effect, which is suitable for industrial wastewater treatment.
Patent Information
- Application Number
- CN202311715239.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-14
- Publication Date
- 2025-12-09
- Estimated Expiration
- 2043-12-14
AI Technical Summary
Existing anaerobic biological decolorization methods suffer from problems such as strict biological culture environment, low decolorization efficiency, and complex biological systems.
Decolorization was performed using Shewanella onychomycosis MR-1 under aerobic conditions using a specific decolorization culture medium, including sodium lactate, glucose, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and yeast extract. This aerobic dye decolorization method simplifies the biological system and improves decolorization efficiency.
It achieves efficient dye degradation under aerobic conditions, with a degradation efficiency of 96.2%, reducing the stringent requirements for environmental pH and oxygen concentration, simplifying the biological system, lowering waste treatment costs, and making it suitable for industrial wastewater treatment.
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Figure CN117776405B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of sewage treatment, and particularly relates to a biological mediation aerobic dye decolorization method and application thereof. BACKGROUND
[0002] Dye decolorization is a method widely used in many fields such as textile, printing, papermaking, sewage treatment and food industry. The traditional dye decolorization method is usually a chemical method, such as bleaching agent or reducing agent, but these methods produce a large amount of harmful waste and by-products in the dye removal process, causing serious pollution to the environment and consuming a large amount of energy.
[0003] At present, a method of using biology for decolorization has been explored, and most of the existing technologies need to be carried out under anaerobic conditions, and these technologies rely on the electrons released by microorganisms, such as bacteria or fungi, to degrade or reduce dye molecules; at the same time, in the state of isolating oxygen, oxygen and dye are prevented from competing for electrons, so that all the electrons are used for the reduction of dye. The anaerobic biological decolorization technology in the prior art has the technical problems of strict biological culture environment, low decolorization efficiency and complex biological system participating in the decolorization process. SUMMARY
[0004] To solve the technical problems of strict biological culture environment, low decolorization efficiency and complex biological system participating in the decolorization reaction in the prior art anaerobic biological decolorization method, the present application provides a biological mediation aerobic dye decolorization method and application thereof.
[0005] One of the purposes of the present application is to provide a biological mediation aerobic dye decolorization method, which comprises the following steps: inoculating Shewanella bacterial liquid into a decolorization medium, and culturing under aerobic conditions to decolorize the dye.
[0006] Further limitation, the Shewanella is Shewanella oneidensis MR-1.
[0007] Further limitation, the preparation process of the bacterial liquid is as follows: using LB medium, culturing Shewanella oneidensis MR-1 under aerobic conditions at 30℃ for 20h to the stable period, then centrifuging the bacterial body precipitate at 8500rpm for 3min, and finally washing the bacterial body precipitate with phosphate buffer for 3 times to obtain the bacterial liquid.
[0008] Further limitation, the composition of the decolorization medium is as follows: 0.3g / L sodium lactate, 0.3g / L glucose, 10g / L 12 water disodium hydrogen phosphate, 3g / L potassium dihydrogen phosphate, 0.5g / L sodium chloride and 0.2g / L yeast extract.
[0009] Further limited, the culture condition is 30±0.5℃, 130±5rpm.
[0010] Further limited, the additive amount of the dye is 90mg / L, 150mg / L, 210mg / L or 270mg / L.
[0011] Further limited, the dye comprises methyl orange.
[0012] The second object of the present application is to provide a sewage treatment method, which comprises a decolorization treatment step, and the decolorization treatment step adopts the above-mentioned biological mediated aerobic dye decolorization method.
