Preparation method of bionic silicon mineralizing bacteria and its application in treating phenol wastewater

Abstract

The present invention discloses a method for preparing biomimetic silicon mineralizing bacteria, the preparation method comprising: (1) collecting a bacterial suspension by centrifugation, adding a cationic polyelectrolyte aqueous solution, depositing the cationic polyelectrolyte on the bacterial surface by low-speed stirring, then centrifuging and discarding the supernatant to obtain bacteria with modified surface charge; (2) adding the surface-modified bacteria prepared in step (1) to an aqueous solution of a mineralizing precursor solution containing silicon elements that has been hydrolyzed overnight, stirring at a low speed, then centrifuging and discarding the supernatant to obtain bacteria with biomimetic silicon mineralization on the surface. The present invention also discloses the use of the biomimetic mineralizing bacteria prepared by the above preparation method in the treatment of phenol wastewater. The present invention can successfully modify a nano-silicon dioxide shell on the bacterial surface through a one-step silicon mineralizing bacteria method, protect the bacteria to have good survival ability in harsh environments, and can be applied to the treatment of phenol wastewater problems.

Description

Technical Field

[0001] The invention relates to the technical field of cell surface interface engineering, and in particular to a preparation method of bionic silicon mineralizing bacteria and application thereof in treating phenol wastewater. Background Art

[0002] Phenol is a toxic and harmful organic compound in soil and industrial wastewater, which has caused many serious environmental problems and endangered human health. Due to the high toxicity and stable chemical properties of phenol, it is particularly important to expand effective ways to prevent and control its pollution. There are many physical and chemical technologies for treating phenol in wastewater, such as activated carbon adsorption, ultraviolet radiation, electrochemical oxidation, ozonation and hydrogen peroxide oxidation. Among these commonly used methods, biodegradation is more economical and feasible, environmentally friendly, and practical for large amounts of wastewater. Several bacteria such as Rhodococcus, Bacillus, Pseudomonas, and Acinetobacter have the ability to degrade phenol in non-extreme environments. Among them, based on the extensive metabolic activities of Rhodococcus, they can be applied to the treatment of phenolic wastewater. However, the application of these biological processes is largely limited due to the weak survival and biological activity of bacteria in highly toxic environments. Although cell immobilization can protect bacteria from harmful conditions in biodegradation, its mass transfer limitation reduces the biodegradation rate at low concentrations of phenol. Therefore, a biocompatibility approach is needed that can both improve the bacterial tolerance in harsh environments and enhance the biodegradation rate over a wide range of concentrations.

[0003] In the process of biological evolution in nature, living organisms can form various inorganic minerals on their surface or inside through spontaneous biomineralization, such as teeth, bones, shells, etc. These biominerals have important functions, including mechanical support, signal transmission, and protection. For example, diatoms form a silica shell with a special structure on their surface to protect them from harmful external environments and improve their photosynthetic efficiency. However, not all living organisms have the ability of biomineralization.

[0004] Scientists have proposed that non-mineralized organisms can be regulated by artificially introducing macromolecules related to biomineralization, inducing their mineralization to achieve the functionalization of organisms, and this is called bionic mineralization. Among them, silica shells have shown excellent performance in many organisms. Bionic silicon mineralized shells can be realized through cell surface interface engineering. Specifically, polyelectrolytes, polypeptide proteins, etc. are modified by using the characteristics of the cell surface, so that the cell surface has a bionic mineralization template, thereby inducing the formation of silica shells on the cell surface. At present, silica shells have been successfully constructed on the surfaces of various organisms, such as viruses, fungi, bacteria, plant cells, animal cells, etc., using bionic mineralization strategies. However, in the process of constructing bionic silicon mineralized shells, most of them use layer-by-layer self-assembly technology to alternately deposit polyelectrolytes, polypeptide proteins, etc. on the cell surface. This complex process will affect the cell activity of the organism.

