Preparation method of bacterial culture carrier and bacterial culture carrier

Abstract

The invention provides a preparation method of a bacterial culture carrier and the bacterial culture carrier. The preparation method of the bacterial culture carrier comprises the following steps: step (i), preparing an electrostatic spinning solution and embedded particles; and (ii) electrospinning the embedded particles with an electrospinning solution. Wherein in step (i), an electrostatic spinning solution is prepared by extracting cellulose and / or hemicellulose from seaweed; each embedded particle comprises a core part containing denitrifying bacteria and a shell layer which is formed by cross-linking of a cross-linking agent and coats the core part. The bacterial culture carrier can be widely applied to sewage nitrogen and phosphorus removal, organic matter purification and pollutant removal processes and sludge anaerobic fermentation and aerobic fermentation processes, the yield of strains is increased, and the bacterial culture carrier can adapt to a worse environment.

Description

Technical Field

[0001] The invention belongs to the field of sewage and sludge biological treatment, and in particular relates to a preparation method of a bacteria cultivation carrier and the bacteria cultivation carrier. Background Art

[0002] Harmful substances in sewage can directly or indirectly harm human health, pollute natural water bodies, and destroy ecological balance. Sewage sludge contains organic matter, heavy metals, and other harmful substances. In particular, the long-term accumulation of heavy metals may cause potential toxicity and ecological risks to the soil and ecosystem. Therefore, sewage sludge needs to be treated.

[0003] However, there are still many problems with the treatment of sewage sludge by existing technologies. For example, the high content of nitrogen source pollution such as ammonia nitrogen and total nitrogen in sewage will make it difficult to treat sewage. At present, nitrogen source pollution in sewage is generally removed through nitrification-denitrification, and heavy metals and other harmful substances in sludge are often treated by aerobic composting, anaerobic digestion and other treatment technologies. However, the technologies used are limited in their wide application in terms of operability, operating costs, environmental pH range, microbial flora growth metabolism and activity.

[0004] Therefore, it is necessary to improve the existing technology.

[0005] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present invention, and therefore it may contain information that does not constitute the prior art known to those skilled in the art. Summary of the invention

[0006] In order to solve one or more of the above problems existing in the prior art, the present invention aims to provide a method for preparing a bacteria cultivation carrier and a bacteria cultivation carrier.

[0007] The method for preparing a bacteria culture carrier of the present invention comprises the following steps: step (i): preparing an electrospinning solution and embedded particles; and step (ii): electrospinning the embedded particles using the electrospinning solution. In the step (i), the electrospinning solution is prepared by extracting cellulose and / or hemicellulose from seaweed; the embedded particles comprise a core containing denitrifying bacteria and a shell formed by cross-linking a cross-linking agent and covering the core.

[0008] According to an embodiment of the present invention, in the above step (i), the above cross-linking agent can be any one of polyvinyl alcohol-sodium alginate, polyethylene glycol, polyvinyl alcohol, and water-based polyurethane, or a combination of several of them.

[0009] According to one embodiment of the present invention, the embedded particles can be prepared by the following method: polyvinyl alcohol and sodium alginate are used as the cross-linking agent in a mass ratio of 3:1 to 5:1, the cross-linking agent is dissolved in water at a concentration of 50 g / L to 150 g / L as a cross-linking agent solution, denitrification sludge is added to the cross-linking agent solution at a volume ratio of the cross-linking agent solution to denitrification sludge of 1:2 to 3:2, activated carbon is added and mixed evenly, and CaCl is added to the obtained mixed solution. 2 The embedded particles are obtained after cross-linking and immobilization reaction in aqueous solution, wherein the activated carbon is added in an amount of 0.5 g to 2 g per 100 mL of the denitrification sludge, and the CaCl 2 The mass concentration of the aqueous solution is 20g / L to 80g / L. 2 The volume ratio of the aqueous solution to the denitrification sludge is 4:1 to 10:1.

