CN110760506B

CN110760506B - Phenol-reducing bacterium immobilized spherical particle and preparation method and application thereof

Info

Publication number: CN110760506B
Application number: CN201911252683.9A
Authority: CN
Inventors: 王雪青; 房玉婷; 张安龙; 陈朵; 罗清; 王哲毅
Original assignee: Shaanxi University of Science and Technology
Current assignee: Shaanxi University of Science and Technology
Priority date: 2019-12-09
Filing date: 2019-12-09
Publication date: 2021-07-09
Anticipated expiration: 2039-12-09
Also published as: CN110760506A

Abstract

The invention provides phenol-reducing bacteria immobilized spherical particles and a preparation method and application thereof. The phenol-reducing bacteria mixed immobilized spherical particles formed by the method have strong mechanical property, small aperture and large specific surface area, make up for the defect of bacterial leakage, and are beneficial to improving the mass transfer rate of a system, so that the adhesion rate of sufficient bacteria is ensured, and the phenol-reducing performance and the recycling efficiency of the phenol-reducing bacteria immobilized spherical particles are improved.

Description

Phenol-reducing bacterium immobilized spherical particle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of environmental protection, and relates to a phenol-reducing bacterium immobilized spherical particle, and a preparation method and application thereof.

Background

The phenol-containing wastewater mainly refers to phenol organic pollutant wastewater generated in industries such as coking, oil refining, papermaking, plastics, ceramics, textile and the like. It is used as one of industrial waste water, has wide source and has great influence on environmental pollution. Phenol and its derivatives belong to Aromatic (Aromatic) compounds, are protoplasmic poisons, have a poisoning effect on organisms, and are difficult to degrade. Phenolics have been listed by the U.S. national environmental protection agency as one of a black list of 129 priority control pollutants. Phenol-containing wastewater has been classified as one of the harmful wastewater to be mainly solved in the water pollution control in China.

At present, compared with a physical and chemical method, the biological method for treating the phenol-containing sewage is more economical and efficient, has larger treatment capacity and has the advantage of no secondary pollution. In a reaction system for treating wastewater by microorganisms, inoculated microorganisms mainly have two forms of suspension and immobilization. The suspended microorganisms are easy to run off along with the effluent, so that the biomass in the reaction system is reduced, and the efficiency of removing pollutants is greatly reduced. The immobilized microorganism technology can obviously increase the concentration of microorganisms and has the advantages of high degradation efficiency, strong environmental adaptability, easy solid-liquid separation, repeated cyclic utilization and the like. It is therefore important to find a good means for immobilizing microorganisms.

Patent CN10117767A reports that the performance of the embedded particles obtained by the microorganism immobilization technology is significantly improved. The embedding method is the most common and widely studied method in the cell immobilization technology at present, and is to trap microbial cells in a network space of water-insoluble gel polymer gaps, prevent leakage of the cells and simultaneously allow substrate infiltration and product diffusion.

The existing embedding immobilization carriers are mainly divided into organic synthetic polymer carriers and natural polymer gel carriers. The organic synthetic polymer carrier has the advantages of good antimicrobial decomposability, high mechanical strength, stable chemical performance and the like; however, the formation conditions of the carrier polymer network are severe, the microbial cells are damaged greatly, and the cost is relatively high. The natural polymer gel is a substance separated and extracted from nature, and has the advantages of high biocompatibility, good mass transfer performance, easy formation, high microorganism density and activity and the like. Agar is a polysaccharide extracted from seaweed, is a natural polymer gel, and is a commonly used carrier in embedding technology. Agar is in hot water solution, is in viscous disordered water sol shape, and is gathered into a cavity when being cooled, and the cavity contains a large amount of water. The agar hydrogel has stronger elasticity, high stability and good biocompatibility, and is an ideal immobilized biological carrier. However, after the agar hydrogel is formed into balls, bacteria are easy to leak due to overlarge pore diameter, and on the other hand, the mechanical strength of the agar hydrogel is low; therefore, the degradation performance and the cycle performance of the microorganism-loaded organic fertilizer on pollutants are not high.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides phenol-reducing bacteria immobilized spherical particles and a preparation method and application thereof. The phenol-reducing bacteria mixed immobilized spherical particles formed by the method have strong mechanical property, small aperture and large specific surface area, make up for the defect of bacterial leakage, and are beneficial to improving the mass transfer rate of a system, so that the phenol-reducing performance and the recycling efficiency of the phenol-reducing bacteria immobilized spherical particles are improved.

