Method for immobilizing rhodococcus and application thereof
By immobilizing Rhodococcus sp.LH-2 combined with end-hydroxyl polybutadiene glue blocks or polyurethane foam, the limited adsorption capacity and energy consumption of RDX and Octor waste liquids in the prior art are solved. High-quality problems have achieved efficient and environmentally friendly degradation effects.
Patent Information
- Application Number
- CN202411625132.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-14
- Publication Date
- 2025-02-11
AI Technical Summary
The prior art has problems such as limited adsorption capacity, high energy consumption, unstable degradation efficiency, high oxidant cost and harsh reaction conditions when dealing with high-energy explosives RDX and Octor waste liquids, resulting in high treatment costs and environmental pollution.
The immobilized Rhodococcus sp.LH-2 is used to improve the growth effect and stability of microorganisms by combining with end-hydroxyl polybutadiene blocks or polyurethane foam, thereby achieving efficient degradation of RDX and Octor today.
Under adverse environmental conditions, the efficient growth and cyclic degradation of RDX and Octor now has a degradation rate of 74.34%, and the method consumes low energy, is pollution-free and has low operating costs.
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Figure CN119410620A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial immobilization, and in particular relates to a method for immobilizing cocci and application thereof. Background Art
[0002] After years of production, manufacturing, use and destruction, the environmental pollution caused by RDX (trimethylene trinitramine) has attracted widespread attention. RDX is a cyclic nitro compound, mainly used as a high-energy explosive and widely used worldwide. RDX has significant biological toxicity and poses a risk of poisoning to humans, animals and plants. Therefore, it is particularly important to find a reasonable method for treating RDX waste liquid.
[0003] The treatment methods for RDX-containing waste liquid mainly include physical method, chemical method and biological method. The physical method mainly includes activated carbon adsorption method, extraction method, membrane separation method, flocculation method, etc. This method is simple to operate, but the cost is high, and the residue after treatment is still a pollutant or hazardous substance, which requires further treatment. Commonly used chemical methods (such as Fenton reagent oxidation method, supercritical water oxidation method, ozone method, electrolysis method, etc.) to treat RDX-containing waste liquid are costly and incomplete. The biodegradation method is a treatment method that uses the metabolism of organisms to transform and stabilize waste RDX pollutants to make them harmless. Because its process for treating waste RDX materials has the advantages of low energy consumption, no pollution, compatibility with existing biochemical pools, and low operating costs, the biodegradation method has become a more promising treatment method.
[0004] Octogen (HMX) is a high-energy explosive, chemical name is cyclotetramethylene tetranitramine, physical method defects:
[0005] The adsorption capacity of the adsorption method is limited: the adsorption capacity of the adsorbent material for octogen is limited. When it reaches saturation, it cannot continue to adsorb. The adsorbent material needs to be frequently replaced or regenerated, which increases the treatment cost and operation complexity. For example, when organic macroporous resin is used to treat wastewater containing octogen, the adsorption capacity will gradually decrease as the treatment volume increases.
[0006] High-temperature pyrolysis method, high energy consumption: High-temperature pyrolysis requires a lot of energy to maintain a high-temperature environment, which makes the processing cost very high. For example, to decompose octogen at high temperatures, a special high-temperature furnace or heating equipment is required, and a large amount of fuel or electricity is continuously consumed, which may cause secondary pollution: During the high-temperature pyrolysis process, octogen may not be completely decomposed, and some intermediate products or by-products may be produced. These substances may have certain toxicity or pollution and need further treatment. For example, some harmful gases such as nitrogen oxides and carbon oxides may be produced. If not handled properly, it will cause secondary pollution to the environment.
[0007] Ultrasonic degradation method, unstable degradation efficiency: The effect of ultrasonic degradation is affected by many factors, such as ultrasonic power, frequency, treatment time, and the nature of the solution, which makes the degradation efficiency unstable. In practical applications, it is difficult to find a universal optimal operating condition, and a large number of experiments and optimizations are required for different wastewater systems, which increases the difficulty and cost of treatment.
