CN114540337A

CN114540337A - Preparation method and application of microorganism immobilized composite particles

Info

Publication number: CN114540337A
Application number: CN202210181583.7A
Authority: CN
Inventors: 项雪珠; 黄明智; 易晓辉; 郑婉冰
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-05-27

Abstract

The invention relates to the field of environmental bioremediation, in particular to a preparation method and application of microorganism immobilized composite particles. The preparation method comprises the following steps: s1, washing, drying and heating a biomass material for reaction, washing with inorganic strong acid, drying, grinding and sieving to obtain biochar; s2, culturing a Chryseobacterium strain, centrifuging, washing and resuspending to obtain a bacterial suspension, and adding the biochar to obtain a mixed solution; heating, dissolving and sterilizing polyvinyl alcohol and sodium alginate, and adding the mixed solution to obtain an embedding solution; dissolving calcium chloride and boric acid in water to obtain a cross-linking solution; and S3, dripping the embedding liquid into the cross-linking liquid, reacting, and washing to obtain the microorganism immobilized composite particles. The microorganism immobilized composite particles prepared by the invention have the advantages of good mass transfer performance, good mechanical performance, large specific surface area and the like, and have good degradation effect on neonicotinoid pesticides in water.

Description

Preparation method and application of microorganism immobilized composite particles

Technical Field

The invention relates to the field of environmental bioremediation, in particular to a preparation method and application of microorganism immobilized composite particles.

Background

Neonicotinoid pesticide is a systemic neuroactive pesticide. The insect is killed by blocking the normal conduction of the central nervous system of the insect, is widely applied to the pest control of vegetables, fruits, grain crops, human living environment and the like, and is the most widely applied pesticide in the whole world. Neonicotinoid pesticides have persistence, migration and bioaccumulation properties, can have extremely serious influence on endocrine, respiratory, reproductive and nervous systems of non-target organisms (such as bees, birds, aquatic organisms and humans), and have potential carcinogenicity, mutagenicity and teratogenicity. The neonicotinoid pesticide can enter a sewage treatment system or be directly discharged to the environment through runoff rainwater, domestic sewage and other ways in the using process, has stable property, is not easy to be converted and degraded, can be migrated and permanently enriched along with sewage and sludge, and brings harm to aquatic ecology and environmental health of a receiving water body.

Microbial degradation is considered to be the most effective, most environmentally friendly, in situ remediation route. However, the biological method has the defects of small bacterial liquid amount in unit volume, slow reaction rate, easy loss, great influence of environmental factors and the like, and most of degradation strains are difficult to be directly used for repairing polluted environments. As one of the potential applications of pollutant degradation, the immobilized microorganism technology has the advantages of high biological concentration, good treatment efficiency, stable operation, simple solid-liquid separation, less sludge production, contribution to the development of a novel reactor and the like. The immobilized cell is used for treating refractory organic substances, besides the advantages, high-efficiency strains can be artificially selected and maintained, and the bearing capacity and the degradation capacity of the cell on toxic substances are obviously improved after the cell is immobilized. In real-world applications, how to safely and economically remove neonicotinoid insecticides in situ is a significant research under the influence of different environmental factors.

The immobilization technology for neonicotinoid pesticide treatment is a composite immobilization technology mainly developed according to embedding immobilization and adsorption immobilization technologies, the composite immobilization technology integrates the advantages of embedding immobilization and adsorption immobilization technologies, the microbial density is guaranteed, meanwhile, the microbial leakage can be effectively prevented, embedded particles are well formed, the mechanical strength is high, and the service cycle is long. However, immobilization techniques based on the embedding technique often cause problems in oxygen and mass transfer due to the mass transfer resistance of the carrier. Polyvinyl alcohol (PVA) is a widely used immobilized embedding carrier and has the advantages of low toxicity, wide application, good mechanical strength and the like. However, when pure polyvinyl alcohol is used for encapsulation, problems such as particle adhesion and poor mass transfer performance are often caused.

Therefore, the research and development of a novel microorganism immobilized composite particle are urgently needed, the technical defects existing in the prior art are overcome, the mass transfer performance of an immobilized strain is enhanced, and the efficiency of degrading anabasine pesticides is improved.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a preparation method of microorganism immobilized composite particles. The immobilization technology used by the invention can form uniform holes in the carrier, and form a mass transfer channel on the surface of the carrier, thereby greatly improving the mass transfer performance of particles; the polyvinyl alcohol is used in combination with Sodium Alginate (SA) in a mixing way, so that the problems of polyvinyl alcohol adhesion and poor mass transfer performance can be solved, and the preparation of the sphere and the immobilized particle is facilitated; meanwhile, the biochar is added, so that the mechanical property of the carrier can be improved, the specific surface area and the adsorption sites of the carrier are increased, and the degradation effect on the neonicotinoid pesticides is greatly improved.