The invention is realized by the following technical scheme:

a preparation method of the phenol-reducing bacterium immobilized spherical particles comprises the following steps:

(1) culturing the phenol-reducing bacteria in an MPYE liquid culture medium to a logarithmic phase, carrying out solid-liquid separation to remove a supernatant, collecting bacteria, adjusting the concentration of the bacteria by using sterile water, and carrying out centrifugal concentration to obtain a phenol-reducing bacteria concentrated solution, wherein the mass concentration of the bacteria in the phenol-reducing bacteria concentrated solution is 3-3.2 mg/L;

(2) adding agar and carrageenan into water, heating and dissolving to obtain an immobilized solution A, wherein the mass ratio of the agar to the carrageenan is 1 (1-3), and the mass concentration of the agar in the immobilized solution A is 1.5-3%;

(3) adding the phenol-reducing bacteria concentrated solution into the immobilized solution A, and uniformly stirring and mixing to obtain a solution B, wherein the volume ratio of the immobilized solution A to the phenol-reducing bacteria concentrated solution is 15 (2-3);

(4) dissolving potassium chloride in sterile water to prepare a potassium chloride aqueous solution, and then dripping the solution B obtained in the step (3) into the potassium chloride aqueous solution to form the norphenol bacterium immobilized spherical particles;

(5) filtering the phenol-reducing bacteria immobilized spherical particles by using sterile filter cloth, and washing the particles by using normal saline.

Preferably, in the step (1), the phenol-reducing bacteria are preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 16041.

Preferably, in the step (4), the mass concentration of the potassium chloride aqueous solution is 1-2%.

Preferably, the immobilized particles are washed with physiological saline in the step (5), and the mass concentration of the physiological saline is 0.8%.

The phenol-degrading bacterium immobilized spherical particles obtained by the preparation method.

The phenol-degrading bacteria immobilized spherical particles are applied to degrading phenol in water.

Compared with the prior art, the invention has the following beneficial technical effects:

carrageenan is a coagulant extracted from algae, has strong gel-forming and high-viscosity characteristics, satisfactory elasticity, transparency and solubility, and strong stability, and does not reduce the gel strength and viscosity even after being placed for a long time. Carrageenan contains more sulfate bonds, and negatively charged groups repel each other, so that carrageenan is the root cause of stronger colloid stability. The invention can obviously change the characteristics of the agar gel after mixing the agar and the carrageenan, leads the agar gel to tend to be rich in elasticity and improves the mechanical strength. Observing that the surface of the immobilized carrier has a very meshed pore channel through a scanning electron microscope (see figure 2), providing attachment points for microorganisms, increasing the attachment amount of the microorganisms due to the improvement of the surface roughness after the microorganisms are captured by the immobilized carrier, and observing that short rod-shaped microorganisms well grown are distributed in the pore channel, and the shape is uniform (see figure 1); more importantly, the aperture of the mixed carrier of the agar and the carrageenan is smaller than that of a single agar used as a carrier (see figure 2), and is more uniform, so that the leakage rate of bacteria is reduced, and the uniform gap enables the spherical particles to transfer substances more easily, so that the degradation of phenol by the bacteria is more direct and efficient. After the microorganisms are recycled for many times, the inside of the pore canal of the microorganisms is full of the microorganisms, the microorganisms are gathered together to form a string shape, and the immobilized carrier forms a biological protection niche, so that the microorganisms are prevented from being influenced by harmful environments such as toxic substances and the like. After repeated use for many times, the immobilized spherical particles do not break and float upwards, the properties are still stable, and the immobilized particles still keep higher strength. After being mixed, the agar and the carrageenan are used as immobilized carriers, and the loaded phenol-reducing bacteria have better phenol-reducing performance and stability compared with the pure agar carrier loaded phenol-reducing bacteria.