[0013] The beneficial effects of the present application are:
[0014] The present application provides a biological mediated aerobic dye decolorization method, which reduces the strict requirements for environmental pH value, temperature and oxygen concentration compared with the traditional anaerobic biological decolorization method, and only utilizes Shewanella oneidensis in the decolorization method, which simplifies the composition of the biological system and is easy to operate; and in the method, the use of the specific decolorization medium is beneficial to the degradation of the dye by the MR-1 strain under aerobic conditions, and further improves the decolorization efficiency, which can make the decolorization efficiency of 270mg / L of methyl orange reach 96.2% within 160min, while the traditional method makes the degradation rate of 0.015g of methyl orange dye reach 95.11% within 600min; in addition, studies have shown that the MR-1 strain completely degrades 200mg / L of methyl orange dye under anaerobic conditions for about 9h, which proves that the decolorization method provided by the present application has high decolorization efficiency; the present application uses biological strains to treat dyes, which reduces the generation of harmful waste compared with the traditional chemical dye decolorization method, improves the decolorization efficiency of biological anaerobic decolorization, reduces the waste treatment cost, and the aerobic microorganisms can also be recycled, which achieves the purpose of environmental protection and low cost, and is suitable for large-scale application in various industrial productions. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 It is a biological mediated aerobic dye decolorization principle diagram; wherein Flavin is riboflavin, Mtrc, MtrB and Mtr A are ten ferriheme c-type cytochromes, OmcA is a Mtrc homolog protein, OM is an extracellular membrane, IM is an intracellular membrane, CymA is a c-type cytochrome on the inner membrane, MQH2 is oxidized methyl naphthoquinone, MQ is reduced methyl naphthoquinone, CO2 is carbon dioxide, e - It is an electron;
[0016] Figure 2 It is an absorbance comparison diagram of samples with different decolorization reaction times in Example 1, wherein the vertical coordinate Absorbance is absorbance;
[0017] Figure 3 Absorbance ratio plot for different methyl orange concentration samples in Example 1, where the ordinate C t / C0 is the absorbance ratio at the time of dye degradation to the initial time;
[0018] Figure 4 Absorbance ratio plot for different decolorization reaction medium samples in Example 2, where the ordinate C t / C0 is the absorbance ratio at the time of dye degradation to the initial time;
[0019] Figure 5 Absorbance ratio plot for different oxygen concentration decolorization reaction samples in Example 3, where the ordinate C t / C0 is the absorbance ratio at the time of dye degradation to the initial time;
[0020] Figure 6 Absorbance ratio plot for different riboflavin concentration decolorization reaction samples in Example 4, where the ordinate C t / C0 is the absorbance ratio at the time of dye degradation to the initial time;
[0021] Figure 7 Absorbance ratio plot for different hexavalent chromium decolorization reaction samples in Example 5, where the ordinate C t / C0 is the absorbance ratio at the time of dye degradation to the initial time. DETAILED DESCRIPTION
[0022] Those skilled in the art can improve the process parameters according to the content herein. In particular, it should be pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present application, to realize and apply the present application technology.
[0023] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments and the drawings of the specification. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.
[0024] The configuration method of the culture medium used in the present application is as follows:
[0025] LB medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L;
[0026] Decolorization medium: 0.3 g / L sodium lactate, 0.3 g / L glucose, 10 g / L 12 water disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride and 0.2 g / L yeast extract.
[0027] Example 1
[0028] S1: First, the Shewanella oneidensis MR-1 stored in glycerol was streaked on a solid plate LB medium and cultured overnight at 30℃, and a single colony was picked up with an inoculation loop and inoculated in 200 mL of liquid LB medium and cultured at 30℃ and 130 rpm for 20 h to the stationary phase, and the bacterial cell precipitate was collected by centrifugation at 8500 rpm for 3 min, and the bacterial cell precipitate was washed with phosphate buffer for 3 times to obtain a bacterial solution.
[0029] S2: Methyl orange powder was dissolved in deionized water to prepare a 3 g / L methyl orange mother liquor.