[0005] Therefore, a method with good biocompatibility is needed to construct a biomimetic silicon mineralized shell on the surface of bacteria to protect the biodegradation ability of single cells in harsh environments, which has great application prospects in the treatment of phenol wastewater. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing bionic silicon mineralizing bacteria and its application in treating phenol wastewater. Through this one-step silicon mineralizing bacteria method, a nano-silicon dioxide shell can be successfully modified on the surface of the bacteria to protect the bacteria from having good survival ability in harsh environments, so that it can be applied to the treatment of phenol wastewater problems.

[0007] The present invention provides the following technical solutions:

[0008] A method for preparing bionic silicon mineralizing bacteria, the preparation method comprising:

[0009] (1) collecting the bacterial suspension by centrifugation, adding a cationic polyelectrolyte aqueous solution, depositing the cationic polyelectrolyte on the bacterial surface by low-speed stirring, and then centrifuging and discarding the supernatant to obtain bacteria with modified surface charge;

[0010] (2) adding the surface-modified bacteria prepared in step (1) to an aqueous solution of a mineralization precursor solution containing silicon that has been hydrolyzed overnight, stirring at a low speed, and then centrifuging and discarding the supernatant to obtain bacteria with biomimetic silicon mineralization on the surface.

[0011] Preferably, in step (1), the cationic polyelectrolyte is polyethyleneimine or polydimethyldiallylammonium chloride.

[0012] Preferably, in step (2), the mineralization precursor solution containing silicon element is selected from sodium silicate, ethyl orthosilicate or 3-mercaptopropyltrimethoxysilane.

[0013] In the present invention, in order to maintain good biological activity of bacteria during the biomimetic mineralization process, the concentration and pH of the polyethyleneimine aqueous solution and the sodium silicate aqueous solution in the preparation method must be within an appropriate range, and the concentration of bacteria in the reaction and the reaction time with each solution must also be optimized.

[0014] Preferably, in step (1), the concentration of the cationic polyelectrolyte aqueous solution is 100-200 mg / L, and the pH is adjusted to 7.0-7.5, the low-speed centrifugation speed is 300-500 rpm, and the bacterial concentration is 2×10 6 -5×10 6 The stirring reaction time is 10-15 min.

[0015] Preferably, in step (2), the concentration of the silicon-containing mineralization precursor solution is 5-10 mmol / L, and the pH is adjusted to 7.0-7.5, the low-speed centrifugation speed is 300-500 rpm, and the stirring reaction time is 20-30 min.

[0016] Further preferably, in step (1), the concentration of the cationic polyelectrolyte aqueous solution is 100 mg / L, and the pH is adjusted to 7.0, the low-speed centrifugation speed is 300 rpm, and the bacterial concentration is 2×10 6 / mL, and the stirring reaction time is 10min.

[0017] Further preferably, in step (2), the concentration of the silicon-containing mineralization precursor solution is 5 mmol / L, and the pH is adjusted to 7.0, the low-speed centrifugation speed is 300 rpm, and the stirring reaction time is 20 min.

[0018] It is more appropriate to select the concentration of cationic polyelectrolyte aqueous solution at 100 mg / L. Too low a concentration will result in too little cationic polyelectrolyte deposition on the bacterial surface, resulting in the inability to form a silica shell. Too high a concentration will significantly reduce the biological activity of the bacteria. It is best to use a 5 mmol / L concentration of a mineralization precursor solution containing silicon. If the concentration is too low, only a few silica particles will be deposited on the bacterial surface. If the concentration is too high, the silica shell will be too thick, affecting the material exchange of the bacteria themselves. The best bacterial concentration is 2×10 6 / mL, which can ensure that the surface of a single bacterium can be completely biomimetic mineralized. In order to ensure the activity of mineralized bacteria, the pH of the cationic polyelectrolyte aqueous solution is maintained at 7.0, and the reaction time is not too long, the optimal time is 10 minutes. In order to promote the hydrolysis of the sodium aqueous solution, ensure the integrity of bacterial mineralization and maintain the activity of bacteria before and after mineralization, the pH of the silicon-containing mineralization precursor aqueous solution is adjusted to 7.0, and the optimal reaction time is 20 minutes.

[0019] The present invention also provides an application of bionic mineralizing bacteria in treating phenol wastewater, wherein the bionic mineralizing bacteria is obtained by the above-mentioned preparation method.