[0010] According to one embodiment of the present invention, the embedded particles are prepared by the following method: polyethylene glycol is used as the cross-linking agent, and the cross-linking agent is dissolved in water at a concentration of 90 g / L to 120 g / L as a cross-linking agent solution, and denitrification sludge is added to the cross-linking agent solution at a volume ratio of the cross-linking agent solution to the denitrification sludge of 1:2 to 3:2, and then activated carbon, N,N,N,N-tetramethylenediamine and potassium persulfate are added to carry out a cross-linking reaction to obtain the embedded particles, wherein the amount of the activated carbon added is 0.5 to 5 g of the activated carbon per 100 mL of the denitrification sludge, and the mass ratio of the polyethylene glycol, N,N,N,N-tetramethylenediamine and potassium persulfate is: 15:1:1 to 30:1:1.

[0011] According to one embodiment of the present invention, the electrospinning solution is prepared by the following method: 10 to 20 parts by mass of hemicellulose and / or cellulose extracted from the seaweed is dissolved in water at a concentration of 400 g / L to 800 g / L, 0.15 to 0.25 parts by mass of ammonium persulfate and 0.15 to 0.25 parts by mass of anhydrous sodium sulfite are added to react, and then 2 to 3 parts by mass of acrylic acid are added to react to obtain a seaweed spinning solution, and the viscosity of the seaweed spinning solution is adjusted to 7 to 10 Pa·s with anhydrous ethanol to obtain the electrospinning solution.

[0012] According to one embodiment of the present invention, in the above step (i), the above hemicellulose is prepared by the following method: the above seaweed is mechanically crushed to obtain seaweed powder, and the above seaweed powder is dissolved in a solution containing 4.8 g / L NaOH and 1% by volume H at a concentration of 40 g / L to 60 g / L. 2 O 2The extract is centrifuged after stirring to obtain an extract, and after removing the protein from the extract, anhydrous ethanol is added to precipitate the solid, which is then dried to obtain the hemicellulose.

[0013] According to one embodiment of the present invention, in the above step (i), the above cellulose is prepared by the following method: the seaweed is mechanically crushed to obtain seaweed powder, the seaweed powder is defatted in methanol, bleached, and alkali treated, then washed to neutrality, filtered and dried to obtain the above cellulose.

[0014] According to one embodiment of the present invention, in the above step (ii), the electrospinning liquid is sprayed on the outside of the above embedded particles using an electrospinning machine, wherein the spraying speed of the electrospinning liquid is 0.4 mL / h to 0.6 mL / h and the spinning temperature is 20°C to 25°C.

[0015] The present invention also provides a bacteria cultivation carrier prepared by the preparation method of the bacteria cultivation carrier.

[0016] The present invention also provides an application of a bacteria cultivation carrier in sewage and / or sludge treatment, wherein the bacteria cultivation carrier is prepared by the preparation method of the bacteria cultivation carrier.

[0017] Beneficial effects:

[0018] The bacterial culture carrier of the present invention can be widely used in sewage denitrification and phosphorus removal, organic matter purification, pollutant removal processes, and sludge anaerobic fermentation and aerobic fermentation processes, and can increase the yield of bacterial strains and enable them to adapt to more severe environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of this specification or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the embodiments of this specification. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0020] Figure 1 is a diagram showing a spinning membrane according to Example 1 and Example 3 of the present invention, wherein, Figure 1 a is the electrospun membrane of Example 1; Figure 1 b is the electrospinning microscope image of Example 1; Figure 1 c is the electrospun membrane of Example 3; Figure 1 d is the electrospinning microscope image of Example 3.

[0021] Figure 2 The photographs are diagrams showing the bacterial culture carriers according to Examples 1 to 3 of the present invention. Figure 2a is a photograph of the bacterial culture carrier of Example 1; Figure 2 b is a photograph of the bacterial culture carrier of Example 2; Figure 2 c is a photograph of the bacterial culture carrier in Example 3.

[0022] Figure 3 It is a graph showing the long-term stable operation of Examples 1 to 3.

