A carbon-based adsorption / microbial aerobic degradation risk control composite material and its application

Abstract

The present invention discloses a carbon-based adsorption / microbial aerobic degradation risk control composite material and its application, which belongs to the field of groundwater remediation technology. The composite material has a core-shell structure, including an aerobic degradation bacteria shell and a functional material core. The functional material of the core is composed of adsorption-type biochar, oxygen slow-release agent calcium peroxide and recovery-promoting factor, which are fixed by sodium alginate and calcium chloride. The aerobic shell is composed of petroleum hydrocarbon aerobic degradation bacteria Rhodococcus pyridinus, which is naturally formed on the surface of microspheres through biofilm. The material is used for aerobic bacteria to construct a local aerobic habitat in the groundwater environment, Rpf is used to improve the degradation activity of aerobic bacteria, and oxygen slow-release agents are used to overcome the inhibitory effect of groundwater oxygen deficiency on the activity of aerobic bacteria. The composite material prepared by the present invention can be used to repair groundwater contaminated by petroleum hydrocarbons, and this material has the advantages of high efficiency, long-term effect, low cost, no secondary pollution, and simple preparation method, and has the potential for large-scale promotion and application.

Description

Technical Field

[0001] The present invention relates to the field of groundwater remediation, and more particularly to a carbon-based adsorption / microbial aerobic degradation risk management composite material having an aerobic degradation bacteria shell and a functional material core, and its application in the remediation of petroleum hydrocarbon-contaminated water bodies. Background Art

[0002] In the process of urbanization and industrialization, groundwater pollution is a common problem and is getting worse. Organic pollution is a difficult problem in groundwater pollution control, which seriously affects the quality of groundwater. Due to the large-scale aggregation of chemical parks and the long history of some enterprises, there are serious problems of organic groundwater pollution around the parks. Petroleum hydrocarbons are very common organic pollutants in groundwater of chemical raw material manufacturing enterprises. Due to its concealment, complexity, and difficulty in control and treatment, groundwater pollution is very difficult to control and restore once it is polluted, and the cost of repair is huge.

[0003] A biobarrier is a method of groundwater pollution treatment and site pollution control that fills a permeable barrier with biological materials to degrade or transform and detoxify pollutants. At present, the research and practical application cases of biobarriers are mainly concentrated in the reduction and remediation of groundwater nitrates and high-valent toxic heavy metals. They are widely used abroad with good application effects, and there are very few demonstration and application studies in China. In general, there are relatively few studies and applications of biobarriers for the remediation of organic pollutants and site risk control. For organic pollutants such as petroleum hydrocarbons, oxidative biobarriers are suitable.

[0004] The primary material of the biological reaction wall is the adsorption material. Since the time of microbial reaction is longer than that of chemical reaction, the adsorption material is needed to adsorb the pollutants on the microbial coexistence matrix to form a microbial reactor for degradation or transformation. For oxidative degradation biological reaction walls, microorganisms mineralize organic pollutants through aerobic degradation and co-metabolism. In addition to oxidative degradation bacteria, the required materials also include oxygen releasers or other non-oxygen electron acceptor matrices, necessary carbon sources, nitrogen sources, phosphorus sources and other nutrient matrices. At present, there is a shortage of oxidative degradation biological reaction wall materials used in actual groundwater. In the long-term operation process, the oxidative degradation biological reaction wall materials mainly have short-term problems such as inactivation of electron acceptor matrices such as oxygen release, instability of skeleton materials, and easy dormancy of degradation bacteria. Therefore, the strategy of combining microorganisms with adsorption materials can effectively solve this bottleneck. Summary of the invention

[0005] In view of the fact that aerobic biological reaction wall materials used for repairing petroleum hydrocarbons in groundwater are prone to short-term problems such as insufficient oxygen and dormancy of degrading bacteria, the present invention provides a core-shell structure material with an aerobic degrading bacteria shell and a functional material core, which has a significant removal and control effect on petroleum hydrocarbons in groundwater.