The invention aims to provide a preparation method of microorganism immobilized composite particles, which comprises the following steps:

s1, washing and drying a biomass material, heating for reaction, cooling, washing with inorganic strong acid, drying, grinding and sieving to obtain biochar;

s2, culturing a Chryseobacterium strain in a culture medium, centrifuging, washing, carrying out heavy suspension to obtain a strain suspension, and adding the biochar to obtain a mixed solution;

heating, dissolving and sterilizing polyvinyl alcohol and alginate, and adding the mixed solution to obtain an embedding solution;

dissolving calcium chloride and boric acid in water to obtain a cross-linking solution;

s3, carrying out ultrasonic treatment on the embedding liquid, then, dripping the embedding liquid into the cross-linking liquid, reacting, and washing to obtain the microorganism immobilized composite particles;

s3, blending the bacterial suspension and the embedding liquid, dripping the mixture into the crosslinking liquid, reacting, and washing to obtain the microorganism immobilized composite particles;

wherein the biomass material is selected from one of rice husk, moso bamboo and rice bran.

Further, in step S1, the heating reaction is performed in an inert gas atmosphere, the reaction temperature is 650 to 850 ℃, and the reaction time is 2 to 4 hours.

Further, in step S2, the OD600 value of the bacterial suspension is 0.75-1.0.

Further, in step S2, the adding amount of the biochar is 0.01-0.02 g per ml of the bacterial suspension.

Further, in step S2, the mass ratio of the polyvinyl alcohol to the sodium alginate is (6-8): 1.

Further, in step S2, the heating temperature is 90-100 ℃.

Further, in step S2, the sterilization time is 20-30 min.

Further, in step S2, the mass ratio of the calcium chloride to the boric acid is 3 (4-5).

Further, in step S3, the reaction is: the reaction is carried out for 10-12 h at-20 to-18 ℃ and then for 10-12 h at 2-4 ℃.

The invention also aims to provide the application of the microorganism immobilized composite particles prepared by the preparation method of the microorganism immobilized composite particles in removing neonicotinoid pesticides in water.

The invention has the following beneficial effects:

1. the surface structure of the microorganism immobilized composite particle prepared by the invention is rough and has a large number of micro pores, the specific surface area of the immobilized carrier is increased by the structure, and the adsorbed active sites are increased. In addition, the surface of the biochar is provided with a large number of irregular bulges, which is beneficial to the external nutrient substances and water to enter the inner side of the pellet, provides sufficient nutrient supply for microorganisms, and is beneficial to the discharge of metabolites, thereby being beneficial to the growth of the microorganisms;

2. according to the invention, polyvinyl alcohol is used in combination with sodium alginate, and a large number of uniform pore structures exist in the central part of the microorganism immobilized composite particles, so that a colonization site can be provided for microorganisms. Because the pores exist in the carrier, macromolecular pollutants can still well migrate in the immobilized particles, and the blocking effect of the immobilized carrier on the transfer of the macromolecular substances is reduced through the raised mass transfer channels on the surface and the cavities in the particles, so that the defect of poor mass transfer effect of a common embedding method on the macromolecular substances is overcome;

3. according to the invention, polyvinyl alcohol, sodium alginate and biochar are used as composite carriers, and Chryseobacterium is used as a microorganism functional component, so that the adsorption and decomposition of pollutants are promoted, the degradation rate of Thiamethoxam (THM) can reach more than 98%, and the removal effect of neonicotinoid pesticides is greatly improved.

Drawings

FIGS. 1(a) to (d) are scanning electron micrographs of the microorganism-immobilized composite particles prepared in example 1;

wherein the content of the first and second substances,

FIG. 1(a) is a view showing the structure of the surface of a microorganism-immobilized composite particle;

FIG. 1(b) is a view showing the center structure of a microorganism-immobilized composite particle;

FIGS. 1(c) and 1(d) are views showing the internal structure of the microorganism-immobilized composite particle.

Fig. 2 shows a time-THM removal rate curve of the THM removal performance test in test example 1.

FIGS. 3(a) - (d) show the THM degradation kinetics fitted curve in test example 2;

wherein the content of the first and second substances,

FIG. 3(a) is a zero order kinetic degradation rate fitted curve of a comparative example;

FIG. 3(b) is a first order kinetic degradation rate fitted curve of a comparative example;

FIG. 3(c) is a zero order kinetic degradation rate fitted curve of example 1;

FIG. 3(d) is a first order kinetic degradation rate fitted curve of example 1.