[0023] Figure 4 It is a diagram showing the analysis of Examples 1 to 3 at the microbial phylum level. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined, separated, interchanged and / or rearranged with each other in the absence of conflict. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] In addition, when the exemplary embodiments can be implemented differently, the specific process order can be performed in a different order than described. For example, two consecutively described processes can be performed substantially simultaneously or in a reverse order to the described order.

[0026] In addition, the terms used in this specification are for the purpose of describing specific embodiments, and are not intended to be restrictive. As used herein, unless the context clearly indicates otherwise, the singular forms "one (kind, person)" and "said (the)" are also intended to include plural forms. In addition, when the terms "comprise" and / or "include" and their variations are used in this specification, it is explained that there are stated features, integral bodies, steps, operations, parts, assemblies and / or their groups, but one or more other features, integral bodies, steps, operations, parts, assemblies and / or their groups are not excluded from existence or addition. It should also be noted that, as used herein, the terms "substantially", "approximately" and other similar terms are used as approximate terms and not as degree terms, so that they are used to explain the inherent deviations of the measured values, calculated values ​​and / or the values ​​provided that will be recognized by those of ordinary skill in the art.

[0027] The inventor of the present invention unexpectedly discovered during the experiment that by using electrospinning technology to spin and spray seaweed fibers on the outside of embedded particles, microbial flora can be enriched and grown at a high concentration, and the embedded particles after electrospinning can still maintain a high bacterial activity even under acidic or alkaline conditions. Based on this, the present invention was completed.

[0028] Specifically, the preparation method of the bacteria culture carrier of the present invention comprises preparing an electrospinning solution and embedded particles; and electrospinning the embedded particles using the electrospinning solution.

[0029] Wherein, the raw material for extracting cellulose and / or hemicellulose adopts seaweed. Seaweed is rich in a large amount of cellulose, hemicellulose, minerals, etc., and is a kind of high-quality raw material for extracting cellulose and hemicellulose. The seaweed used in the present invention can be common algae such as Enteromorpha, Yellow Silk Algae, and Laminaria japonica, wherein Enteromorpha is preferably used. Considering aspects such as large output, low price, and harmless treatment of biological waste, Enteromorpha is preferably used as raw material to extract cellulose and hemicellulose, and electrostatic spinning technology is used to spin it.

[0030] In addition, the embedded particles of the present invention include a core containing denitrifying bacteria and a shell formed by cross-linking agent and coating the core. The cross-linking agent can be any one or more combinations of polyvinyl alcohol-sodium alginate (PVA-SA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), and waterborne polyurethane (WPU). The cross-linked layer formed by polyvinyl alcohol-sodium alginate (PVA-SA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), and waterborne polyurethane (WPU) has the advantages of good mechanical strength, good biocompatibility, good internal spatial structure, and low toxicity. The embedding bacteria can make the internal reaction mild, which is conducive to bacterial growth and enrichment of high concentration activity. However, these polymer materials have poor swelling properties during long-term stable operation. Therefore, the present invention electrostatically spins seaweed extract to outer layers such as PVA-SA, PEG, PVA, and WPU to enhance its swelling properties and mechanical strength, and is not easily damaged during long-term stable operation.

[0031] The bacterial cultivation carrier thus formed forms a double-layer structure outside the core containing denitrifying bacteria, the inner layer is a coating layer (also called a cross-linking layer) obtained by ionic cross-linking, and the outer layer is a porous mesh structure seaweed spinning layer prepared using electrostatic spinning technology. Accordingly, the bacterial cultivation carrier prepared by the preparation method of the bacterial cultivation carrier of the present invention is conducive to the growth and development of bacteria during sewage and sludge treatment, so that the microorganisms have good activity and high concentration enrichment, and also have excellent mechanical properties, high specific surface area and swelling and expansion properties. The bacterial cultivation carrier of the present invention has excellent acid and alkali resistance, and can well adapt to the impact of external environmental conditions on it. Under acidic or alkaline conditions, the bacterial cultivation carrier can not only maintain the activity of the internal flora, but also ensure the stability of its own structure, is not easy to dissociate and break, and can be continuously reused.