[0006] The technical solutions specifically adopted in the present invention are as follows:

[0007] In a first aspect, the present invention provides a carbon-based adsorption / microbial aerobic degradation risk management composite material, wherein the composite material is composed of an aerobic degradation bacteria shell and a functional material core to form a core-shell structure;

[0008] The inner core is composed of functional materials such as adsorption-type biochar, oxygen slow-release agent calcium peroxide and resuscitation-promoting factor (Rpf), which are fixed by sodium alginate and calcium chloride to form the inner core of the microsphere; the aerobic outer shell is a biofilm of petroleum hydrocarbon aerobic degradation bacteria Rhodococcus pyridinivorans attached to the surface of the microsphere.

[0009] As a preferred embodiment of the first aspect, the adsorption-type biochar is biochar obtained by subjecting sawdust raw materials to oxygen-limited pyrolysis at 650 to 750° C. for 5 to 7 hours.

[0010] As a preferred embodiment of the above-mentioned first aspect, the aerobic petroleum hydrocarbon degrading bacteria, Rhodococcus pyridinovus, was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on December 11, 2023, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.29278.

[0011] As a preference of the first aspect above, the particle size of the core-shell structure is 0.1 to 0.2 cm.

[0012] In a second aspect, the present invention provides a method for preparing a carbon-based adsorption / microbial aerobic degradation risk management composite material as described in any one of the schemes of the first aspect, characterized in that it comprises:

[0013] S1. A 1wt.% sodium alginate solution at 40-50°C is used as a mother solution, and adsorption-type biochar with a final concentration of 50 g / L, Rpf with a final concentration of 20 mg / L, and calcium peroxide CaO2 with a final concentration of 2wt.% are added and mixed, and the mixed solution is added dropwise to a 1wt.% CaCl2 solution through a droplet forming device to form microspheres;

[0014] S2. The microspheres are added to LB medium inoculated with Rhodococcus pyridinovus for overnight culture, and a Rhodococcus pyridinovus biofilm is grown on the surface of the microspheres, thereby obtaining a core-shell structure composite material with the microspheres as the functional material core and the Rhodococcus pyridinovus biofilm as the aerobic degradation bacteria shell.

[0015] As a preferred embodiment of the second aspect, the LB medium inoculated with Rhodococcus pyridinovorax needs to be pre-recovered and cultured to an OD of 600 Reach 1.7~1.8.

[0016] In a third aspect, the present invention provides a carbon-based adsorption / microbial aerobic degradation risk management composite material prepared by the preparation method described in the second aspect above.

[0017] In a fourth aspect, the present invention provides a bioreaction wall using the carbon-based adsorption / microbial aerobic degradation risk control composite material as filler as described in any one of the first or third aspects above.

[0018] In a fifth aspect, the present invention provides an application of the carbon-based adsorption / microbial aerobic degradation risk management composite material as described in any one of the first aspect or the third aspect in the remediation of petroleum hydrocarbon contaminated water bodies.

[0019] As a preferred embodiment of the fifth aspect above, the water body is groundwater.

[0020] The present invention provides an oxidative degradation type bioreaction wall composite material with an inner layer of functional materials and an outer layer of aerobic bacteria. The composite material is used to construct a local aerobic habitat for aerobic bacteria in a groundwater environment, and Rpf is used to improve the degradation activity of aerobic bacteria. An oxygen slow-release agent is used to overcome the inhibitory effect of lack of oxygen in groundwater on the activity of aerobic bacteria. The composite material of the present invention has a significant removal effect on petroleum hydrocarbons in groundwater. The carbon-based adsorption / microbial aerobic degradation risk control composite material and a single carbon-based material have purification capacities of 15.7 mg / g and 9.8 mg / g for typical hydrocarbon organic pollutants on the site, respectively. The carbon-based adsorption / microbial aerobic degradation risk control composite material of the present invention has a purification capacity of 60.2% higher than that of a single carbon-based material for typical hydrocarbon organic pollutants on the site, and can be used for in-situ microbial remediation of groundwater. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The morphology numbers of the composite materials corresponding to different times in the embodiments of the present invention.

