NL2032420B1

NL2032420B1 - Preparation method of immobilized microbial bead, and use thereof

Info

Publication number: NL2032420B1
Application number: NL2032420A
Authority: NL
Inventors: Zhang Xiaoling; Zeng Taotao; Song Xin; Wang Liangqin; Wang Guohua; Liu Yingjiu; Zhou Yulin; Liu Jinxiang
Original assignee: Univ South China
Priority date: 2021-12-21
Filing date: 2022-07-07
Publication date: 2023-06-29
Also published as: CN114231455A

Abstract

The present disclosure relates to a microbial bead, an immobilized microsphere, a preparation method, and use, which belongs to the technical field, of wastewater treatment. The microbial bead, is prepared, by embedding multiple microorganisms with sodium alginate; where the multiple microorganisms include Enterobacteriaceae, Alcaligenes, Terrisporobacter, Pararaclostridium, and Shewanella. The microbial bead is capable of adsorbing and reducing sodium selenite without a carbon source to obtain the immobilized, microsphere; and biological selenium nanoparticles (Se(0) and CdSe) generated, on a surface of the immobilized microsphere continues to remove cadmium. The microbial bead has a removal efficiency of not less than 97% for selenium in polluted, water, and the immobilized, microsphere has a removal efficiency of not less than 99% for cadmium in the polluted water, with a desirable prospect for use.

Description

P1445 /NLpd

PREPARATION METHOD OF IMMOBILIZED MICROBIAL BEAD, AND USE THEREOF

TECHNICAL FIELD

The present disclosure relates to the technical field of wastewater treatment, in particular to a microbial bead, an immo- bilized microsphere, a preparation method, and use.

BACKGROUND ART

Selenium is a natural trace element that has received great attention in high-tech products in recent years. Selenium com- pounds are mainly used in the production of glass and semiconduc- tor materials. In addition, selenium nanoparticles, due to the de- sirable electrical conductivity and catalytic properties, as well as lower biotoxicity than the selenium compounds, are widely used in electronics, optics and medicine. However, selenium in nature often exists in an oxidized state, with high solubility and tox- icity. A slight increase in the concentration of selenium may pose a great threat to human health and the ecological environment.

With the development of human activities such as mining, smelting and agricultural irrigation, selenium pollution has become in- creasingly serious in local water bodies.

Generally, excessive selenium in water has a small range of concentration, and selenium coexists with other heavy metals. This complicates the traditional physical and chemical treatment of se- lenium-containing wastewater with large energy consumption. Bio- logical methods have environmental protection and low cost, and can reduce the oxidized selenium into biological selenium nanopar- ticles with low biological toxicity. At present, various microor- ganisms such as Microbacteria, Pseudomonas and Bacillus each are proved to have high selenium removal ability. However, in the bio- logical treatment process, selenium removal efficiency is affected by factors such as temperature, carbon source, and pH value; mi- croorganisms are easily lost from the reactor; and an external carbon source is required to maintain growth of the microorgan- isms. These defects limit use of the biological treatment process-

es for heavy metal removal.

SUMMARY

An objective of the present disclosure is to provide a micro- bial bead. The microbial bead can effectively remove selenium without an external carbon source, and continue to remove cadmium to generate nano cadmium selenide particles. Therefore, selenium and cadmium are effectively removed.

To achieve the above objective, the present disclosure pro- vides the following technical solutions.

The present disclosure provides a microbial bead, prepared by embedding multiple microorganisms with sodium alginate; where the multiple microorganisms include Enterobacteriaceae, Alcaligenes,

Terrisporobacter, Pararaclostridium, and Shewanella.

The present disclosure further provides use of the microbial bead in removing selenium and cadmium in polluted water.

The present disclosure further provides an immobilized micro- sphere, obtained from biological selenium nanoparticles adhered on a surface of the microbial bead.

The present disclosure further provides a preparation method of the immobilized microsphere, including the following step: mix- ing the microbial bead with a sodium selenite solution for reac- tion to obtain the immobilized microsphere.