Drawings

FIG. 1 is an electron microscope photograph of the immobilized carrier-supported bacteria of the present invention, (a) agar, (b) 2% agar + 2% carrageenan.

FIG. 2 is an electron micrograph (magnification × 400) of the immobilizing material of the present invention. (a) 4% carrageenan granules, (b) 3% agar granules, (c) 2% agar and 2% carrageenan granules.

FIG. 3 shows the phenol-reducing effect of immobilized bacteria of different materials in the present invention.

FIG. 4 shows the phenol-reducing effect of immobilized bacteria with mixed carriers in different proportions.

FIG. 5 storage modulus of various supports.

FIG. 6 loss moduli of different carriers.

Figure 72% agar + 2% carrageenan immobilized bacteria recycled 20 times phenol degradation rate.

FIG. 82% agar + 2% carrageenan immobilized bacteria were recycled for 20 times of cell viability.

Figure 9 different proportions of mixed carriers provide a balling effect.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The phenol reducing bacteria adopted by the invention are novel strains of Klebsiella Pneumoniae, are named as ZS01 and are strains which are separated, purified and stored in a laboratory. The preservation number of the strain preserved in the China general microbiological culture Collection center is CGMCC No. 16041.

The sterile water adopted by the invention is deionized water sterilized by high-pressure steam, the treatment temperature is 121 ℃, and the treatment time is 30min under the condition of 0.105 MPa.

1. Preparation of the Cross-linking solution

1% KCl solution: dissolving 1g of potassium chloride in distilled water, diluting to a constant volume of 100mL, sterilizing at 121 ℃ for 30min, cooling to normal temperature after sterilization, and placing the solution under an ice bath condition to reduce the temperature to 0 ℃ for later use. A2% KCl solution was prepared in the same manner.

2. Preparation of culture Medium

MPYE liquid medium: 3g of yeast extract, 3g of peptone, 1M MgCl₂ 1.6mL，1M CaCl₂Adding 1mL of the mixture into 1000mL of distilled water, stirring the mixture fully and uniformly by magnetic force, adjusting the pH value to 7 by using 10% of NaOH and 10% of HCl in mass fraction, sterilizing the mixture at the high temperature of 121 ℃ for 30min, and cooling the mixture for later use.

Culture solution with phenol concentration of 1500 mg/L: glutamic acid 0.58g/L, 10% NaCl 10.0mL, nutrient solution [ nitrilotriacetic acid 10.0g, MgSO₄·7H₂O 29.5g，CaCl₂·2H₂O3.335g，FeSO₄·7H₂O 0.099g，(NH₄)6Mo₇O₂₄·4H₂0.0093g of O, 50.0mL of trace element liquid and 950.0mL of distilled water]20.0mL/L, adding phosphate buffer (KH) after sterilizing₂PO₄68.05g，K₂HPO₄87.09g, 1L distilled water) 20.0mL/L, vitamin solution (0.1 mg/mL nicotinic acid, 0.05mg/mL thiamine hydrochloride, 1.0 μ g/mL biotin) 10.0mL/L, phenol 1500mg/L, pH adjusted to 7 with 1mol/L NaOH and HCl.

Example 1

1. Preparation of immobilization solution

2% agar + 2% carrageenan: weighing 2g of agar and 2g of carrageenan, mixing, dissolving with distilled water, diluting to 100mL, sterilizing at 121 ℃ for 30min, and cooling to 40-45 ℃.

2. Enrichment culture of phenol-reducing bacteria

Transferring the phenol-reducing bacteria preserved in the 3-ring solid culture medium into a conical flask filled with 100mL of sterilized MPYE liquid culture medium by using an inoculating ring, uniformly mixing, sealing the periphery of the conical flask opening by using a sealing film, placing in a shaking table, culturing at the temperature of 30 ℃ and the rotating speed of 200r/min for 24h, and obtaining the phenol-reducing bacteria enriched culture solution.