[0022] Figure 2 The change of the outer layer of the core-shell structure material of the present invention over time.

[0023] Figure 3 The change of the activity of the outer layer of the core-shell structure material of the present invention.

[0024] Figure 4 The core-shell structure material of the present invention has a removal effect on petroleum hydrocarbons in simulated groundwater. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings, which are intended to explain the present invention rather than to limit it. The methods used in the following examples are all conventional methods unless otherwise specified, and the reagents used can all be existing commercially available products unless otherwise specified.

[0026] The two microbial strains involved in the following embodiments of the present invention are both existing strains or preserved strains. Among them, the aerobic degradation bacteria of petroleum hydrocarbons is Rhodococcus pyridinivorans, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on December 11, 2023. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.29278. Micrococcus luteus IAM 14879 is a commercially available bacterial agent.

[0027] The specific preparation process and application effect of the above-mentioned carbon-based adsorption / microbial aerobic degradation risk management composite material of the present invention are demonstrated through examples below.

[0028] Embodiment 1:

[0029] In this embodiment, the preparation process of a carbon-based adsorption / microbial aerobic degradation risk management composite material according to the present invention is mainly demonstrated, as follows:

[0030] (1) Culture of bacterial strains

[0031] Cultivation of Rhodococcus pyridinosus: The Rhodococcus pyridinosus strain with the deposit number of CGMCC NO.29278 was inoculated into 20 mL LB medium at a 1% inoculation rate (v / v), and placed in a dark shaker at 30°C for recovery culture at a speed of 180 rpm. After culturing for 24 to 48 hours, the recovery solution was inoculated into a new medium at a 1% inoculation rate (v / v), and placed in a dark shaker at 30°C for expansion culture at a speed of 180 rpm. The strain was cultured with LB medium until its OD 600 Reach 1.7~1.8.

[0032] It should be noted that the LB medium formula used in this embodiment is as follows: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, add 1000 mL of distilled water to dissolve, adjust pH=7, and sterilize at high temperature and high pressure. The LB medium formula used in subsequent embodiments is also the same and will not be repeated here.

[0033] Preparation of Rpf: The amount of Rpf protein secreted by Micrococcus luteus itself is low, and it is difficult to extract directly. Therefore, when the protein is used, its rpf gene is connected to a plasmid vector and transformed into Escherichia coli to prepare a genetically engineered bacterium, which can quickly and efficiently obtain a large amount of Rpf protein. The specific strain of Micrococcus luteus is not limited, as long as it can produce Rpf. In this embodiment, the rpf gene of Micrococcus luteus IAM 14879 is selected as the object, and its base sequence length is 672bp. The rpf expression strain Escherichia coli pET28a-RPF is prepared by plasmid transduction, and its rpf gene length is 500-750bp. After the transduced expression strain is cultured in 37°C LB for 24 hours, the Rpf protein can be obtained by crushing it at a low temperature of 4°C. The Rpf protein expressed by E.coli pET28a-RPF carries a histidine tag and can be combined with agarose medium of nitrilotriacetic acid chelated nickel ions (Ni-NTA). The impurities are washed with a low concentration of imidazole solution (10mM), and then eluted with an appropriate concentration of imidazole solution (100mM) to obtain the target protein. The flow-through of each step in the protein purification process is collected. The target protein has high purity, and the band is between 25-33kD, about 26kD.

[0034] However, it should be noted that the above-mentioned preparation method of Rpf is only a feasible method in this embodiment and is not necessarily limited. The present invention can also adopt other existing technical methods to prepare Rpf protein, and the theoretical effect is similar.