Preferably, the reaction may be conducted at a pH value of 4 to 7.

Preferably, the microbial bead and the sodium selenite solu- tion may have a mass-to-volume ratio of (0.25-6.0) g: 1 L.

Preferably, the sodium selenite solution may have a concen- tration of 3.95 mg/L to 39.50 mg/L.

The present disclosure further provides use of the immobi- lized microsphere or the preparation method in removing cadmium in polluted water.

The present disclosure further provides a method for removing selenium and cadmium in polluted water, including the following steps: mixing the microbial bead with the polluted water to adsorb selenium to obtain an immobilized microsphere; and allowing the immobilized microsphere to have reaction with cadmium in the pol- luted water to remove selenium and cadmium in the polluted water.

Preferably, no additional carbon source may be added during removing selenium and cadmium in the polluted water.

The present disclosure provides the microbial bead obtained by embedding the multiple microorganisms enriched from soil of a mining area by the sodium alginate. The microbial bead can adsorb and reduce sodium selenite without a carbon source to obtain an immobilized microsphere. The biological selenium nanoparticles (Se (0) and CdSe) generated on the surface of the immobilized mi- crosphere can remove cadmium. It has been confirmed by experiments that a removal efficiency of selenium in the polluted water by the microbial bead reaches not less than 97%, and a removal efficiency of cadmium in the polluted water by the immobilized microspheres reaches not less than 99%, with a desirable prospect for use in removing selenium and cadmium in the polluted water.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a process flow of removing selenium and cadmium in polluted water in the present disclosure;

FIG. 2 shows exploration of influencing factors for reduction of sodium selenite by a microbial bead; where (a) shows an effect of the reaction time on reduction of Se (IV) by the microbial bead; (b) shows an effect of the dosage on reduction of Se (IV) by the microbial bead; (c) shows an effect of the initial Se (IV) concen- tration on reduction of Se (IV) by the microbial bead; and (d) shows an effect of the pH on reduction of Se (IV) by the microbial bead;

FIG. 3 shows a cadmium removal effect of an immobilized mi- crosphere; where (a) shows an effect of the reaction time on

Cd{(II) removal characteristics; and (b) shows an effect of the different Se (IV) concentrations on the Cd (II) removal characteris- tics;

FIG. 4 shows a cycle experiment of removing selenium and re- ducing cadmium with the immobilized microsphere;

FIG. 5 shows macroscopic and microscopic structures of the microbial bead before and after reaction; where (a) and (b) show macroscopic and microscopic results of the microbial bead before the reaction; (d) and (e) show macroscopic and microscopic results of the microbial bead exposed to 7.9 mg/L Se(IV) for 168 h; (gq) and (h) show macroscopic and microscopic results of the immobi- lized microsphere exposed to 11.2 mg/L Cd(II) for 10 h; and arrows in (h) represent particulate matters;

FIG. 6 shows morphology and elemental composition of the mi- crobial bead before and after the reaction; where (¢) shows the morphology and elemental composition of the microbial bead before the reaction; (f) shows morphology and elemental composition of the microbial bead exposed to 7.9 mg/L Se (IV) for 168 h; and (i) shows morphology and elemental composition of the immobilized mi- crosphere exposed to 11.2 mg/L Cd(II) for 10 h;

FIG. 7 shows a particle size of particles generated after re- moving selenium and a particle size of particles generated after continuing to remove cadmium; where (a) shows the particle size of the particles generated after removing selenium; and (b) shows the particle size of the particles generated after continuing to re- move cadmium after removing selenium;

FIG. 8 shows an Fourier transform infrared spectroscopy (FTIR) spectra of the microbial bead before and after reaction; and

FIG. 9 shows an element valence state of the microbial bead before and after removing Se (IV) and Cd (II); where (a) shows an ¥X- ray photoelectron spectroscopy (XPS) full spectrum; {b} shows a fine spectrum of Se 3d; and (c¢) shows a fine spectrum of Cd 3d.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Terrisporobacter, Pararaclostridium, and Shewanella.