Detailed Description

In order to more clearly illustrate the technical solution of the present invention, the following examples are given, but the present invention is not limited thereto.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.

The rice hulls in the examples of the invention were purchased from Harbin, Heilongjiang province.

The mao bamboo in the embodiment of the invention is purchased from Huai nan city of Anhui province.

The rice bran of the present example was purchased from Harbin, Heilongjiang province.

The golden yellow bacillus in the embodiment of the invention is obtained by early screening of an inventor, and comprises the following specific steps:

activated sludge in aerobic zone was collected from a sewage treatment plant, 20mL of the activated sludge was centrifuged (x 4000g, 10min), washed 2 times with phosphate buffer solution (1 x), placed in a 250mL Erlenmeyer flask containing 100mL of inorganic salt medium (MSM), cultured in a shaker at 30 ℃ and 160rpm, sampled, and the residual amount of THM was measured by high performance liquid chromatography. Samples with the ability to degrade THM were inoculated twice in succession into fresh MSM solution with THM added. Streaking and purifying in thiamethoxam MSM solidified medium containing 2% agar. The isolated single colonies were cultured individually. The separated sample was stored in a-80 ℃ refrigerator. The strain is identified through morphological, physiological and biochemical tests and 16S rDNA gene sequence analysis, and the result shows that the strain belongs to the genus Flavobacterium.

The LB liquid culture medium in the embodiment of the invention is: each liter of LB liquid culture medium contains 5.00g of beef extract, 10.00g of peptone and 5.00g of NaCl, and the pH value is 7.0.

The polyvinyl alcohol in the examples of the invention was a 1788 type polyvinyl alcohol (analytical grade).

The sodium alginate, the calcium chloride and the boric acid in the embodiment of the invention are all analytically pure.

The inorganic salt culture medium in the test example of the invention is: the inorganic salt culture medium contains 14mg of THM and 1.5g K per liter₂H₂PO₄、0.5g KH₂PO₄、0.20g MgSO₄·7H₂O, 1.0g NaCl and 10mL trace elements; each liter of trace elements contains 0.40g of CaCl₂·2H₂O、0.30g H₃BO₃、0.04g CuSO₄·5H₂O、0.10g KI、0.20g FeSO₄·7H₂O、0.40g MnSO₄·7H₂O、0.20gNaMoO₄·2H₂O、10.0mLHCl。

The THM in the test examples of the present invention was analytically pure and purchased from Aladdin (Shanghai, China).

Example 1

A preparation method of microorganism immobilized composite particles comprises the following steps:

s1, washing rice hulls with deionized water, drying, placing the rice hulls into a crucible, placing the crucible into a tubular furnace, reacting for 3 hours at 700 ℃ in an argon atmosphere, cooling to room temperature, taking out the rice hulls, and then using 0.1 mol.L^-1HCl and HNO of₃Washing the mixed liquor until the washing liquor is neutral, drying the rice hulls, and then grinding and sieving the rice hulls with a 100-mesh sieve to obtain biochar;

s2, inoculating a golden yellow bacillus strain into 100mL of LB liquid culture medium, placing the golden yellow bacillus strain into a shaking table for constant-temperature culture (30 ℃ and 160rpm), culturing to the late stage of logarithmic phase, centrifuging the culture medium (x 4000g and 10min) at room temperature, washing with sterile water, suspending in 0.9% physiological saline to obtain a bacterial suspension with an OD600 value of 0.75, adding 0.6g of charcoal into 50mL of the bacterial suspension, and standing for 2 hours to obtain a mixed solution;

adding 8g of polyvinyl alcohol and 1g of sodium alginate into a beaker, uniformly mixing, stirring, adding deionized water, dissolving in a water bath at 90 ℃, then sterilizing with high-pressure steam at 121 ℃ for 20min, cooling to room temperature, adding the mixed solution, and preparing into 100mL of embedding solution;

adding 3g of calcium chloride and 4g of boric acid into a beaker, adding 100mL of sterile water, and dissolving to obtain a cross-linking solution;

s3, carrying out ultrasonic treatment on the embedding liquid for 15min, then dripping the embedding liquid into 200mL of the cross-linking liquid by using an injector, reacting for 12h at-20 ℃, then transferring to 4 ℃ for reacting for 12h, washing the prepared particles for 3 times by using sterile water to obtain the microorganism immobilized composite particles, and soaking the microorganism immobilized composite particles in 0.9% physiological saline at 4 ℃ for storage for later use.