[0035] (2) Preparation process of carbon-based adsorption / microbial aerobic degradation risk management composite materials

[0036] In this embodiment, the carbon-based adsorption / microbial aerobic degradation risk control composite material is a core-shell structure composed of an aerobic degradation bacteria shell and a functional material core, the core is composed of adsorption-type biochar, oxygen slow-release agent calcium peroxide and resuscitation-promoting factor (Rpf) to form a functional material, and the microsphere core is fixed by sodium alginate and calcium chloride; and the aerobic shell is a biofilm of petroleum hydrocarbon aerobic degradation bacteria Rhodococcus pyridinivorans attached to the surface of the microsphere. Therefore, the composite material in this embodiment is a Rhodococcus+Rpf+CaO2 core-shell structure material, and its design principle is based on: (1) Rhodococcus pyridinivorans has an Rpf response pathway, can be stimulated to grow by Rpf, and improve activity; (2) CaO2 can improve the degradation effect of aerobic bacteria by increasing the local oxygen content. In the oxidative degradation type biological reaction wall composite material of the functional material inner layer-aerobic bacteria outer layer finally prepared, the active ingredient is Rhodococcus pyridinivorans.

[0037] The preparation of the core-shell sphere structure of the composite material in this embodiment is described in detail below:

[0038] (2.1) Preparation of functional material core: First prepare a 1 wt.% sodium alginate solution at 40-50°C as the mother liquor, add WBC700 adsorptive biochar with a final concentration of 50 g / L, Rpf protein with a final concentration of 20 mg / L (prepared in the above step (1)) and calcium peroxide CaO2 with a final concentration of 2 wt.% to the mother liquor, mix well, and then add the mixed solution to a 1 wt.% CaCl2 solution through a droplet forming device to form microspheres.

[0039] The WBC700 adsorption biochar used above is obtained by oxygen-limited pyrolysis of sawdust at 700°C for 6 hours. The specific method is: the dried sawdust is put into a crucible and compacted, the crucible is covered with a lid and cracked in a muffle furnace, the temperature is increased to 700°C at a rate of 5°C / min, and the constant temperature is maintained for carbonization for 6 hours, then the heating is stopped, the grayish white part of the surface of the cracked sample is removed, and the carbonaceous component is retained to obtain the biochar.

[0040] (2.2) Preparation of aerobic degradation bacteria shell: The microspheres prepared above are used as the core of the functional material, and the microspheres are added to the LB medium of Rhodococcus pyridinivorans (prepared in the above step (1)) for overnight culture, so that a biofilm is formed on the surface of the microspheres in the core, and the biofilm is the shell of the aerobic degradation bacteria. Thus, a Rhodococcus+Rpf+CaO2 core-shell structure material with microspheres as the core of the functional material and Rhodococcus pyridinivorans biofilm as the shell of the aerobic degradation bacteria is obtained, and the particle size is 0.1 to 0.2 cm.

[0041] The morphological characteristics of the Rhodococcus+Rpf+CaO2 core-shell structure material prepared above were observed by optical microscope. Figure 1 As shown, it can be seen that the aerobic degradation bacteria + Rpf + CaO2 composite material exhibits an obvious core-shell multilayer structure under an optical microscope.

[0042] Embodiment 2:

[0043] In this example, the carbon-based adsorption / microbial aerobic degradation risk management composite material prepared in Example 1 was applied to the remediation of petroleum hydrocarbon pollution in groundwater to demonstrate its specific application effect.

[0044] In this example, the prepared composite material was used in simulated petroleum hydrocarbon contaminated groundwater, where the concentration of petroleum hydrocarbon in the simulated groundwater was 40 mg / L. The composite material was added to the simulated groundwater, and a single biochar control group was set up at the same time. The final simulation results are shown in Figure 2. Figure 2 to Figure 4 shown.