[0008] Chemical method, oxidation degradation method has high oxidant cost: the cost of oxidants such as ozone and Fenton's reagent is relatively high, especially the preparation of ozone requires special equipment and energy, and the ferrous ions and hydrogen peroxide in Fenton's reagent also need to be continuously added, which increases the treatment cost. For example, when the ozone oxidation method is used to treat wastewater containing octogen, the preparation and consumption cost of ozone accounts for a large part of the total treatment cost.
[0009] Harsh reaction conditions: Oxidative degradation methods usually require certain pH values, temperatures, oxidant concentrations, and other conditions to achieve good degradation effects, which requires high control of reaction conditions. If the reaction conditions are not appropriate, it may lead to waste of oxidants or poor degradation effects. For example, the Fenton oxidation method works better under acidic conditions, but in actual wastewater treatment, adjusting the pH value of wastewater requires a large amount of acid and alkali reagents, and may cause secondary pollution.
[0010] Reduction degradation method, difficult to select reducing agent: It is difficult to select a suitable reducing agent. Some reducing agents may have strong reducing properties, but they are also dangerous or toxic, such as sulfides. During use, the amount of reducing agent and reaction conditions need to be strictly controlled to avoid safety problems and secondary pollution.
[0011] Complex treatment of degradation products: The products after reduction degradation may need further treatment, because some products may still be harmful or difficult to discharge directly. For example, after reducing the nitro group in Octogen to amino group, the treatment of amino compounds also requires special methods and equipment.
[0012] Hydrolysis degradation method, slow reaction rate: The reaction rate of hydrolysis degradation is usually slow, and a long reaction time is required to achieve a good degradation effect. This makes the treatment equipment occupy a large area and the treatment efficiency is low, which is not conducive to large-scale wastewater treatment.
[0013] Due to the defects of the above methods, the existing methods of degradation by microorganisms can overcome the above defects, but due to the stress of RDX and octogenin on microorganisms, the growth effect of microorganisms is general and cannot be reused; it is urgent to find a method that enables microorganisms to grow better, degrade RDX and octogenin faster and more efficiently under the stress of adverse environments. Summary of the invention
[0014] The purpose of the present invention is to provide a rhodococcus with an immobilization effect and capable of efficiently and cyclically degrading RDX and octogen, as well as a method for cyclically utilizing the rhodococcus.
[0015] The present invention provides a method for immobilizing Rhodococcus sp., wherein the mixing ratio of Rhodococcus sp. LH-2, the hydroxyl-terminated polybutadiene rubber block and the Rhodococcus sp. LH-2 is 0.5 cm 3 × (10-20) / 200ml bacterial solution or the mixing ratio of polyurethane foam and Rhodococcus LH-2 is 1cm 3 × (5-15) / 200ml bacterial liquid, the Rhodococcus LH-2 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the preservation date is July 15, 2024, and the preservation number is: CGMCC No.31308.
[0016] Further, the mixing ratio of Rhodococcus LH-2 and the hydroxy-terminated polybutadiene rubber block to Rhodococcus LH-2 is 0.5 cm 3 × 12 / 200ml bacterial solution or the mixing ratio of polyurethane foam and Rhodococcus LH-2 is 1cm 3 ×8 cells / 200ml bacterial liquid.
[0017] The present invention provides an immobilized Rhodococcus obtained by the above method.
[0018] The invention provides a microbial preparation of the above-mentioned immobilized Rhodococcus.
[0019] The present invention provides an application of the immobilized Rhodococcus or the microbial preparation in resisting RDX.
[0020] The present invention provides an application of the immobilized Rhodococcus or the microbial preparation in resisting Octogen.
[0021] The present invention provides an application of the immobilized Rhodococcus or the microbial preparation in resisting octogene and RDX.
[0022] The invention provides a method for degrading RDX. The method comprises adding the above-mentioned immobilized rhodococcus into a sample containing RDX, and fermenting the sample at 37°C for 72 hours.
[0023] The invention provides a method for degrading octochrome, wherein the immobilized rhodococcus is added into a sample containing roxygen, and fermented at 37°C for 72 hours.
[0024] It is further defined that the nitrogen sources for the fermentation are RDX and octogen.