[0032] Therefore, the bacterial culture carrier of the present invention can be widely used in sewage denitrification and phosphorus removal, organic matter purification, pollutant removal processes, and sludge anaerobic fermentation and aerobic fermentation processes, and can increase the yield of bacterial strains and make them adapt to more severe environments. Therefore, it has important application prospects in sewage and sludge biological treatment processes.

[0033] In the present invention, when polyvinyl alcohol and sodium alginate are used as crosslinking agents, the embedded particles can be prepared by the following method: polyvinyl alcohol and sodium alginate are mixed at a mass ratio of 3:1 to 5:1 as a crosslinking agent, the crosslinking agent is dissolved in water at a concentration of 50 g / L to 150 g / L as a crosslinking agent solution, denitrification sludge is added to the crosslinking agent solution at a volume ratio of the crosslinking agent solution to the denitrification sludge of 1:2 to 3:2, activated carbon is added and mixed evenly, and the prepared mixed solution is dripped with CaCl by, for example, a syringe. 2 The embedded particles are obtained after cross-linking and immobilization reaction in aqueous solution. The addition amount of activated carbon is preferably 0.5 g to 2 g per 100 mL of the above denitrification sludge, and CaCl 2 The mass concentration of the aqueous solution is preferably 20 g / L to 80 g / L, and CaCl 2 The volume ratio of the aqueous solution to the denitrification sludge is preferably 4:1 to 10:1.

[0034] More specifically, for example, 4 parts by mass of polyvinyl alcohol and 1 part by mass of sodium alginate as a crosslinking agent can be placed in distilled water at a concentration of 10 g / L, heated and dissolved at 95°C to form a crosslinking agent solution, and after cooling, an equal volume of denitrification sludge is added to the crosslinking agent solution, and activated carbon is added at a ratio of 1 g per 100 mL of denitrification sludge, and then 40 g / L of CaCl 2 The solution was fixed, and after refrigerated cross-linking reaction for 12 hours, the PVA-SA embedded particles were obtained after washing with deionized water.

[0035] In the present invention, when polyethylene glycol is used as a cross-linking agent, embedded particles can be prepared by the following method: the cross-linking agent is dissolved in water at a concentration of 90 g / L to 120 g / L as a cross-linking agent solution, denitrification sludge is added to the cross-linking agent solution at a volume ratio of the cross-linking agent solution to the denitrification sludge of 1:2 to 3:2, and then activated carbon, N,N,N,N-tetramethylenediamine as a promoter and potassium persulfate as an initiator are added to perform a cross-linking reaction to obtain embedded particles, wherein the amount of activated carbon added is 0.5 to 5 g of activated carbon per 100 mL of denitrification sludge, and the mass ratio of polyethylene glycol, N,N,N,N-tetramethylenediamine and potassium persulfate is 15:1:1 to 30:1:1.

[0036] More specifically, 2 parts by mass of polyethylene glycol can be heated and dissolved in deionized water at a concentration of 100 g / L as a cross-linker solution, and after cooling, an equal volume of denitrification sludge is added to the cross-linker solution, and activated carbon is added at a ratio of 5 g per 100 mL of denitrification sludge. Then, 0.1 parts by mass of a promoter (N,N,N,N-tetramethylenediamine, analytical grade) and 0.1 parts by mass of an initiator (potassium persulfate, analytical grade) are added, and the gel polymerization process is completed after a few minutes. The resulting gel is cut into cubes of about 5×5×5 mm to obtain PEG-encapsulated particles.

[0037] In addition, the present invention can extract hemicellulose from seaweed by the following method: mechanically crush the seaweed to obtain seaweed powder, dissolve the seaweed powder at a concentration of 40 g / L to 60 g / L in a solution containing 4.8 g / L NaOH and 1% by volume H 2 O 2 The extract is centrifuged after stirring to obtain an extract, and after removing the protein from the extract, anhydrous ethanol is added to precipitate the solid, which is then dried to obtain the hemicellulose.