In the present disclosure, the multiple microorganisms in- clude the Enterobacteriaceae, the Alcaligenes, the Terrisporobac- ter, the Pararaclostridium, and the Shewanella; where, the Entero- bacteriaceae has an abundance ratio of preferably 40% to 663, the

Alcaligenes has an abundance ratio of preferably 20% to 36%, the

Terrisporobacter has an abundance ratio of preferably 1% to 8%, the Pararaclostridium has an abundance ratio of preferably 1% to 4%, and the Shewanella has an abundance ratio of preferably 1% to 33. The multiple microorganisms are preferably obtained by screen- 5 ing soil near a lead-zinc mining area; in a specific example, the multiple microorganisms are more preferably obtained by screening from soil near a typical lead-zinc mining area in Hunan Province.

There is no special limitation on a screening method; in a specif- ic example, a screening method of the multiple microorganisms in- cludes preferably the following steps: mixing soil with sterile water and shaking for 24 h, and separating to obtain the multiple microorganisms using a plate streaking method. A medium used for the plate streaking method includes preferably: 5 g/L to 8 g/L of a beef extract, 8 g/L to 12 g/L of peptone, and 15 g/L to 20 g/L of agar, with a pH value of preferably 7.0 to 7.2.

In the present disclosure, the microbial bead is obtained by embedding the multiple microorganisms with sodium alginate. There is no special limitation on an embedding method; in a specific ex- ample, an embedding method includes preferably the following steps: mechanically stirring sodium alginate mixed with sterile water, and adding bacterial cells of the multiple microorganisms; adding a homogeneous "multiple microorganisms-sodium alginate" mixture dropwise to a 2% CaCl; solution; and conducting cross- linking, filtering and washing at room temperature to obtain the microbial bead. There is no special limitation on a mechanical stirring and filtering method, and conventional mechanical stir- ring or filtering methods in the art can be used.

The present disclosure further provides use of the microbial bead in removing selenium and cadmium in polluted water. The mi- crobial bead has an adsorption effect on Se (IV) and is reduced af- ter adsorption; meanwhile, the microbial bead can directly adsorb

Cd(II) in the solution to remove cadmium, and can generate nano- selenium and nano-cadmium selenide.

In the present disclosure, the immobilized microsphere is ob- tained by mixing the microbial bead with a sodium selenite solu- tion for reaction; and a surface of the immobilized microsphere is adhered with biological selenium nanoparticles. The microbial bead and the sodium selenite solution have a mass-to-volume ratio of preferably (0.25-6.0) g: 1 L, more preferably (2-5) g: 1 L. The sodium selenite solution has a concentration of preferably 3.95 mg/L to 39.50 mg/L, more preferably 7.9 mg/L. The reaction is con- ducted for preferably 1 h to 168 h, more preferably 72 h to 144 h at a pH value of preferably 4 to 7, more preferably 5 to 6.

A specific procedure is shown in FIG. 1.

In the present disclosure, no additional carbon source is added during removing selenium and cadmium in the polluted water.

The reaction of the immobilized microsphere with cadmium in the polluted water is conducted for preferably 1 h to 144 h, more preferably 10 h.

In the present disclosure, an LB medium includes: 5 g/L of a beef extract, 10 g/L of tryptone, and 10 g/L of sodium chloride, with a pH value of 7.0 to 7.2; the LB medium is autoclaved at 121°C for 20 min.

In the present disclosure, the raw materials, reagents and equipment used are all known products, and conventional commer- cially available products can be used.

In the present disclosure, "0", "II" and "IV" represent a va- lence of a corresponding element.

In order to further illustrate the present disclosure, the technical solutions provided by the present disclosure are de- scribed in detail below in connection with examples, but these ex- amples should not be understood as limiting the claimed scope of the present disclosure.

Example 1

Acquisition of multiple microorganisms

The multiple microorganisms were isolated from soil near a typical lead-zinc mining area in Hunan Province by a plate streak- ing method.