3. Preparation of phenol-reducing bacterium concentrated solution

Centrifuging the phenol-reducing bacteria enriched culture solution at high speed for 5min at 8000r/min, discarding supernatant, collecting thallus, and regulating the bacterial concentration OD with sterile water₆₀₀At 0.8, 25mL OD₆₀₀Centrifuging the 0.8 bacterial solution for 5min at 8000r/min, discarding 2mL of the rest supernatant, and mixing to obtain the phenol-reducing bacteria concentrated solution with the mass concentration of 3.1 mg/L.

4. Preparation of phenol-reducing bacterium immobilized spherical particles

2% agar + 2% carrageenan: adding 2mL of the norphenol bacteria concentrated solution into 15mL of the cooled immobilized solution, uniformly mixing to obtain a solution B, slowly dripping the solution B into a 1% KCl solution by using a syringe type injector with a 16G needle to form immobilized spherical particles with the particle size of 3-4 mm, filtering the immobilized spherical particles by using sterile filter cloth, and then washing the particles by using 0.8% physiological saline for later use.

Example 2

1. Preparation of immobilization solution

1.5% agar + 1.5% carrageenan: weighing 1.5g of agar and 1.5g of carrageenan respectively, mixing, dissolving with distilled water, diluting to 100mL, sterilizing at 121 ℃ for 30min, and cooling to 40-45 ℃.

2. Enrichment culture of phenol-reducing bacteria

3. Preparation of phenol-reducing bacterium concentrated solution

The phenol-reducing bacteria enriched culture solution is put into a high-speed centrifuge at a high speed of 8000r/minCentrifuging for 5min, discarding supernatant, collecting thallus, and adjusting bacterial concentration OD with sterile water₆₀₀At 0.8, 25mL OD₆₀₀Centrifuging the 0.8 bacterial solution for 5min at 8000r/min, discarding 2mL of the rest supernatant, and mixing to obtain the phenol-reducing bacteria concentrated solution with the mass concentration of 3.1 mg/L.

4. Preparation of phenol-reducing bacterium immobilized spherical particles

1.5% agar + 1.5% carrageenan: adding 2mL of the norphenol bacteria concentrated solution into 15mL of the cooled immobilized solution, uniformly mixing to obtain a solution B, slowly dripping the solution B into a 1% KCl solution by using a syringe type injector with a 16G needle to form immobilized spherical particles with the particle size of 3-4 mm, filtering the immobilized spherical particles by using sterile filter cloth, and then washing the particles by using 0.8% physiological saline for later use.

Example 3

1. Preparation of immobilization solution

1% agar + 3% carrageenan: weighing 1g of agar and 3g of carrageenan, mixing, dissolving with distilled water, diluting to 100mL, sterilizing at 121 ℃ for 30min, and cooling to 40-45 ℃.

2. Enrichment culture of phenol-reducing bacteria

3. Preparation of phenol-reducing bacterium concentrated solution

4. Preparation of phenol-reducing bacterium immobilized spherical particles

1% agar + 3% carrageenan: adding 2mL of the norphenol bacteria concentrated solution into 15mL of the cooled immobilized solution, uniformly mixing to obtain a solution B, slowly dripping the solution B into a 1% KCl solution by using a syringe type injector with a 16G needle to form immobilized spherical particles with the particle size of 3-4 mm, filtering the immobilized spherical particles by using sterile filter cloth, and then washing the particles by using 0.8% physiological saline for later use.

Comparative example 1

Agar gel method: adding 2mL of the above phenol-reducing bacteria concentrated solution into 15mL of agar gel solution with content of 3%, mixing well, sucking with 20mL syringe, extruding, dropping into ice-bath sterile water, and coagulating at 4 deg.C for 3 hr.