[0025] The present invention provides an application of the immobilized rhodococcus or the microbial preparation in the cyclic degradation of anti-octogen and RDX.
[0026] Beneficial effects: The present invention fermented and separated a high-concentration octogen-tolerant and degrading strain Rhodococcus sp. LH-2 from the seed-maintaining bacteria of Rhodococcus sp. LMD. Compared with the disclosed degradation strains, the octogen-tolerant concentration of the red coccus Rhodococcus sp. LH-2 provided by the present invention is higher than the highest concentration reported in the literature (6 mg / L), can tolerate high-concentration octogen wastewater (19.23 mg / L, i.e., 0.0649 mmol / L) and grow and reproduce in an inorganic salt culture medium with octogen as the sole nitrogen source, and can tolerate 18 mg / L of RDX wastewater, and can cyclically degrade RDX or octogen. The strain attached to hydroxyl-terminated polybutadiene and polyurethane foam at a temperature of 37°C, pH of about 7, and a rotation speed of 220 r / min for a period of 72 hours. The fixation effect of polyurethane foam was poor, while the fixation effect of hydroxyl-terminated polybutadiene rubber block was very good. However, polyurethane foam was better at overcoming stress and the growth effect of Rhodococcus was better. The number of bacteria attached to each plane of the rubber block could reach 1.7×10 4 The degradation rate can reach 74.34%.
[0027] The hydroxy-terminated polybutadiene block has excellent viscoelasticity, gas permeability, biodegradability, and low cytotoxicity, and has potential application value in microbial and drug carriers, and can be compared with the immobilization effect of polyurethane foam. Therefore, this study uses immobilized microbial technology and uses hydroxy-terminated polybutadiene as an immobilized carrier to enable free Rhodococcus to improve its own activity and stability, thereby providing a theoretical basis and data support for its practical application and bioremediation.
[0028] [Biological Deposit Information]: A strain of Rhodococcus sp., the name of which is Rhodococcus sp. LH-2, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is July 15, 2024, and the deposit number is: CGMCC No.31308. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 This is the result of the isolation and identification of Rhodococcus sp.LH-2;
[0030] Figure 2This is the colony image of Rhodococcus sp.LH-2;
[0031] Figure 3 The growth curves of Rhodococcus sp. LH-2 in immobilized solution with RDX and Octogen as the sole nitrogen source, respectively, wherein A is the growth curve with Octogen as the nitrogen source, the horizontal axis is time, and the vertical axis is biomass; B is the growth curve with RDX as the nitrogen source, the horizontal axis is time, and the vertical axis is biomass;
[0032] Figure 4 These are the immobilized images of Rhodococcus sp.LH-2, wherein A is the image of Rhodococcus immobilized on polyurethane material; B is the image of Rhodococcus not immobilized on polyurethane material; C is the image of Rhodococcus not immobilized on hydroxy-terminated polybutadiene material; and D is the image of Rhodococcus immobilized on hydroxy-terminated polybutadiene material. DETAILED DESCRIPTION
[0033] The polyurethane foam was purchased from Henan Tianyi Environmental Protection Materials Company, item number polyurethane sponge 98 (porosity greater than 98%);
[0034] Hydroxyl-terminated polybutadiene is a liquid prepolymer developed in the 1960s. Through chain extension and cross-linking curing reactions, it can be made into an elastomer with a three-dimensional network structure. Because it has the same properties as solid rubber, some people also call it liquid rubber. The cured product has excellent mechanical properties, especially hydrolysis resistance, acid and alkali resistance, wear resistance, low temperature resistance and excellent electrical insulation. Hydroxyl-terminated polybutadiene (HTPB), referred to as butyl hydroxyl rubber, is a type of telechelic liquid rubber that can be used as a structural material for the production of cast elastomers and automobile tires. Hydroxyl-terminated polybutadiene is mainly used as an adhesive for solid rocket propellants.
[0035] Example 1. Method for isolating and identifying Rhodococcus
[0036] Fermentation medium: 0.31 g potassium dihydrogen phosphate, 0.99 g potassium hydrogen phosphate, 146 μL glycerol, 0.18 g glucose, 0.294 g sodium citrate, 200 mL RO water, 200 μL trace elements, pH adjusted to 7.2, autoclave and cool, then add the sole nitrogen source octogen or 0.063 g potassium nitrite.