Specifically, a screening method of the multiple microorgan- isms included: 1. 1 g of soil was placed in 50 mL of sterile water and shak- en for 24 h. 2. Melting of a medium: the medium included 6 g/L of a beef extract, 10 g/L of peptone, and 18 g/L of agar, and was heated in a water bath until melted. 3. Pouring to a plate: after being cooled to 50°C, the medium was poured to 2 plates in an ultra-clean bench according to an aseptic operation method; the plates were laid flat for solidify- ing. 4. Selecting of bacteria-containing samples: a small amount of bacteria were selected using a flat and smooth inoculation ring according to the aseptic operation method. 5. The plate was placed upside down next to a gas lamp, a bottom of the dish was held with the left hand and try to make the plate perpendicular to a table top, with the medium facing the gas lamp (at this time, a lid of the dish was facing up and still be- side the gas lamp), the inoculation loop was held with the right hand to draw first 4 continuous parallel streaks. After streaking, residual bacteria on the ring were burned immediately to prevent from affecting a separation effect due to excessive bacteria. 6. Constant-temperature culture: the streaked plate was in- verted, culture was conducted at 37°C (or 28°C), followed by ob- servation after 24 h; dominant flora, including Enterobacteri- aceae, Alcaligenes, Terrisporobacter, Pararaclostridium, and She- wanella were obtained and stored at 4°C for later use.

Example 2

Acquisition of a microbial bead

The bacterial flora obtained and stored in Example 1 were in- oculated into an LB medium, and cultured for 48 h in a constant- temperature shaker at 30°C and 150 rpm/min. Centrifugation was conducted at 8,000 rpm/min for 10 min in a high-speed refrigerated centrifuge (ESCO Versari T1000R, Singapore), and a supernatant was discarded; an obtained bacterial cell precipitate was washed three times with sterile water to remove the residual medium, and stored at 4°C for later use. 1 g of sodium alginate was added to 50 mL of sterile water, and subjected to mechanical stirring for 15 min; 1.5 g of the stored bacterial cells were added, and mechanical stirring was continued for 15 min; a homogeneous "multiple microorganisms- sodium alginate" mixture was added dropwise to a 2% CaCl, solution, and cross-linked at room temperature for 24 h; after filtration, a resulting product was washed three times with the sterile water to obtain an immobilized microbial bead, and stored in a normal sa- line at 4°C for later use. Meanwhile, a control bead without the multiple microorganisms was prepared by a same process.

Example 3

Acquisition of an immobilized microsphere 5 g of the microbial bead prepared in Example 2 was mixed with 7.9 mg/L of a sodium selenite solution; the immobilized mi- crosphere was obtained by incubation for 144 h in a constant- temperature shaking incubator at 150 rpm/min at a pH value of 4.

Example 4

I. Batch experiments

To analyze the characteristics of removing Se (IV) using the microbial bead by different factors, batch experiments were con- ducted with reaction time, microbial bead dosage, pH and initial

Se(IV) concentration as research objects. To deeply analyze the characteristics and mechanism of removing Se(IV) using the micro- bial bead, a Se(IV)}) solution around the microbial bead was blotted with a filter paper, and the microbial bead was continued to be used for treating a Cd(II)-containing solution; after a certain time of reaction, filtration was conducted with a 0.22 um filter membrane to determine remaining concentrations of the Se (IV) and the Cd{II}) in the solution separately. All experiments were re- peated three times, and a control experiment was conducted with a bead without the multiple microorganisms as a control group.