Comparative example 2

A carrageenan gel method: adding 2mL of phenol-reducing bacteria concentrated solution into 15mL of 4% carrageenan solution, mixing uniformly, sucking with a 20mL syringe, dripping into 1% KCl solution, and crosslinking at 4 ℃ for 3h for later use.

Comparative example 3

1. Enrichment culture of phenol-reducing bacteria

2. Preparation of suspension cells

Centrifuging the phenol-reducing bacteria enriched culture solution at high speed for 5min under the condition that the rotation speed of a high-speed centrifuge is 8000r/min, discarding supernatant, collecting thallus, and adjusting the bacteria concentration of the bacteria solution with sterile water_OD600Measuring 25mL of bacterial liquid as suspension cells for standby when the cell concentration is 0.8.

First, degradation efficiency test

Degradation efficiency tests were performed on the norfolacin immobilized spherical particles prepared in examples 1 to 3 and comparative examples 1 to 3, respectively. Inoculating the phenol-reducing bacteria immobilized spherical particles and 25mL of suspended cells into a culture solution with the phenol concentration of 1500mg/L, placing the culture solution into a constant-temperature shaking table for shaking culture at 30 ℃ and 150r/min, and measuring the degradation condition of phenol at regular time.

From comparison example 3 and comparison examples 1 and 2, it can be seen from FIG. 3 that the removal rate of phenol by immobilized bacteria has a shorter lag phase and higher superiority than that of free bacteria. Bacteria in pure agar-immobilized form degrade phenol 100% in 60 hours, while free cells (non-immobilized cells) take longer to completely remove phenol. Compared with agar, because of the lower porosity inside the carrageenan particles, the direct action of phenol and microorganisms is hindered, the mass transfer efficiency is limited, and the utilization rate of phenol in the carrageenan particles is lower.

Examples 1, 2 and 3 were conducted to investigate the effect of different ratios of agar and carrageenan on phenol degradation efficiency. As can be seen from fig. 4: the 2% agar and 2% carrageenan ratio has a clear advantage over other ratios, as shown in figure 1 b: the mixed matrix provides a larger surface area for the attachment of bacteria, and besides, the rough surface compensates the problem of low bacterial adhesion rate caused by the excessively smooth surface of the agar; on the other hand, it makes the larger pore size of the agar smaller and more uniform, thereby reducing the leakage rate of bacteria. The smaller and uniform voids allow the spherical particles to more easily transfer substances, thereby allowing bacteria to act directly on phenol.

From examples 1 to 3, it can be seen that: after agar and carrageenan of different concentrations are combined, the immobilized microorganism presents different phenol degradation efficiencies, and after 2% agar and 2% carrageenan are mixed, the aperture is reduced, the specific surface area is increased, leakage is reduced, mass transfer is increased, and the phenol degradation efficiency is increased. The degradation rate of phenol reaches 100 percent in 55 h.

Second, rheological Property test

The rheological properties of the immobilized spherical particles of the norfolacin prepared in examples 1 to 3 and comparative examples 1 to 2 were respectively measured by a rheometer, and a 25mm flat plate was used to perform temperature slope shaking at 20 to 40 ℃ with a plate interval of 1 mm.

The gel characteristics of 3 percent agar, 4 percent agar and mixed agar with different proportions are determined by an oscillatory rheology method, and the storage modulus and the loss modulus are measured in a temperature range of 25-40 ℃. The storage modulus and loss modulus values in all the tested hydrogels increased with decreasing temperature, which is typical of gel formation. The results show that the storage modulus values are much larger than the values of the loss modulus, which is why they exhibit a solid form in this temperature range. When the temperature is around 25 ℃, as shown in fig. 5 and 6, the gel strength (storage modulus and loss modulus) of agar is lower than that of carrageenan, probably because no ionic interaction is formed between agar, and the formation of gel is promoted by the presence of potassium ions in carrageenan and mixed carrier. There was a significant difference in gel strength between agar, carrageenan and mixed vehicle, which was probably due to the content of anhydrogalactose-linked sulfate ester salts. The results show that: the 2% agar and 2% carrageenan had the best gelling properties. Therefore, when the optimal addition amount of the carrageenan is 2%, the gel strength of the mixed carrier can be obviously improved.