[0037] LB liquid medium: 1 g sodium chloride, 1 g tryptone, 0.5 g yeast extract, 100 mL RO water.
[0038] LB solid medium: 2 g sodium chloride, 2 g tryptone, 1 yeast extract, 3 g agar powder, 100 mL RO water.
[0039] The pH of the above three culture media is about 7. They were sterilized by high pressure at 121°C for 30 min before inoculation.
[0040] Screening, separation and purification of degrading bacteria: The seed-keeping bacteria Rhodococcus sp. LMDx of Rhodococcus sp. LH-2 was inoculated at a 5% inoculation rate, the inoculated bacterial liquid was centrifuged, the supernatant was discarded and the bacteria were retained, and inoculated into 200 mL fermentation medium containing 24 mg / L octoquinone as the only nitrogen source, and the culture was cultured at 220 r / min in a constant temperature shaker at 37°C. After the fermentation was completed, the fermented Rhodococcus was obtained, which can tolerate and degrade supersaturated high-concentration octoquinone solution, and the seed was preserved in a -80°C refrigerator.
[0041] The test results showed that the strain was a Gram-positive bacillus that did not form spores. Figure 2 This is the colony formed by the strain in LB solid culture medium. It can be seen that the colony is round, arched, orange-yellow, opaque, with a smooth and moist surface and complete edges.
[0042] The physiological and biochemical characteristics of this strain are: Gram-positive, polymorphic bacteria, may form rudimentary mycelium, immediately break into irregular cells, generally light orange-yellow, pink, orange or red, with colorless variants.
[0043] Identification of degrading bacteria: The present invention uses 16S rDNA to perform homology analysis on the gene sequence of the screened strains, and compares the sequences through the NCBI (National Center for Biotechnology Information) database to obtain a chart.
[0044] Results: The 16S rDNA (sequence https: / / www.ncbi.nlm.nih.gov / nuccore / MK351311.1?from=1&to=1471) gene molecular biological identification results of the strain were 100% homologous to Rhodococcus ruberstrain R1 chromosome. The isolation and identification results of the strain are shown in Figure 1 The obtained Rhodococcus was named Rhodococcus sp.LH-2.
[0045]
[0046] Example 2. Degradation of Octogen and RDX
[0047] 1. The seed-keeping bacteria of Rhodococcus sp. LH-2 obtained in Example 1 was inoculated at a rate of 5%, the inoculated bacterial solution was centrifuged, the supernatant was discarded and the bacterial body was retained, and it was inoculated into 200 mL of fermentation medium containing 24 mg / L octogen as the sole nitrogen source, and the culture was placed in a 37°C constant temperature shaker at 220 r / min. The control group used potassium nitrite as the sole nitrogen source, and the OD value was measured by an ultraviolet spectrophotometer every 12 hours to observe the changes in the bacterial solution, and the fermentation liquid was taken before and after the inoculation of the bacteria to determine the concentration of octogen before and after degradation.
[0048] The Rhodococcus containing 24 mg / L octoquinone entered the logarithmic growth phase after 24 hours of culture. As the nutrients in the culture medium were continuously consumed, the growth rate of the strain slowed down, the activity gradually decreased, and the overall growth effect was average, indicating that octoquinone had a certain stress effect on Rhodococcus. The degradation rate of octoquinone in the solution is shown in the following table. When the experimental group used octoquinone as the only nitrogen source, the results showed that 19.23 mg / L octoquinone was tolerated.
[0049] 2. RDX degradation test: The Rhodococcus sp. LH-2 obtained in Example 1 was inoculated with 5% of the inoculum into an inorganic salt culture medium containing different concentrations of RDX (54 mg / L, 36 mg / L, and 18 mg / L), and cultured in a constant temperature shaker at 30-37°C at 220 r / min. The changes in the bacterial solution were observed, and the culture solution was taken every 12 hours to determine the growth of the strain and the residual amount of RDX in the solution. Results showed that the strain could tolerate 18 mg / L of RDX wastewater.