The specific steps were as follows: (1) Reaction time: 20 mL of a Se (IV) solution with a concen- tration of 7.9 mg/L was added to a 50 mL conical flask, and the microbial bead had a dosage of 5 g/L; at a pH value of 4, incuba- tion was conducted in a constant-temperature shaking incubator at 150 rpm/min, and a concentration of remaining Se (IV) in the solu- tion was measured after 0 h to 168 h of reaction. (2) Dosage: after obtaining an optimum adsorption time, 20 mL of the Se (IV) solution with a concentration of 7.9 mg/L was added to the 50 mL conical flask; at the pH value of 4, a dosage of the microbial bead was changed, and incubation was conducted in the constant-temperature shaking incubator at 150 rpm/min; after 6 d, the concentration of remaining Se (IV) in the solution was meas- ured. (3) Initial concentration: after obtaining optimum adsorption time and dosage, 20 mL of solutions of different Se (IV) concentra- tions were added into the 50 mL conical flask; at a pH value of 4 and a dosage of 5 g/L, incubation was conducted in the constant- temperature shaking incubator at 150 rpm/min; after 6 d, the con- centration of remaining Se (IV) in the solution was measured. (4) pH: after obtaining optimum adsorption time, dosage and initial concentration, 20 mL of the Se (IV) solution with a concen- tration of 7.9 mg/L was added to the 50 mL conical flask; a pH value of the solution was changed, and incubation was conducted in the constant-temperature shaking incubator at 150 rpm/min; after 72 h and 144 h, concentrations of remaining Se (IV) in the solution were measured separately.

II. Experimental result 1. Batch experiment of reduction of sodium selenite by micro- bial bead 1.1. Influence of reaction time

At a concentration of Se(IV}) of 7.9 mg/L, a dosage of 5 g/L, and a pH value of 4, the effect of reaction time is explored on the reduction of Se(IV) by microbial bead. The results are shown in FIG. 2{(a).

It can be seen that the reaction time has a great influence on the reduction of Se(IV) by the microbial bead. Within 24 h be- fore the reaction, the microbial bead has a higher adsorption rate of Se(W), and a highest removal efficiency of 57.94%; at 48 h, the removal efficiency does not increase significantly; from 72 h, the removal efficiency begins to show an upward trend, reaching a max- imum of 98.79%; and at 168 h, the effluent concentration of Se (IV) is 0.095 mg/L. From the kinetic point of view, at an initial stage of the reaction, the microbial bead may have physical adsorption to Se(W), which is not efficient but can guickly adsorb the Se (IV }; with an increase of the reaction time, the bacteria in the mi- crobial bead gradually adapt to the environment, begin to biore- duce the Se (IV), and slowly diffuse inside the microspheres, grad- ually turning red. This proves that the Se (lV) is reduced. 1.2. Influence of dosage

The reaction is conducted for 6 d under a concentration of

Se (IV) of 7.9 mg/L and a pH value of 4, and the effect of the dos- age of microbial bead (0.25 g/L to 6.0 g/L) on the reduction of

Se (IV) is investigated. The results are shown in FIG. 2 (b).

It can be seen from the figure that when the dosage is 0.25 g/L to 1.0 g/L, the dosage has a great influence on the reduction of Se (IV) by the microbial bead; however, when the dosage of mi- crobial bead is kept at 2.0 g/L to 6.0 g/L, the effect of dosage is small, and the removal efficiency is not less than 90%. There- fore, the dosage of 2 g/L can keep the removal efficiency high enough. 1.3. Influence of initial concentration

The reaction is conducted for 6 d at a Se(IV) concentration of 3.95 mg/L to 39.50 mg/L, a pH value of 4, and a dosage of 5 g/L, and characteristics of the reduction of different Se (IV) con- centrations by microbial bead are investigated. The results are shown in FIG. 2{(c).

It can be seen that the microbial bead has a removal effi- ciency of not less than 27% to the Se(IV) not more than 7.9 mg/L, within a concentration range of most selenium-containing wastewater. This shows that the microbial bead has a desirable po- tential for use. 1.4. Influence of pH on reduction efficiency of sodium sele- nite by microbial bead

At a concentration of Se (IV) of 7.9 mg/L and a dosage of 5 g/L, the reaction is conducted for 72 h and 144 h separately, to explore the effect of pH (4 to 7) on the reduction of Se(IV) by microbial bead. The results are shown in FIG. 2{(d).