[0030] S3: The decolorization and degradation reaction was carried out in a 100 mL conical flask, 50 ml of the decolorization medium was poured into each conical flask, and the bacterial solution obtained in S1 was added, and the bacterial cell OD value in the medium was 1.0; at the same time, a test treatment group (adding MR-1 bacterial solution and methyl orange with a final concentration of 90 mg / L, 150 mg / L, 210 mg / L or 270 mg / L), a control group A (only adding 90 mg / L methyl orange), a control group B (adding inactivated MR-1 bacterial solution and 90 mg / L methyl orange), and five parallel tests were set up for each group; under the conditions of 30℃ and 130 rpm aerobic shaking culture, the samples of each group were sampled, centrifuged at 8500 rpm for 3 min, and the absorbance of methyl orange (465 nm) was measured by using a UV-visible spectrophotometer, and the decolorization efficiency was calculated, and the calculation formula was as follows:
[0031] Decolorization efficiency (%) = 100 x (1-C t / C0)
[0032] Wherein, C0 refers to the initial absorbance of the dye, C t refers to the absorbance measured during degradation, and each test was repeated five times.
[0033] The reaction principle of the decolorization method provided in the embodiment is as shown in Figure 1 The absorbance of the sample was measured after adding 90 mg / L methyl orange and bacterial solution to the decolorization and degradation reaction for 0 min, 15 min, 30 min, 45 min, 60 min, 75 min and 90 min, and the results are shown in Figure 2 The methyl orange was completely degraded after 60 min of decolorization and degradation reaction, indicating that the biological decolorization method provided in the embodiment is not limited by oxygen.
[0034] The absorbance of each group of samples was measured, and the results are shown in Table 1. Figure 3 As shown in Table 1, compared with the control groups A and B, the MR-1 strain can still rapidly degrade the dye as the concentration of methyl orange dye increases, and the decolorization efficiency of 270 mg / L of methyl orange can reach 96.2% within 160 min, proving that the decolorization method of the present embodiment has high decolorization efficiency.
[0035] Example 2:
[0036] S1: First, the Shewanella oneidensis MR-1 stored in glycerol was streaked on a solid plate LB medium and cultured overnight at 30°C. A single colony of activated bacteria was picked up with an inoculation loop and inoculated into 200 mL of liquid LB medium. The bacteria were cultured at 30°C and 130 rpm for 20 h to the stationary phase, and then centrifuged at 8500 rpm for 3 min to collect the bacterial precipitate. The bacterial precipitate was washed with phosphate buffer for 3 times to obtain a bacterial solution.
[0037] S2: Methyl orange powder was dissolved in deionized water to prepare a methyl orange stock solution with a concentration of 3 g / L.
[0038] S3: The decolorization and degradation reaction process was carried out in a 100 mL conical flask. 50 ml of decolorization medium was poured into each conical flask, and the bacterial solution obtained in S1 was added. The bacteria in the medium were cultured until the OD value reached 1.0. MR-1 bacterial solution and methyl orange with a final concentration of 90 mg / L were added. Five parallel tests were set up for each group. The samples were cultured at 30°C and 130 rpm under aerobic conditions. The samples were centrifuged at 8500 rpm for 3 min, and the absorbance of methyl orange (465 nm) was measured using a UV-visible spectrophotometer. The decolorization efficiency was calculated, and the calculation formula is as follows:
[0039] Decolorization efficiency (%) = 100 x (1-C t / C0)
[0040] Wherein, C0 refers to the initial absorbance of the dye, C t refers to the absorbance measured during degradation, and each test was repeated five times.
[0041] The absorbance of each group of samples was measured, and the results are shown in Table 1. Figure 4 As shown in Table 1, compared with 1 / 2LB and LB medium, the MR-1 strain has the highest degradation rate of methyl orange dye in the decolorization medium, thus proving that the decolorization medium provided by the present application is beneficial to the degradation of the MR-1 strain, and can achieve the purpose of rapidly degrading the dye.
[0042] Example 3:
[0043] S1: First, streak Oneida Shewanella MR-1 preserved in glycerol onto solid LB agar plates and incubate overnight at 30°C. Use an inoculation loop to pick up a single activated colony and inoculate it into 200 mL of liquid LB agar. Incubate at 30°C and 130 rpm for 20 h until the stationary phase. Centrifuge at 8500 rpm for 3 min to collect the bacterial pellet. Wash the bacterial pellet three times with phosphate buffer to obtain the bacterial culture.