[0050] Example 3. A method for immobilizing Rhodococcus
[0051] Rhodococcus immobilization test: Rhodococcus sp. LH-2 obtained in Example 1 was inoculated with 5% of the inoculation amount into a fermentation medium containing potassium nitrite (0.31 g potassium dihydrogen phosphate, 0.99 g potassium dihydrogen phosphate, 146 μL glycerol, 0.18 g glucose, 0.294 g sodium citrate, 200 mL RO water, 200 μL trace elements, pH adjusted to 7.2, autoclaved and cooled, and then the only nitrogen source 0.063 g potassium nitrite was added), placed in a 37°C constant temperature shaker at 220 r / min for culture, and 12 0.5 3 cm hydroxyl-terminated polybutadiene rubber block cube (hydroxyl polybutadiene group, experimental group 1), less than 10 1 3cm hydroxy polybutadiene rubber block cube, insufficient immobilization, and 8 1 3 cm polyurethane foam cube (polyurethane foam group, experimental group 2), less than 5 1 3 The polyurethane foam cube of cm was not fully immobilized. The changes of bacterial solution were observed and the culture solution was taken every 12 hours to determine the growth of the strain. After 72 hours of fermentation, the immobilized material was continued to be placed in the fermentation medium with octoquinone or RDX as the only nitrogen source. The bacteria could continue to grow and continue to cycle and degrade octoquinone or RDX.
[0052] The results are as follows Figure 3 , 4, as shown in Tables 1-4 (Tables 1 and 3 show the effects of the first degradation of immobilized Rhodococcus, and Tables 2 and 4 show the effects of the first degradation of repeated recycling): through the control group (nitrite as nitrogen source, the experimental group with HMX as nitrogen source, the other culture medium components of the two groups are the same, the experimental group and the control group need to be inoculated with Rhodococcus, nitrite group), polyurethane foam group (experimental group 2) and terminal hydroxyl polybutadiene group (experimental group 1) Figure 3 and Figure 4 As shown in the figure, the growth curve analysis of the experimental group 1 showed that the vitality of the red coccus in the two experimental groups was higher than that of the blank control in the late growth stage, indicating that immobilization is indeed beneficial to the reproduction and growth of the colony compared with liquid culture, making the growth state of the bacteria better. However, the experiment found that the effect of enriching red coccus with terminal hydroxyl polybutadiene was not as good as that of polyurethane foam. Polyurethane foam had a stronger ability to adsorb and fix red coccus during the culture of bacteria. However, through morphological analysis of the two experimental groups, it was found that the effect of immobilizing red coccus in the hydroxyl polybutadiene group after the experimental treatment was better than that of polyurethane foam. The number of fixed red coccus with terminal hydroxyl polybutadiene was larger, and the bacteria were more easily found on the surface of the terminal hydroxyl polybutadiene rubber block during the scanning electron microscopy process, while the polyurethane foam group was rarely found. Moreover, it was found through calculation that each plane could adsorb up to 1.7×10 4 The results showed that the attachment and fixation effect of Rhodococcus was quite good. Finally, the degradation rate of the hydroxy-terminated polybutadiene group reached 74.34%, and that of the polyurethane group reached 60.73%, indicating that the immobilization of Rhodococcus by hydroxy-terminated polybutadiene material was indeed beneficial to the degradation of Octogen.
[0053] The formula for the number of attached bacteria on each plane is: S (specific surface area) (immobilization image) × 3 × 0.48 / S (hydroxyl-terminated polybutadiene rubber block plane);
[0054] Degradation rate formula: (C-C0) / C×100% (C: initial octogenin or RDX concentration C0: octogenin or RDX concentration at the end of fermentation);
[0055] Calculation of attachment rate: After forming, red coccus is generally round, oval or oblong in shape, with slightly bright edges and a size of about 0.8-5μm. Figure 4 A in the figure is a picture of Rhodococcus immobilized on polyurethane material; Figure 4 B in the figure is a picture of Rhodococcus not immobilized on polyurethane material; Figure 4 C in the figure is the hydroxy-terminated polybutadiene material, and the image of Rhodococcus not immobilized; Figure 4 D in the figure is the picture of Rhodococcus immobilized on hydroxy-terminated polybutadiene material.