It can be seen that a lower pH value leads to a higher con- centration of H' in the solution, and a greater adsorption and binding efficiency of H' and oxygen anions. After 3 d of reaction, the pH has no significant effect on the reduction of Se (IV) by mi- crobial bead, indicating that the microbial bead does not remove

Se (IV) by electrostatic adsorption; after 6 d of reaction, the re- moval efficiency can be kept not less than 80%; and the changes of pH value has a certain influence on the removal of Se (IV), when the

DH is 5, a maximum removal efficiency is 92.36%. It can be seen that the microbial bead has a relatively stable effect on Se (IV) in an acidic range (a pH value of 4 to 6), indicating that the mi- crobial bead can be used for the treatment of acidic selenium- containing wastewater. 2. Cadmium removal effect of immobilized microsphere 2.1. Influence of time on cadmium removal

The microbial bead was taken out after reacting for 144 h at a Se{IV) concentration of 7.9 mg/L and a pH value of 5, and con- tinued to treat 11.2 mg/L of Cd(II} at a dosage of 5 g/L. The ef- fect of reaction time on Cd(II) removal characteristics was inves- tigated. The results are shown in FIG. 3(a).

It can be seen from the figure that the immobilized micro- sphere reacted with Se (IV) has an efficiency of Cd(II) removal im- proving with the increase of time during the removal of Cd(II); after adsorption for 10 h, the removal efficiency of Cd(II) can reach 99.15%, indicating that the immobilized microsphere has a desirable effect in removing Cd (II). 2.2. Influence of different selenium concentrations on cadmi- um removal

The microbial bead was taken out after reacting for 144 h at

Se (IV) concentrations of 3.95 mg/L to 39.50 mg/L and a pH value of 5, and continued to treat 11.2 mg/L of Cd(II)-containing wastewater at a dosage of 5 g/L for 10 h. The effect of different

Se (IV) concentrations on Cd(II) removal characteristics was inves- tigated. The results are shown in FIG. 3(b).

As can be seen from FIG. 2{(c) and FIG. 3(b), when the Se (IV) concentration increases from 3.95 mg/L te 23.79 mg/L, a bioreduced

Se (IV) content in the system increases, and a content of SeNPs in a product increases, thereby improving an adsorption efficiency of ¢cd(H). When the concentration of Se (IV) continues to increase, the removal efficiency of Cd(II) remains not less than 99%, and the

Cd (II) is basically completely removed. Therefore, the reduction of Se (IV) by immobilized microsphere can further enhance the ad- sorption and removal of Cd(II). 2.3. Repeated experiments of removing selenium and reducing cadmium

The microbial bead was taken out after reacting for 144 h at a Se(lV) concentration of 7.9 mg/L and a pH value of 5, and treated an aqueous solution containing 7.9 mg/L of Se(W) and 11.2 mg/L of cd(Ï) at a dosage of 5 g/L; taking 24 h as a cycle, and 7 cycles were repeated to explore the reusability of microbial bead. The results are shown in FIG. 4.

Combining FIG. 4 and FIG. 2(a), it can be seen that within 24 h, the microbial bead may only physically adsorb Se (IV) on the surface, and the bicadsorption and reduction effect starts after 48 h. In the first two cycles, the removal efficiency of Cd(l) reaches not less than 97%; with an increase of the cycle, the re- moval efficiency of Cd([) decreases gradually. This is because that an adsorption capacity of the SeNPs generated in the early stage has been saturated, and the microorganisms do not continu- ously reduce Se (IV) to produce SeNPs in the subsequent cycle.

Therefore, prolonging the cycle can maintain a desirable removal effect of Se (IV) and Cd(ITI).

Example 5 1. SEM-EDS and nanoparticle size

A SEM-EDS method included: the microbial bead before and af- ter reaction was frozen at -60°C for 8 h, freeze-dried in a freeze dryer to obtain a powder, and subjected to gold spraying. At 3.00 kV, the morphology and elemental changes of the prepared samples were observed using Zeiss Sigma300, and the macroscopic and micro- scopic morphologies and elemental composition of the microbial bead before and after the reaction were obtained as shown in FIG. and FIG. 6.