[0044] S2: Dissolve methyl orange powder in deionized water to prepare a methyl orange stock solution with a concentration of 3 g / L.
[0045] S3: The decolorization and degradation reaction was carried out in 100mL Erlenmeyer flasks. 50mL of decolorization medium was added to each flask, along with the bacterial suspension obtained in S1. The mixture was cultured until the bacterial OD value reached 1.0. Simultaneously, MR-1 bacterial suspension and methyl orange at a final concentration of 90mg / L were added. Five parallel experiments were conducted for each group. The mixture was cultured at 30℃ with shaking at 130rpm, 150rpm, 170rpm, and 190rpm. Samples from each group were taken and centrifuged at 8500rpm for 3min. The absorbance of methyl orange (465nm) was measured using a UV-Vis spectrophotometer, and the decolorization efficiency was calculated using the following formula:
[0046] Decolorization efficiency (%) = 100 × (1 - C) t / C0)
[0047] Where C0 refers to the initial absorbance of the dye, C t The absorbance measured during degradation is used as a reference, and each experiment is repeated five times.
[0048] In this embodiment, the absorbance of each group of samples was measured. The higher the rotation speed, the higher the oxygen content. Figure 5 As shown, when the present invention is under high oxygen conditions, the rate of degradation of methyl orange by strain MR-1 decreases, indicating that the high oxygen environment inhibits the degradation of methyl orange by strain MR-1. However, conventional conditions are not high oxygen environments, and the process of microbial degradation of azo dyes in the prior art is all under anaerobic conditions, which have strict requirements for environmental conditions and high costs. Therefore, the biological decolorization method provided by the invention can carry out efficient decolorization under aerobic conditions, with lower environmental requirements and lower costs.
[0049] Example 4:
[0050] S1: First, the Shewanella oneidensis MR-1 stored in glycerol was streaked on solid LB medium and incubated at 30°C overnight. The activated single colony was picked up with an inoculation loop and inoculated into 200 mL liquid LB medium. The culture was incubated at 30°C, 130 rpm for 20 h to the stationary phase. The bacterial pellet was collected by centrifugation at 8500 rpm for 3 min and washed with phosphate buffer for 3 times to obtain the bacterial solution.
[0051] S2: Methyl orange powder was dissolved in deionized water to prepare a methyl orange stock solution with a concentration of 3 g / L.
[0052] S3: The decolorization and degradation reaction was carried out in a 100 mL conical flask. 50 mL of decolorization medium was added to each conical flask, and the bacterial solution obtained in S1 was added. The culture was incubated until the OD value of the bacterial solution in the medium reached 1.0. MR-1 bacterial solution and methyl orange with a final concentration of 90 mg / L were added, and riboflavin was added at 0 μM, 0.2 μM, 0.5 μM and 0.8 μM, respectively. Each group had five parallel tests. The samples were incubated at 30°C, 130 rpm, and then centrifuged at 8500 rpm for 3 min. The absorbance of methyl orange (465 nm) was measured by UV-visible spectrophotometer, and the decolorization efficiency was calculated. The formula is as follows:
[0053] Decolorization efficiency (%) = 100 x (1-C t / C0)
[0054] Wherein, C0 refers to the initial absorbance of the dye, C t refers to the absorbance measured during degradation. Each test was repeated five times.
[0055] The absorbance of each group of samples was measured in this example, and the results are shown in Figure 6 Under the condition of adding 90 mg / L methyl orange, as the concentration of riboflavin increases, the time for MR-1 strain to degrade methyl orange also gradually shortens. When the concentration of riboflavin is 0.8 μM, compared with the experimental group of adding MR-1 bacterial solution and 90 mg / L methyl orange in Example 1, the time for MR-1 strain to degrade methyl orange is accelerated by about 20 min, and the decolorization rate is increased by about 44%.