[0038] More specifically, the seaweed can be mechanically crushed into seaweed powder with a particle size of 26-38 μm, and the seaweed powder is dissolved in a solution containing 4.8 g / L NaOH and 1% by volume H 2 O 2 The extract is heated (e.g., 92°C) and stirred for 5 hours, and then centrifuged to obtain an extract. The extract is adjusted to a neutral pH with 1 mol / L hydrochloric acid, and then the protein is removed by the Sevage method. After static extraction, anhydrous ethanol is added overnight to precipitate the hemicellulose semi-finished product, and the hemicellulose powder is obtained after centrifugal freeze drying. In the Sevage method, the volume ratio of chloroform to n-butanol is 5:1, and the extraction is repeated 4 times. The volume of anhydrous ethanol can be 3 times that of the extract.

[0039] In addition, the present invention can extract cellulose from seaweed by the following method: mechanically crushing the seaweed to obtain seaweed powder, defatting the seaweed powder in methanol, bleaching and alkali treatment, washing to neutrality, filtering and drying to obtain cellulose.

[0040] More specifically, for example, the seaweed can be mechanically crushed into seaweed powder with a particle size of 26-38 μm, 10-20 g of the seaweed powder can be repeatedly defatted in methanol at room temperature for 8 days, and then placed in a 1 L containing 36 g NaClO 2 The cellulose was bleached in an acetic acid buffer at 60°C for 3 hours, washed to neutrality, treated with 0.5 mol / L sodium hydroxide aqueous solution at 60°C overnight, and washed to neutrality with deionized water. Finally, the light yellow sample was filtered and freeze-dried to obtain cellulose powder.

[0041] In addition, in the present invention, 10 to 20 parts by mass of the hemicellulose and / or cellulose prepared above can be dissolved in water at a concentration of 400 g / L to 800 g / L, 0.15 to 0.25 parts by mass of ammonium persulfate and 0.15 to 0.25 parts by mass of anhydrous sodium sulfite are added to react, and then 2 to 3 parts by mass of acrylic acid are added to react to obtain a seaweed spinning solution, and the viscosity of the seaweed spinning solution is adjusted to 7 to 10 Pa·s with anhydrous ethanol to obtain the electrospinning solution of the present invention.

[0042] Specifically, 10 g of hemicellulose and / or cellulose can be dissolved in 15 mL of distilled water, 0.15 g of ammonium persulfate and 0.15 g of anhydrous sodium sulfite can be added, and 2 g of acrylic acid can be added and stirred after heating in a water bath at 80° C. for 5 minutes, and then kept in a constant temperature water bath at 80° C. for 3 hours to obtain a seaweed spinning solution. The seaweed spinning solution is adjusted to a viscosity of about 8 Pa·s with anhydrous ethanol to obtain the electrospinning solution of the present invention.

[0043] In addition, the present invention can use an electrospinning machine to spray the electrospinning liquid on the outside of the embedded particles. The electrospinning liquid ejection rate is preferably 0.4mL / h to 0.6mL / h, the spinning temperature is preferably 20°C to 25°C, and the spinning humidity is preferably 21%.

[0044] The present invention is further described in detail below in conjunction with specific examples, but the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry.

[0045] Example 1

[0046] Seaweed (Enteromorpha)

[0047] Preparation of hemicellulose: Enteromorpha was mechanically crushed to obtain powder. 10 g of Enteromorpha powder was placed in a solution containing 0.96 g NaOH and 2 mL H 2 O 2 The extract was stirred in 200 mL of aqueous solution and then centrifuged to obtain an extract. The pH of the extract was adjusted to neutral with 1 mol / L hydrochloric acid and the protein was removed by the Sevage method (wherein the ratio of chloroform to n-butanol was 5:1). After static extraction, 3 times the volume of anhydrous ethanol was added to the extract overnight to precipitate hemicellulose, and hemicellulose powder was obtained after centrifugal freeze-drying.