[0020] Preferably, the bacteria is of the genus Rhodococcus, Bacillus, Pseudomonas or Acinetobacter.

[0021] Further preferably, the bacteria is of the genus Rhodococcus.

[0022] Further preferably, the pH value of the phenol wastewater is 3-6.

[0023] In the present invention, when polyethyleneimine and sodium silicate solution are used, the technical principle of the present invention is:

[0024] Polyethyleneimine is used as a bridging molecule for one-step modification of the bacterial surface. It is deposited on the negatively charged bacterial surface through electrostatic interaction and provides nucleation sites for subsequent biomimetic silicon mineralization. Sodium silicate aqueous solution is used as a silicon source for biomimetic silicon mineralization on the bacterial surface, and its hydrolysis reaction is as follows:

[0025]

[0026] By adding hydrochloric acid solution, the sodium hydroxide in reaction formula 1 reacts with hydrochloric acid to undergo a neutralization reaction, and the reaction formula is as follows:

[0027] NaOH + HCl = NaCl + H 2 O Reaction 2

[0028] The equilibrium of reaction formula 1 proceeds in the positive direction to obtain a silicic acid solution. After modification with cationic polyelectrolyte, the polyethyleneimine exposed on the bacterial surface acts as a catalytic template, and in situ silicon mineralization occurs on the bacterial surface under the induction of silicic acid conditions.

[0029] The technical concept of the present invention is to select a cationic polyelectrolyte with good biocompatibility (which can be polyethyleneimine, polydimethyldiallyl ammonium chloride, etc.), and deposit it on the surface of bacteria with negative surface charge through electrostatic interaction. The means of charge modification of the bacterial surface provides nucleation sites for subsequent biomimetic mineralization. Subsequently, a mineralization precursor solution containing silicon elements (which can be sodium silicate, ethyl orthosilicate, 3-mercaptopropyltrimethoxysilane, etc.) is selected to provide a silicon source for its biomimetic mineralization, and a nano-scale silica shell is formed on the surface of the bacteria modified by the cationic polyelectrolyte. Using this biomimetic mineralization strategy, bacteria with a silica shell have good survival activity and the ability to degrade organic matter in a strongly acidic phenol solution.

[0030] The method of constructing an inorganic nano-silica shell on the bacterial surface by biomimetic mineralization provided by the present invention can improve the survival ability of bacteria in harsh environments, and thus be applied to the biodegradation of phenol-containing wastewater, thereby solving the limitation of biodegradation being affected by the environment in sewage treatment, especially the problem of low bacterial biodegradation efficiency in a strongly acidic environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Scanning electron micrographs of unmineralized bacteria (a) and bionic silicon-mineralized bacteria (b); X-ray electron spectra of unmineralized bacteria (a) and bionic silicon-mineralized bacteria (b).

[0032] Figure 2 Transmission electron micrographs of unmineralized bacteria (a), bacteria modified with polyethyleneimine (PEI) (b), and bionic silicon-mineralizing bacteria (c); high-resolution transmission electron microscopy X-ray spectroscopy elemental image analysis of bionic silicon-mineralizing bacteria (df).

[0033] Figure 3 Zeta potential analysis of unmineralized bacteria (a), bacteria modified with polyethyleneimine (PEI) (b), and biomimetic silicon mineralized bacteria (c).

[0034] Figure 4 Degradation rates of unmineralized bacteria and biomimetic silicon-mineralizing bacteria in phenol solutions at pH = 3 (a), pH = 4 (b), pH = 5 (c) and pH = 6 (d). DETAILED DESCRIPTION

[0035] To make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0036] Example 1: Construction of biomimetic silicon mineralized shell on the surface of Rhodococcus erythroxysporum using cell surface modification

[0037] Rhodococcus rubervillei cultured in the basal medium was collected by centrifugation at 5000 g for 5 min and washed three times with PBS buffer. The cells were resuspended in polyethyleneimine solution (100 mg / L, pH 7.0) and diluted to 2 × 10 Rhodococcus rubervillei per ml. 6 After incubation with continuous shaking for 10 minutes, the Rhodococcus ruberii was collected by centrifugation at 5000g for 5 minutes and washed twice with PBS buffer. The Rhodococcus ruberii was dispersed in a pre-prepared 5mmol / L sodium silicate aqueous solution (pH 7.0), stirred for 20 minutes, washed three times with PBS buffer, and collected by centrifugation at 5000g for 5 minutes to obtain biomimetic silicon-mineralized Rhodococcus ruberii.