Nanoparticle size acquisition: 5 mL to 10 mL of liquid sam- ples after two reaction stages of microbial bead with sodium sele- 5 nite and microbial bead with cadmium chloride were taken, respec- tively, ethanol was used as a dispersant, and a particle size of a product was measured with a nanoparticle analyzer (Malvern

Zetasizer Nano ZS90). The results are shown in FIG. 7.

It can be seen from FIG. 5 to FIG. 7 that before the reac- tion, the surface of microbial bead is smooth, pale yellow, and uniform in size; when exposed to 7.9 mg/L Se (lV) for 168 h, the surface of microbial bead turns red and has many red particles generated around, with a microscopic surface not significantly changed; after continuous exposure to 11.2 mg/L Cd(I) for 10 h, the red microbial bead becomes lighter, with the microscopic sur- face clearly layered, and has uniform and regular folds, and par- ticles are formed. Combined with EDS results, it is found that the microbial bead does not contain Se element before the reaction, and has a relatively high content of organic elements such as C, O and P; after exposure to Se(IV), the Se element content is 0.03%.

It is proved that the microbial bead has an adsorption effect on

Se (IV), but the Se (IV) may be reduced after adsorption, resulting in low content of Se on the surface. After continued exposure to

Cd (ITI), the contents of Se and Cd are 1.43% and 1.60%, respective- ly. It is proved that the immobilized microsphere can remove sele- nium and cadmium to generate nano-selenium and nano-cadmium sele- nide, with average particle sizes of 344 nm and 479 nm, respec- tively. 2. FTIR

FTIR spectra of the microbial bead before and after reaction were observed with a Fourier transform infrared spectrometer in a range of 4,000 cmt to 400 cmt, and the results are shown in FIG. 8.

It can be seen from the FTIR results that a main mechanism for the reduction of selenium and the coupled adsorption of cadmi- um by the microbial bead is coordination and complexation of func-

tional groups such as -0H, aliphatic, amide, and nitro groups. 3. XPS

The valence state before and after the removal of Se (IV) and

Cd (ITI) with microbial bead was analyzed by XPS, and the results are shown in FIG. 9.

It can be seen that a Se 3d peak appears at a binding energy of 57.08 eV after the reduction of Se (IV) by the microbial bead; after continuing the reaction to remove Cd(II), a Cd 3d peak ap- pears at a binding energy of 411.99 eV, which further proves that the microbial bead can reduce Se (IV) and adsorb Cd(II) to form na- noparticles.

Meanwhile, it can be seen that after the reduction of Se(IV), a fine spectrum of Se 3d fits two peaks of Se(-II) and Se (0). The peaks at binding energies of 54.66 eV and 55.26 eV correspond to

Se (-II), indicating that the microbial bead can reduce Se (IV) to

Se (-II), thus providing an opportunity for the subsequent removal of Cd(II) to form CdSe nanoparticles. The peaks at binding ener- gies 55.60 eV and 56.20 eV are considered to be elemental selenium

Se (0). In addition, there is no peak corresponding to Se (IV) in the results, indicating that Se (IV) adsorbed on the surface has been completely converted into intracellular or extracellular com- pounds by microorganisms at an initial stage of the reaction of microbial bead. After continuing to remove Cd (II), the correspond- ing peak binding energies of Se(-II}) and Se(0) change; XPS semi- quantitative analysis finds that the content of Se(-II) decreases from 31.97% to 21.61%, and the content of Se{0) increases from 68.03% to 78.39%, indicating that the addition of Cd(II} promotes the reduction of Se (IV) to Se (0) by microorganisms in the microbi- al bead, and a small part of Se (IV) is reduced to Se(-II). The corresponding peaks at 412.20 eV and 405.42 eV of the fine binding energy of Cd 3d are CdSe, indicating that the microbial bead can indeed synthesize nanoparticles CdSe. The peaks at 405.67 eV and 412.17 eV are attributed to Cd(II), indicating that the microbial bead can directly adsorb Cd(II) in the solution.