[0045] In this example, SYTO9 staining combined with laser confocal microscopy was used to characterize the distribution of microorganisms on the composite material. Figure 2 The morphological changes of the core-shell structure material in the present invention in simulated groundwater and the changes of the outer layer of pyridinium-eating Rhodococcus over time are shown. A1, A3, and A5 are the morphologies of the composite materials under the laser confocal bright field on the 0th day, the 2nd day, and the 7th day, respectively. A2, A4, and A6 are the composite materials corresponding to the green fluorescence field of the laser confocal on the 0th day, the 2nd day, and the 7th day, respectively. The green fluorescence in the figure is the aerobic degradation bacteria, i.e., pyridinium-eating Rhodococcus. The results show that the outer layer of the composite material shows obvious green fluorescence characteristics, while the inner core area of ​​the composite material has less green fluorescence, indicating that the aerobic degradation bacteria are loaded on the surface of the biochar material (A2). The composite material was added to simulate the actual petroleum hydrocarbon contaminated groundwater for cultivation experiments to investigate the morphological changes of the material. The results show that the number of green colonies in the outer layer of the microspheres gradually increases with the extension of the culture time, the thickness of the green colonies also increases, and the green colonies gradually tend to diffuse to the inner layer (A4 and A6).

[0046] In addition, single cell Raman spectroscopy can well reflect the material composition of cells. When combined with isotope labeling, the water utilization ability of aerobic degrading bacteria cells can be judged by the change of Raman peak position. In this example, the activity of aerobic degrading bacteria on the surface of the composite material was examined by incubating the composite material with heavy water. Figure 3The Raman spectroscopy characterization results of the activity of the outer layer of the core-shell structure material of the present invention are shown. T0, T2, and T7 are the Raman spectra of the single-cell aerobic degrading bacteria on the 0th day, the 2nd day, and the 7th day, respectively. The aerobic degrading bacteria without heavy water incubation are used as negative controls, and the aerobic degrading bacteria in the logarithmic phase incubated with 50% heavy water are used as positive controls. The Raman shift is 2800-3100cm -1 represents CH peak), Raman shift 2040-2300cm -1 Represents the CD peak. The results showed that the aerobic degrading bacteria on the surface of the composite material had an obvious D peak, and the C-Dratio was 31.3%, which was significantly higher than the CD ratio of the aerobic degrading bacteria in the logarithmic phase (red line and blue line). The above results show that the aerobic degrading bacteria of petroleum hydrocarbons on the composite material have good activity. Single-cell Raman spectroscopy showed that on the 2nd and 7th days of incubation, the CD ratio of aerobic degrading bacteria on the surface of the microspheres also gradually increased, from 31.3% on the 0th day to 39.5% on the 7th day. The above results show that in simulated petroleum hydrocarbon contaminated groundwater, aerobic degrading bacteria can use petroleum hydrocarbons as a carbon source for growth. At the same time, with the extension of time, the amount of aerobic degrading bacteria on the surface of the composite material increases, and the activity of aerobic degrading bacteria also gradually increases.

[0047] This example also examines the removal capacity and kinetics of petroleum hydrocarbons by the aerobic degradation bacteria + Rpf + CaO2 composite material through simulation and actual petroleum hydrocarbon contaminated groundwater remediation experiments. Figure 4 The removal effect of the core-shell structure material, the single material group and the control group on petroleum hydrocarbons in simulated groundwater in the present invention is demonstrated, wherein the single material is the biochar treatment group, and the control group is the experimental group without adding materials. From the results, it can be calculated that the purification capacity of the carbon-based adsorption / microbial aerobic degradation risk control composite material and the single biochar material for typical hydrocarbon organic pollutants on the site is 15.7 mg / g and 9.8 mg / g respectively. The carbon-based adsorption / microbial aerobic degradation risk control composite material of the present invention can achieve a petroleum hydrocarbon removal rate of 87.9% on the 10th day, and the purification capacity of typical hydrocarbon organic pollutants on the site is 60.2% higher than that of the single carbon-based material.