[0038] Basic culture medium formula (1L): peptone 10.0g, yeast extract 5.0g, glucose 1.0g, distilled water 1.0L, adjust pH to 6.8-7.0, and sterilize at 121℃ for 20min.

[0039] like Figure 1 As shown in the figure, scanning electron microscopy and X-ray electron spectroscopy can prove that the bacterial surface has been successfully biomimetic silicon mineralized, which is different from the smooth surface of the unmineralized bacterial surface, and there is an obvious silicon element signal on the surface; Figure 2 As shown in Figure 2, observations using a transmission electron microscope and high-resolution transmission electron microscope X-ray energy spectrum elemental image analysis show that the thickness of the silica shell on the bacterial surface is about 40 nm. Figure 3 As shown, from the Zeta potential, it can be seen that the original potential of the bacteria is -40.77±1.07mV, which is converted to 18.07±1.67mV after modification with cationic polyelectrolyte, and then the potential is converted to -9.36±0.74mV after bionic silicon mineralization.

[0040] Example 2 Biodegradation of biomimetic silicon-mineralized Rhodococcus erythroxysporum in acidic phenol solution

[0041] Rhodococcus rubervillei was cultured in a biological shaker at a temperature of 30°C and 150 r / min. The Rhodococcus rubervillei was cultured and activated with a basic culture medium, and the Rhodococcus rubervillei was collected by centrifugation at 5000g for 5 minutes, and dispersed in an inorganic salt culture medium, with phenol as the sole carbon source, and the initial concentration was 100 ppm. The pH of the inorganic salt culture medium was adjusted to 3, 4, 5, and 6 with HCl, and the supernatant was taken at different time points. The absorbance value was measured at 270 nm by an ultraviolet spectrophotometer to determine the concentration change of phenol after degradation by Rhodococcus rubervillei at each time point.

[0042] Inorganic salt culture medium formula (1L): MgSO 4 0.2g; KH 2 PO 4 0.2g; K 2 HPO 4 0.5g; NH 4 Cl 2g; FeCl 3 0.005g; CaCl 2 0.001 g, adjust pH to 7.0, and sterilize at 121°C for 20 min.

[0043] like Figure 4As shown in the figure, from the degradation curve of bacteria to phenol, it can be seen that as the pH decreases, the degradation ability of the unmineralized bacteria decreases significantly, and is almost completely 0 at pH 3, while the bacteria mineralized by bionic silicon can still maintain good activity. This shows that this method can successfully construct a nano-bionic silicon mineralized shell on the surface of bacteria, while maintaining the biological activity of bacteria in an acidic environment, and thus be applied to the treatment of phenol wastewater.

[0044] The specific implementation methods described above provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above is only the most preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of bionic silicon mineralizing bacteria in the treatment of phenol wastewater, It is characterized in that The preparation method of the bionic silicon mineralizing bacteria comprises: (1) The Rhodococcus suspension was collected by centrifugation and diluted to a concentration of 2 × 10 6 / mL, polyethyleneimine was deposited on the bacterial surface by low-speed stirring for 10 min, and then centrifuged at 5000 g for 5 min to collect and discard the supernatant to obtain surface charge-modified bacteria; (2) Add the surface charge-modified bacteria prepared in step (1) to an aqueous solution of a silicon-containing mineralization precursor solution that has been hydrolyzed overnight, wherein the silicon-containing mineralization precursor solution is a 5 mmol / L sodium silicate aqueous solution, and adjust the pH to 7.

0. Stir at a low speed for 20 min, then centrifuge at 5000 g for 5 min, collect and discard the supernatant, and obtain surface bionic silicon mineralizing bacteria.

2. Application of the biomimetic mineralizing bacteria according to claim 1 in treating phenol wastewater, It is characterized in that The pH value of the phenol wastewater is 3-6.

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