FIGS. 1(a) to (d) are scanning electron micrographs of the microorganism-immobilized composite particles prepared in example 1. Fig. 1(a) is a structure diagram of the surface of the microorganism immobilized composite particle, which shows that the unevenness of the outer surface of the microorganism immobilized composite particle has a large number of micro pores and a large number of microorganisms are attached, the structure increases the specific surface area of the immobilized carrier, increases the adsorbed active sites, facilitates the external nutrient substances and water to enter the inner side of the pellet, provides sufficient nutrient supply for the microorganisms, and is beneficial to the discharge of metabolites, thereby promoting the growth of the microorganisms; FIG. 1(b) is a diagram of the center structure of a microorganism immobilized composite particle, which shows that a large number of honeycomb-shaped pore structures exist at the center of the microorganism immobilized composite particle, which can provide a colonization site for microorganisms, and macromolecular pollutants can still migrate well inside the immobilized particle due to the pores inside the carrier, so that the barrier effect of the immobilized carrier on the transfer of the macromolecular substances is reduced through the mass transfer channels on the surface and the pores inside the particle, and the defect of poor mass transfer effect of the conventional embedding method on the macromolecular substances is overcome; fig. 1(c) and fig. 1(d) are internal structure diagrams of the microorganism immobilized composite particle, which show that a large number of mesopores and micropores exist in the immobilized carrier, and a large number of microorganisms are attached to the pores, so that a basic condition is provided for immobilized degradation.

Example 2

s1, washing moso bamboo with deionized water, drying, placing the moso bamboo into a crucible, placing the crucible into a tubular furnace, reacting for 2 hours at 850 ℃ in an argon atmosphere, cooling to room temperature, taking out rice hulls, and then using 0.1 mol.L^-1HCl and HNO of₃Washing the mixed liquor until the washing liquor is neutral, drying the rice hulls, and then grinding and sieving the rice hulls with a 100-mesh sieve to obtain biochar;

s2, inoculating a golden yellow bacillus strain into 100mL of LB liquid culture medium, placing the golden yellow bacillus strain into a shaking table for constant-temperature culture (30 ℃ and 160rpm), culturing to the late stage of logarithmic phase, centrifuging the culture medium (x 4000g and 10min) at room temperature, washing with sterile water, suspending in 0.9% physiological saline to obtain a bacterial suspension with an OD600 value of 0.85, adding 0.8g of charcoal into 50mL of the bacterial suspension, and standing for 2 hours to obtain a mixed solution;

Example 3

s1, washing rice bran with deionized water, drying, placing the rice bran into a crucible, placing the crucible into a tubular furnace, reacting for 4 hours at 650 ℃ in an argon atmosphere, cooling to room temperature, taking out the rice bran, and then using 0.1 mol/L^-1HCl and HNO of₃Washing the mixed liquor until the washing liquor is neutral, drying the rice hulls, and then grinding and sieving the rice hulls with a 100-mesh sieve to obtain biochar;

s2, inoculating a golden yellow bacillus strain into 100mL of LB liquid culture medium, placing the golden yellow bacillus strain into a shaking table for constant-temperature culture (30 ℃ and 160rpm), culturing to the late stage of logarithmic phase, centrifuging the culture medium (x 4000g and 10min) at room temperature, washing with sterile water, suspending in 0.9% physiological saline to obtain a bacterial suspension with an OD600 value of 1.0, adding 1.0g of charcoal into 50mL of the bacterial suspension, and standing for 2 hours to obtain a mixed solution;

Comparative example

The golden yellow bacillus strain is inoculated in 100mL LB liquid culture medium, placed in a shaking table for constant temperature culture (30 ℃, 160rpm), cultured until the late stage of logarithmic phase, the culture medium is centrifuged (x 4000g, 10min) at room temperature, then washed by sterile water and resuspended in 0.9% physiological saline to prepare bacterial suspension with OD600 value of 0.75.

Test example 1

THM removal Performance test

The test method comprises the following steps:

l1, placing the microorganism immobilized composite particles prepared in the example 1 in an inorganic salt culture medium, adding 14ppm of thiamethoxam, placing the mixture in a shaking table for constant temperature culture (30 ℃, 160rpm), adding 14ppm of thiamethoxam again after the thiamethoxam is degraded and consumed, adding 50ppm of thiamethoxam after the degradation effect is stable, repeating the steps, maintaining the substrate concentration, continuously culturing for 3 times, and transferring the microorganism immobilized composite particles and the inorganic salt culture medium to a sterile bottle for storage as activated microorganism immobilized composite particles for later use;

l2, preparing 7 250mL conical flasks, adding 100mL of an inorganic salt culture medium with thiamethoxam concentration of 14mg/L into each flask, adding 5g of the activated microorganism immobilized composite particles into No. 1-3 conical flasks, adding 0.5mL of the bacterial suspension prepared according to the comparative example into No. 4-6 conical flasks, and taking No. 7 conical flasks as blank samples; placing the conical flask into a shaking table for constant-temperature culture (30 ℃, 160rpm), and sampling at regular time to detect the concentration of thiamethoxam.