[0048] Preparation of seaweed spinning solution: Dissolve 10 g of Enteromorpha hemicellulose powder in 15 mL of distilled water, add 0.15 g of ammonium persulfate and 0.15 g of anhydrous sodium sulfite, heat in a water bath for 5 minutes, then add 2 g of acrylic acid and stir, and keep in a constant temperature water bath at 80°C for 3 hours to obtain seaweed spinning solution (hemicellulose spinning solution).

[0049] Preparation of embedded particles: 8g polyvinyl alcohol and 2g sodium alginate were dissolved in 100mL distilled water and heated to dissolve. After cooling, 100mL denitrification sludge was added and 1g activated carbon was added to mix well to obtain a mixed solution. 20g CaCl 2 Prepare CaCl by dissolving in 500 mL of distilled water 2 The mixed solution was added dropwise with a syringe into CaCl 2 The aqueous solution was fixed, refrigerated and cross-linked for 12 hours, and then washed with deionized water to obtain PVA-SA embedded particles.

[0050] Preparation of bacterial culture carrier: The viscosity of the seaweed spinning solution was adjusted to 8 Pa·s with anhydrous ethanol, and the electrospinning machine was set to set the spinning solution ejection speed to 0.5 mL / h, the spinning temperature to 23°C, and the humidity to 21% for electrospinning, and then sprayed on the outside of the PVA-SA embedded particles to finally obtain a bacterial culture carrier (hemicellulose electrospinning embedded particles, EH-PVA-SA). Among them, the hemicellulose formed a thin film after spinning, and its electrospun membrane and microscope image are shown as follows: Figure 1 As shown in a and b in Example 1. Figure 2 As shown in a in .

[0051] Example 2

[0052] Seaweed (Yellow Silk Algae)

[0053] Preparation of hemicellulose: The yellow silk algae was mechanically crushed to obtain powder. 10 g of the yellow silk algae powder was placed in a solution containing 0.96 g NaOH and 2 mL H 2 O 2The extract was stirred in 200 mL of aqueous solution and then centrifuged to obtain an extract. The pH of the extract was adjusted to neutral with 1 mol / L hydrochloric acid, and the protein was removed by the Sevage method (the ratio of chloroform and n-butanol was 5:1). After standing for extraction, anhydrous ethanol was added overnight to precipitate hemicellulose, and hemicellulose powder was obtained after centrifugal freeze-drying.

[0054] Preparation of seaweed spinning solution: Dissolve 10 g of the above hemicellulose powder in 20 mL of distilled water, add 0.2 g of ammonium persulfate and 0.2 g of anhydrous sodium sulfite, heat in a water bath for 5 minutes, then add 2.5 g of acrylic acid and stir, and then keep in a constant temperature water bath at 80°C for 3 hours to obtain seaweed spinning solution (hemicellulose spinning solution).

[0055] Preparation of embedded particles: Dissolve 2 g of polyethylene glycol (PEG) in 20 mL of deionized water, cool and add 20 mL of denitrification sludge, add 1 g of activated carbon, add 0.1 g of promoter (N,N,N,N-tetramethylenediamine, analytical grade) and 0.1 g of initiator (potassium persulfate, analytical grade), let stand to complete gel polymerization, cut the obtained gel into cubes of about 5×5×5 mm to obtain PEG embedded particles.

[0056] Preparation of bacterial culture carrier: The seaweed spinning solution was adjusted to a viscosity of 8 Pa·s with anhydrous ethanol, and the spinning solution was electrospun using an electrospinning machine with a spinning solution ejection rate of 0.5 mL / h, a spinning temperature of 23°C, and a humidity of 21%. The solution was then sprayed on the outside of the PEG-embedded particles to obtain a bacterial culture carrier (EH-PEG). The electrospinning membrane and microscope image of Example 2 are similar to those of Example 1, and are not shown here. The bacterial culture carrier of Example 2 is as shown in FIG. Figure 2 As shown in b.