[0056] Since very few polyurethane foams were found, only a very small number of bacteria were found during the scanning electron microscopy process, and then the polyurethane immobilization image 1 was retained ( Figure 4 A), the rest are similar to polyurethane fixation Figure 2 ( Figure 4 B) in the figure is a structural image of polyurethane, so it cannot be calculated; however, since more bacteria were found in the hydroxyl-terminated polybutadiene rubber block during the scanning electron microscopy process, and basically at least 3 bacteria can be found in each 20μm scanning electron microscopy image of the same scale, it can be calculated by the surface area ratio method. In addition, 17 of the 33 hydroxyl-terminated polybutadiene immobilization images were taken with no bacteria in the structure, and the rest had bacteria. In addition to the 20μm image, there were also 2, 5, 10μm, and 50μm images. It can be seen from the 50μm scale image that its adhesion effect is better ( Figure 4 C and D), by using the appropriate ratio of 20 μm to calculate, the calculation of the remaining ratios will be much larger than the calculation using the maximum ratio. Therefore, after calculating the area ratio and multiplying it by the bacterial ratio of (33-17) / 33=0.48, the surface area of the hydroxy-terminated polybutadiene immobilized image is: 52.4 μm×40 μm, that is, 2096 μm 2 The surface area of each side of the hydroxyl-terminated polybutadiene rubber block is 0.25 cm 2 , each side adsorbs bacteria 0.25cm×3×0.48 / 2096μm, that is, 1.7×10 4 A bacteria.
[0057] Table 1 Liquid phase and degradation results of Octogenin by Rhodococcus sp.LH-2
[0058]
[0059] Table 2 Liquid phase and degradation results of RDX by Rhodococcus sp.LH-2
[0060]
[0061] Table 3 Liquid phase and degradation results of Octogen by Rhodococcus sp.LH-2
[0062]
[0063] Table 4 Liquid phase and degradation results of RDX degradation by Rhodococcus sp.LH-2
[0064]
[0065]
Claims
1. A method for immobilizing Rhodococcus sp., characterized in that: The mixing ratio of Rhodococcus LH-2, hydroxyl-terminated polybutadiene rubber block and Rhodococcus LH-2 is 0.5cm 3 × (10-20) / 200ml bacterial solution or the mixing ratio of polyurethane foam and Rhodococcus LH-2 is 1cm 3 × (5-15) / 200ml bacterial liquid, the Rhodococcus LH-2 is deposited in the General Microbiology Center of China Culture Collection Administration, the deposit date is July 15, 2024, and the deposit number is: CGMCC No.31308.
2. The method according to claim 1, characterized in that The mixing ratio of Rhodococcus LH-2, hydroxyl-terminated polybutadiene rubber block and Rhodococcus LH-2 is 0.5cm 3 × 12 / 200ml bacterial solution or the mixing ratio of polyurethane foam and Rhodococcus LH-2 is 1cm 3 ×8 cells / 200ml bacterial liquid.
3. The immobilized Rhodococcus obtained by the method according to claim 1 or 2.
4. A microbial preparation containing the immobilized Rhodococcus according to claim 3.
5. Use of the immobilized Rhodococcus according to claim 3 or the microbial preparation according to claim 4 in resisting RDX or octoquin.
6. Use of the immobilized Rhodococcus according to claim 3 or the microbial preparation according to claim 4 in resisting octogene and RDX.
7. A method for degrading RDX, characterized in that: The immobilized Rhodococcus described in claim 3 is added to a sample containing RDX, and fermented at 37° C. for 72 hours.
8. A method for degrading octogen, characterized in that: The immobilized Rhodococcus described in claim 3 is added to a sample containing RDX, and fermented at 37° C. for 72 hours.
9. The method according to claim 7 or 8, characterized in that The nitrogen sources for fermentation were RDX and Octogen.
10. Use of the immobilized Rhodococcus described in claim 3 or the microbial preparation described in claim 4 in the cyclic degradation of anti-octogen and RDX.