And (3) testing results:

the test results are shown in table 1 and fig. 2.

Table 1 THM removal performance test results

Fig. 2 shows a time-THM removal rate curve of a thiamethoxam removal performance test, and it can be seen from table 1 and fig. 2 that the microbial immobilized composite particles prepared by the invention greatly improve the removal rate of thiamethoxam, the degradation rate of 96 h can reach 98%, and compared with a comparative example (free bacteria), the microbial immobilized composite particles are improved by 41%, and have good neonicotinoid pesticide removal performance.

Test example 2

Kinetics of THM degradation

The research method comprises the following steps:

respectively adding the microorganism immobilized composite particles prepared in example 1 and the bacterial suspension prepared in the comparative example into a thiamethoxam inorganic salt-containing culture medium with the initial concentration of 14 mg.L < -1 >, sampling at regular time to detect the concentration of THM, and calculating the degradation kinetics of THM according to a Monod equation, wherein the relational expression between the substrate degradation speed and the substrate concentration is

When C > K, γ ═ γ_m，k₀＝γ_mThe reaction follows zero order kinetics,

the formula can be simplified as c ═ a + k₀t；

When C < K,

the reaction conforms to the first-order reaction kinetics,

the formula can be simplified as lnc ═ b + k₁t；

Wherein, γ: specific degradation rate of organic substrate (d)^-1)；γ_m: maximum specific degradation rate of substrate (d)^-1) (ii) a K: a half-saturation constant; c: substrate concentration (mg/L); k is a radical of₀: zero order kinetic degradation rate constant (g/(mg. h)); k is a radical of formula₁: first order kinetic degradation rate constant (g/(mg. h)); a and b are constants.

The research results are as follows:

the kinetic fit results are shown in table 2 and fig. 3:

table 2THM degradation kinetics results

Fig. 3(a) to (d) show THM degradation kinetics fitted curves, in which fig. 3(a) is a zero-order kinetics degradation rate fitted curve of a comparative example, fig. 3(b) is a first-order kinetics degradation rate fitted curve of a comparative example, fig. 3(c) is a zero-order kinetics degradation rate fitted curve of example 1, and fig. 3(d) is a first-order kinetics degradation rate fitted curve of example 1, and it can be found from fig. 3 that the degradation of THM by both free bacteria and microorganism-immobilized composite particles conforms to a first-order kinetics model.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. A method for preparing microorganism immobilized composite particles, which is characterized by comprising the following steps:

2. The method for preparing immobilized microorganism composite particles according to claim 1, wherein in step S1, the heating reaction is carried out in an inert gas atmosphere, the reaction temperature is 650-850 ℃, and the reaction time is 2-4 h.

3. The method of claim 1, wherein the bacterial suspension has an OD600 of 0.75 to 1.0 in step S2.

4. The method for preparing immobilized microorganism composite particles according to claim 1, wherein in step S2, the biochar is added in an amount of 0.01-0.02 g per ml of bacterial suspension.

5. The method for preparing the microorganism-immobilized composite particles according to claim 1, wherein in step S2, the mass ratio of the polyvinyl alcohol to the sodium alginate is (6-8): 1.

6. The method for producing the microorganism-immobilized composite particles according to claim 1, wherein the heating temperature in step S2 is 90 to 100 ℃.

7. The method for preparing immobilized composite microbe particles according to claim 1, wherein the sterilization time in step S2 is 20-30 min.

8. The method for preparing the microorganism-immobilized composite particles according to claim 1, wherein in step S2, the mass ratio of the calcium chloride to the boric acid is 3 (4-5).

9. The method for producing a microorganism-immobilized composite particle according to claim 1, wherein in step S3, the reaction is: the reaction is carried out for 10-12 h at-20 to-18 ℃ and then for 10-12 h at 2-4 ℃.

10. Use of the microorganism-immobilized composite particles prepared by the method for preparing microorganism-immobilized composite particles according to any one of claims 1 to 9 for removing neonicotinoid pesticides from water.