[0057] Example 3

[0058] Seaweed (Enteromorpha)

[0059] Preparation of cellulose: 10-20 g Enteromorpha powder was repeatedly defatted in methanol at room temperature for 8 days and then placed in 1 L of 36 g NaClO 2 The cellulose was bleached in an acetic acid buffer at 60°C for 3 hours and washed to neutral. The cellulose was treated with 0.5 mol / L sodium hydroxide solution at 60°C overnight and washed to neutral with deionized water. Finally, the light yellow sample was filtered and freeze-dried to obtain cellulose (EC) powder.

[0060] Preparation of seaweed spinning solution: Dissolve 10 g of cellulose powder in 20 mL of distilled water, add 0.2 g of ammonium persulfate and 0.2 g of anhydrous sodium sulfite, heat in a water bath for 5 minutes, then add 2.5 g of acrylic acid and stir, and then keep in a constant temperature water bath at 80°C for 3 hours to obtain the seaweed spinning solution.

[0061] Preparation method of embedded particles: dissolve 2g polyethylene glycol (PEG) in 20mL deionized water, cool it, add 20mL denitrification sludge, add 1g activated carbon, add 0.1g promoter (N,N,N,N-tetramethylenediamine, analytical grade) and 0.1g initiator (potassium persulfate, analytical grade), wait for a few minutes to complete the gel polymerization process, cut the obtained gel into cubes of about 5×5×5mm, and obtain PEG embedded particles.

[0062] Preparation of bacterial culture carrier: The viscosity of the seaweed spinning solution was adjusted to 8 Pa·s with anhydrous ethanol, and the electrospinning machine was set to set the spinning solution ejection speed to 0.5 ml / h, the spinning temperature to 23°C, and the humidity to 21% for electrospinning, and then sprayed on the outside of the PEG-embedded particles to finally obtain the bacterial culture carrier (cellulose electrospinning embedded particles, EC-PEG). After cellulose spinning, a thin film was formed, and its electrospun membrane and microscope image are shown in Figure 2. Figure 1 As shown in c and d in Example 3. Figure 2 As shown in c.

[0063] Performance Testing

[0064] For Examples 1 to 3, a control group was set up with PVA-SA embedded particles as the control group bacterial culture carrier, which was prepared as follows: 8g of polyvinyl alcohol and 2g of sodium alginate were dissolved in 100mL of distilled water and heated to dissolve, and then 100mL of denitrification sludge was added after cooling, and 1g of activated carbon was added and mixed to obtain a mixed solution, and 20g of CaCl 2 Dissolve in 500 mL of distilled water to prepare CaCl 2 aqueous solution, add CaCl 2 The aqueous solution was used for immobilization, and after refrigerated cross-linking for 12 hours, the PVA-SA embedded particles (bacterial culture carrier of the control group) were obtained after washing with deionized water.

[0065] The mechanical stability, long-term stability and microbial enrichment of the bacterial culture carriers prepared in Examples 1 to 3 were studied.

[0066] Analytical methods

[0067] 1.Mechanical stability analysis method:

[0068] 1) Comparison of physical and chemical properties of bacterial culture carriers: mechanical strength, expansion coefficient, and swelling performance

[0069] Mechanical strength: Take 20 bacterial culture carriers of similar size and put them into a conical flask and add 500 mL of deionized water. After stirring on a magnetic stirrer at the same speed for 72 hours, calculate the ratio of the number of complete bacterial culture carriers to the original bacterial culture carriers.

[0070] Expansion coefficient: Take 20 bacterial culture carriers of similar size and put them into a conical flask and add 500 mL of deionized water. Use a vernier caliper to measure the diameter of the bacterial culture carrier. Place it in a constant temperature oscillator and slowly oscillate it for 72 hours. Use a vernier caliper to measure the diameter of the bacterial culture carrier after oscillation, and calculate the ratio of the average diameter of the bacterial culture carrier after expansion to the average diameter of the bacterial culture carrier before expansion.