As can be seen from FIG. 9, the combination of agar and carrageenan in different proportions has the best effect of forming spheres by mixing the carrier with 2% agar and 2% carrageenan.

Thirdly, testing the reusability:

the immobilized particles of example two were placed in 1500mg/L phenol solution at 30 deg.C and 150r for min^-1After shaking culture for 3d, the phenol degrading ability was measured. And (3) taking out the immobilized microorganism particles, washing the immobilized microorganism particles for 3 times by using sterile water, performing a phenol degradation experiment for the second time, repeating the phenol degradation experiment of the immobilized particles by analogy of the method, and researching the recycling reusability of the immobilized microorganism particles.

As can be seen from the cyclic repetition experiment of example II, FIG. 7, the immobilized bacteria can achieve a total phenol degradation rate of 100% within 32 hours after 10 consecutive reaction cycles; with the increase of the cycle times, the degradation rate of the phenol is increased, and when the cycle is carried out to the 14 th time, the degradation rate of the immobilized bacteria to the phenol within 27h can reach 100%; on cycling to 20 cycles, the phenol degradation rate decreased slightly, but the total degradation time remained within 33 h. As can be seen from fig. 8, if the initial activity is considered to be 100%, the relative activity is expressed as a percentage thereof from the initial activity. The viability of the cells of the immobilized product is still maintained above 100% at 20 cycles, and the relative viability of the cells reaches a maximum of 113% at 14 th cycle. The relative phenol-reducing activity of the compound is improved due to two reasons: 1. the continuously proliferated bacteria provide enough biomass for the degradation of phenol, form a natural barrier under the protection of the immobilized system carrier and reduce the toxic effect of phenol; 2. the phenol reducing bacteria can be further adaptive to phenol by repeated recycling, so that the phenol reducing efficiency is improved to a certain extent. And when the immobilized bacteria circulate to 20 periods, the particles still have no obvious fragmentation phenomenon and keep complete shapes.

From the above examples it can be seen that: by comparing the phenol degradation performance, the mechanical strength and the reusability of the phenol-reducing bacteria immobilized spherical particles, it is found that when the mass fractions of the agar and the carrageenan are both 2%, the immobilized phenol-reducing bacteria show higher phenol-reducing performance, mechanical performance and stability.

The phenol-reducing bacteria immobilized spherical particles prepared by the invention have the advantages of strong mechanical property and reusability, simple preparation method, mild reaction condition, low price and the like; and because the carrageenan has stronger gelling property and high viscosity characteristic, high elasticity, transparency and solubility, the mechanical property of the agar pellet is favorably enhanced, the mass transfer efficiency is promoted, and the agar is more easily formed into the pellet. On the premise of ensuring the growth activity and sufficient biomass of the phenol-reducing bacteria, the treatment effect of the phenol-reducing bacteria on the organic wastewater is improved.

Several specific illustrations and advantages of the invention:

first, agar is in a hot aqueous solution in the form of a viscous, disordered water sol that, upon cooling, aggregates into a cavity containing a large amount of water. The agar hydrogel has stronger elasticity, high stability and good biocompatibility, and is an ideal immobilized carrier; the carrageenan has strong gelling property and high viscosity characteristic, strong elasticity, high transparency and solubility and outstanding stability, the gel strength and viscosity of the carrageenan cannot be reduced even if the carrageenan is placed for a long time, the carrageenan is very stable at normal temperature, the gelling property of the carrageenan can be obviously improved after the agar and the carrageenan are mixed, the gel tends to be rich in elasticity, and the hydrophilic phenomenon of the carrageenan can be reduced. Carrageenan has a large number of sulfate ester bonds, and negatively charged groups repel each other, thereby increasing colloidal stability. The natural polymer gel is a substance separated and extracted from nature, and has the advantages of no biological toxicity, good mass transfer performance, easy formation, high immobilization density and the like. When the method is applied to industry, the price is low, and the method has good development prospect.