[0056] Example 5:
[0057] S1: First, the Shewanella oneidensis MR-1 stored in glycerol was streaked on solid LB medium and incubated at 30°C overnight. The activated single colony was picked up with an inoculation loop and inoculated into 200 mL liquid LB medium. The culture was incubated at 30°C, 130 rpm for 20 h to the stationary phase. The bacterial pellet was collected by centrifugation at 8500 rpm for 3 min and washed with phosphate buffer for 3 times to obtain the bacterial solution.
[0058] S2: Dissolve methyl orange powder in deionized water to prepare a methyl orange mother liquor with a concentration of 3 g / L.
[0059] S3: The decolorization and degradation reaction process is carried out in a 100 mL conical flask, 50 ml of decolorization medium is poured into each conical flask, and the bacterial liquid obtained in S1 is added, and the OD value of the bacterial body in the medium is cultured to 1.0; MR-1 bacterial liquid and methyl orange with a final concentration of 90 mg / L are added, and 0 mM, 0.02 mM, 0.06 mM, 0.08 mM and 0.12 mM of hexavalent chromium are added respectively, and each group is set with five parallel tests; respectively under the conditions of 30℃ and 130rpm, the samples in each group are sampled, centrifuged at 8500rpm for 3min, and the absorbance of methyl orange (465nm) is measured by using a UV-visible spectrophotometer, and the decolorization efficiency is calculated, and the calculation formula is as follows:
[0060] Decolorization efficiency (%) = 100 x (1-C t / C0)
[0061] Wherein, C0 refers to the initial absorbance of the dye, C t refers to the absorbance measured during degradation, and each test is repeated five times.
[0062] The absorbance of each group of samples is measured in this example, and the results are shown in Table 1. Figure 7 As the content of hexavalent chromium increases, the degradation of methyl orange by MR-1 strain is gradually inhibited, and when the concentration of hexavalent chromium is 0.12 mM, the degradation ability of MR-1 strain is almost completely lost.
[0063] The results of adding electron transfer riboflavin and electron inhibitor hexavalent chromium in examples 4-5 show that the degradation of methyl orange by MR-1 strain is achieved by releasing electrons, rather than using azo reductase on the surface of bacterial cells.
[0064] The decolorization method provided by the application is applied to industrial wastewater treatment, and specifically includes: a sedimentation tank, a filtration system and a wastewater treatment unit, the decolorization treatment is carried out in the wastewater treatment unit, a real-time monitoring system is arranged, parameters such as oxygen concentration, pH value, temperature and microbial activity are measured at the same time, and oxygen supply and reaction conditions can be automatically adjusted to optimize the decolorization effect.
[0065] The content not described in detail in the specification of the application is the technology known to those skilled in the art. Although the application has been disclosed as above with preferred embodiments, it is not intended to limit the application, and anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the application, therefore the protection scope of the application should be defined by the claims.
Claims
1. A bio-mediated aerobic decolorization process of dyes, characterized in that, The method comprises the following steps: inoculating a Shewanella bacterial solution into a decolorization medium, and culturing the dye under aerobic conditions to decolorize the dye; the Shewanella is Shewanella oneidensis MR-1; The decolorization medium comprises 0.3 g / L sodium lactate, 0.3 g / L glucose, 10 g / L 12 water sodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride and 0.2 g / L yeast extract; The culture condition is 30±0.5 ℃ and 130±5 rpm; The dye comprises methyl orange.
2. The method of claim 1, wherein, The preparation process of the bacterial solution is as follows: culturing Shewanella oneidensis MR-1 in an LB medium under aerobic conditions at 30 ℃ for 20 h to the stable phase, centrifuging the bacterial solution at 8500 rpm for 3 min to collect the bacterial precipitate, and then washing the bacterial precipitate with a phosphate buffer for 3 times to obtain the bacterial solution.
3. The method of claim 1, wherein, The additive amount of the dye is 90 mg / L, 150 mg / L, 210 mg / L or 270 mg / L.
4. A method of sewage treatment, characterised in that, The sewage treatment method comprises a decolorization treatment step, and the decolorization treatment step adopts the biological mediated aerobic dye decolorization method according to any one of claims 1-3.