[0048] In addition, it should be noted that the water body to be repaired in the present invention may be other water bodies besides groundwater, such as lakes, rivers, landscape water bodies, etc., and there is no limitation on this.

[0049] The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A carbon-based adsorption / microbial aerobic degradation risk management composite material, characterized in that: The composite material is composed of an aerobic degradation bacteria shell and a functional material core to form a core-shell structure; The inner core is composed of functional materials such as adsorption biochar, oxygen slow-release agent calcium peroxide and resuscitation-promoting factor (Rpf), which are fixed by sodium alginate and calcium chloride to form the inner core of the microsphere; the outer shell of the aerobic degradation bacteria is a petroleum hydrocarbon aerobic degradation bacteria Rhodococcus pyridinus attached to the surface of the microsphere. Rhodococcus pyridinivorans ) biofilm; The petroleum hydrocarbon aerobic degradation bacteria Rhodococcus pyridinosus was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on December 11, 2023, with the deposit address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC NO. 29278; The preparation method of the carbon-based adsorption / microbial aerobic degradation risk management composite material comprises: S1. Take 1 wt.% sodium alginate solution at 40-50℃ as the mother solution, add adsorption biochar with a final concentration of 50 g / L, Rpf with a final concentration of 20 mg / L and calcium peroxide CaO2 with a final concentration of 2 wt.%, mix them evenly, and add the mixed solution to 1 wt.% CaCl2 solution through a droplet forming device to form microspheres; S2. The microspheres are added to LB medium inoculated with Rhodococcus pyridinovus for overnight culture, and a Rhodococcus pyridinovus biofilm is grown on the surface of the microspheres, thereby obtaining a core-shell structure composite material with the microspheres as the functional material core and the Rhodococcus pyridinovus biofilm as the aerobic degradation bacteria shell.

2. The carbon-based adsorption / microbial aerobic degradation risk management composite material according to claim 1, characterized in that: The adsorption-type biochar is the biochar obtained by pyrolysis of sawdust raw materials at 650-750°C with limited oxygen for 5-7 hours.

3. The carbon-based adsorption / microbial aerobic degradation risk management composite material according to claim 1, characterized in that: The particle size of the core-shell structure is 0.1-0.2 cm.

4. A method for preparing the carbon-based adsorption / microbial aerobic degradation risk management composite material as claimed in any one of claims 1 to 3, characterized in that: include: S1. Take 1 wt.% sodium alginate solution at 40-50℃ as the mother solution, add adsorption biochar with a final concentration of 50 g / L, Rpf with a final concentration of 20 mg / L and calcium peroxide CaO2 with a final concentration of 2 wt.%, mix them evenly, and add the mixed solution to 1 wt.% CaCl2 solution through a droplet forming device to form microspheres; S2. The microspheres are added to LB medium inoculated with Rhodococcus pyridinovus for overnight culture, and a Rhodococcus pyridinovus biofilm is grown on the surface of the microspheres, thereby obtaining a core-shell structure composite material with the microspheres as the functional material core and the Rhodococcus pyridinovus biofilm as the aerobic degradation bacteria shell.

5. The preparation method according to claim 4, characterized in that The LB medium inoculated with pyridinium-eating Rhodococcus needs to be revived and cultured to OD 600 Reach 1.7 ~ 1.

8.

6. A biological reaction wall, characterized in that: The carbon-based adsorption / microbial aerobic degradation risk control composite material as described in any one of claims 1 to 3 is used as filler.

7. Use of the carbon-based adsorption / microbial aerobic degradation risk management composite material as described in any one of claims 1 to 3 in the remediation of water bodies contaminated by petroleum hydrocarbons.

8. The use according to claim 7, characterized in that: The water body is groundwater.

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