Second, the mixed carrier has the advantages of: 1. the compression resistance and hardness are high, and the compression resistance and the water impact resistance of the agar spherical particles are favorably enhanced; 2. the obtained particles have uniform size, and can maintain the uniformity of the mechanical strength of spherical particles and the shape of spheres; 3. the porous material has good biocompatibility, has no toxicity to microorganisms, and is beneficial to the attachment and mass transfer process of the microorganisms; 4. the reutilization efficiency is high, and the performance is stable in the fermentation reaction process; 5. the cost of the used raw materials is low, and the preparation process is simple and convenient.

Thirdly, compared with the traditional biotechnology, the immobilized cell can tolerate high-concentration phenol-containing wastewater, and because of the advantage of recycling, the service life of the immobilized cell is prolonged, the treatment efficiency is high, and microorganisms are continuously added with value, so that the reactor can keep higher biomass concentration; the higher the concentration of the microorganisms, the shorter the treatment time, and the smaller the volume of the reactor required, thus contributing to less capital construction and operating costs; the solid-liquid separation is easy to realize, and the microorganisms immobilized by the insoluble carrier are in a highly dense state, are easy to separate from water, are beneficial to the interception and the reutilization of the microorganisms, reduce the loss of the microorganisms to a great extent and are beneficial to the improvement of the effluent quality; because the microorganism is limited in a certain space by immobilization, the sludge bulking phenomenon does not exist, and the loss of some microorganisms which are suspended in the wastewater and have better treatment capacity due to natural sedimentation separation in an activated sludge method is avoided; is suitable for treating waste water containing toxic and harmful substances, and has high microbial stability, high toxicity resistance and high environment adaptability.

Fourthly, potassium chloride (chemical formula: KCl), one of hydrochloride, white crystal or crystalline powder, is easy to dissolve in water and glycerol, difficult to dissolve in alcohol, insoluble in ether and acetone, mild in property, nontoxic, wide in source and low in price. The mixed carrier embedded particles form uniform spherical particles in a liquid potassium chloride solution, the particles are not toxic to microorganisms, potassium chloride serving as a dispersion medium can separate each drop of injected mixed solution, each drop of solution is independently formed into spheres, the particles are not polymerized or agglomerated after being formed, and the formed particles are spherical and have uniform sizes. The potassium chloride has a synergistic effect on the carrageenan, is a salt, and is most strong in the synergistic effect on the carrageenan because the Kappa-carrageenan is sensitive to potassium ions, has the highest potassium ion content and is a strong electrolyte. Generally, within 2 percent of the content of potassium chloride solution, the synergistic effect on the carrageenan gelling is realized, and the synergistic amplitude is reduced along with the increase of the dosage of potassium chloride.

Claims

1. A preparation method of phenol-degrading bacterium immobilized spherical particles is characterized by comprising the following steps:

(5) filtering the phenol-reducing bacteria immobilized spherical particles by using sterile filter cloth, and then washing the particles clean by using normal saline;

wherein in the step (5), the particle size of the obtained phenol-degrading bacterium immobilized spherical particles is 3-4 mm.

2. The method for preparing the phenol-reducing bacteria immobilized spherical particles as claimed in claim 1, wherein in the step (1), the phenol-reducing bacteria is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 16041.

3. The method for preparing the norphenolic fungus immobilized spherical particles as claimed in claim 1, wherein in the step (4), the mass concentration of the potassium chloride aqueous solution is 1-2%.

4. The method for preparing the phenolic bacteria immobilized spherical particles according to claim 1, wherein the immobilized particles are washed with physiological saline solution at a concentration of 0.8% by mass in step (5).

5. The norphenol bacterium immobilized spherical particles obtained by the production method according to any one of claims 1 to 4.

6. The phenol-reducing bacteria immobilized spherical particles of claim 5, which are used for treating phenol-containing wastewater.