Example 6

Analysis of multiple microorganisms

Experimental steps were as follows.

Three stages, a microbial bead, an immobilized microsphere, and an immobilized microsphere after reacting with cadmium chlo- ride, were mixed uniformly separately; a genomic DNA was extracted with an E.Z.N.A Soil DNA Kit, PCR amplification was conducted on bacteria with universal primers 515FomodF/806RmodR, and high- throughput sequencing was conducted on an Illumina MiSeq PE plat- form; obtained sequences were subjected to quality control, and classified into Operational Taxonomic Units (OTUs) with a thresh- old of 97% sequence similarity. Classification was conducted using

RDP Classifier software (version 2.6), and composition and abun- dance of the bacteria were evaluated at phylum and genus levels.

The results are shown in Table 1.

Table 1 Abundance at phylum level

Microbial bead after Re-adsorption of immobi-

Initial micro-

Microorganisms treating Se-containing lized microsphere to re- bial bead wastewater move Cd

Proteobacteria 83.96% 76.21% 96.02%

Bacteroidetes 15.58% 23.78% 3.37%

Others 0.46% 0.01% 0.61%

It can be seen that the microorganisms in the microbial bead and the immobilized microsphere have always been dominated by Pro- teobacteria and Firmicutes; although being slightly affected dur- ing the whole process, the abundance of Proteobacteria has been in a relatively large proportion. At the genus level, the microorgan- isms in the microbial bead are mainly composed of five genera, namely Enterobacteriaceae (40% to 66%), Alcaligenes (20% to 36%),

Terrisporobacter (1% to 83), Paraclostridium (1% to 4%), and She- wanella (1% to 3%).

It can be seen from the above examples that the multiple mi- croorganisms of the present disclosure have desirable resistance to selenium and cadmium; the microbial bead and the immobilized microsphere do not need an external carbon source during removing selenium and cadmium, and can simultaneously remove selenium and cadmium in the polluted water, with removal efficiencys of seleni- um and cadmium up to not less than 97% and not less than 99%, re-

spectively. Therefore, a desirable prospect for use is achieved in the present disclosure.

The above are only the examples of the present disclosure and therefore do not limit the patent scope of the present disclosure.

Any equivalent structure or equivalent process transformation used according to the contents of the specification in the present dis- closure, no matter whether it is directly or indirectly used in any other related technical field, should be included within the scope of patent protection of the present disclosure.

Claims

CONCLUSIONS

1. Microbial granule prepared by embedding multiple microorganisms in sodium alginate; wherein the plurality of microorganisms include Enterobacteriaceae, Alcaligenes, Terrisporobacter, Pararaclostridium and Shewanella.

Use of the microbial granule according to claim 1 for removing selenium and cadmium in contaminated water.

Immobilized microspheres obtained from biological selenium nanoparticles adhered to a surface of the microbial granule according to claim 1.

A method for preparing the immobilized microspheres according to claim 3, comprising the step of: mixing the microbial granule according to claim 1 with a sodium selenite solution for reaction to obtain the immobilized microspheres.

The method of preparation according to claim 4, wherein the reaction is carried out at a pH value of 4 to 7.

The method of preparation according to claim 4, wherein the microbial bead and the sodium selenite solution have a mass to volume ratio of (0.25 to 6.0) g: 1 L.

The method of preparation according to claim 4, wherein the sodium selenite solution has a concentration of 3.95 mg/L to 39.50 mg/L.

Use of the immobilized microspheres according to claim 3 or the method of preparation according to any one of claims 4 to 7 in the removal of cadmium in contaminated water.

A method for removing selenium and cadmium in contaminated water, comprising the steps of: mixing the microbial granule of claim 1 with the contaminated water to adsorb selenium to obtain an immobilized microsphere; and reacting the immobilized microsphere with cadmium in the contaminated water to remove selenium and cadmium in the contaminated water.

10. A method according to claim 9, wherein no extra carbon source is added when removing selenium and cadmium in the polluted water.