[0071] Swelling performance: Take 20 bacterial culture carriers and put them into a conical flask, add 500 ml of artificial wastewater, put it into a constant temperature oscillator and shake it for one week, and observe the swelling and bursting of the bacterial culture carriers.

[0072] The test results are recorded in Table 1.

[0073] 2. Long-term operation stability test:

[0074] The bacterial culture carrier of Example 1 was placed in a neutral solution of distilled water, the bacterial culture carrier of Example 2 was placed in an acidic solution with a pH range of 2 to 5, and the bacterial culture carrier of Example 3 was placed in an alkaline solution with a pH range of 7 to 10 for soaking and culture for one week to observe whether the internal bacterial flora can withstand acid and alkali and whether the bacterial flora has lost its activity. After soaking for one week, they were directly placed into a 5LSBR reactor for a short-range denitrification early start-up test, and then the carbon-nitrogen ratio was adjusted to select the optimal conditions for a long-term stable operation test to observe whether the internal bacterial flora can successfully start the reactor and operate stably for a long time. The results are as follows Figure 3 shown.

[0075] 3. Microbiological analysis methods:

[0076] The samples of bacterial culture carriers after 7 days of culture in acid, alkaline and neutral solutions and 40 days of long-term stable operation of the reactor were selected for 16s rRNA amplification detection to analyze the composition of the microbial community. The samples were compared on the Illumina MiSeq PE300 platform / NovaSeq PE250 platform (Illumina, San Diego, USA) to clarify the microbial structure composition in different samples. The results are shown in Figure 4 shown.

[0077] Results and Discussion

[0078] Table 1 Mechanical stability performance test results of Examples 1 to 3

[0079] Bacteria culture carrier Mechanical strength Expansion coefficient Swelling properties Control group 0.7 1.06 Breakthrough Example 1 1 1.06 Not broken Example 2 1 1.12 Not broken Example 3 1 1.66 Not broken

[0080] Through the test of mechanical properties in Table 1, it was found that the mechanical strength and swelling performance of the bacterial culture carrier in the control group were poor, while the bacterial culture carriers in Examples 1 to 3 showed excellent mechanical strength and swelling performance. During the experiment, the bacterial culture carriers of each example were not damaged, indicating that the electrospinning of seaweed cellulose and hemicellulose enhanced the mechanical properties of the embedded particles, which played a key role in further stabilizing the experiment and microbial cultivation.

[0081] The bacterial culture carriers of Examples 1 to 3 were subjected to a long-term stability test. Figure 3 It was found that during long-term operation, the bacterial culture carriers of Examples 1 to 3 (i.e., embedded particles after electrospinning) had a higher removal rate of nitrate nitrogen and an accumulation rate of nitrite nitrogen, indicating that the bacterial culture carriers cultured under acidic and alkaline conditions did not produce much difference in the start-up of the reactor and the long-term stable operation process compared with the bacterial culture carriers cultured in neutral solutions, and the internal bacterial flora activity was still good, and the reactor could be started quickly. In addition, it was shown that the bacterial culture carrier of the present invention has good acid and alkali resistance, can adapt to the impact of a harsh environment, and can maintain the activity of the internal bacterial flora.

[0082] In addition, during the long-term stable operation test, the bacterial culture carriers prepared in Examples 1 to 3 were not damaged, and still maintained good mechanical properties, swelling properties and expansion properties, and can be repeatedly and sustainably used in subsequent sewage treatment.

[0083] In addition, microbial community analysis was performed on the embedded particles spun from seaweed hemicellulose and cellulose. Figure 4 It can be seen that after the bacteria culture carriers of Examples 1 to 3 were immersed in the culture carriers for one week, live microorganisms were detected, and it was found that the bacteria culture carrier of the present invention still had a relatively high concentration of key bacterial flora, further indicating that the bacteria culture carrier of the present invention can have acid and alkali resistance.

[0084] After 40 days of long-term stable operation, the bacterial cultivation carrier of the present invention was subjected to high-throughput sequencing to analyze the community composition. It was found that the dominant phylum Bacteroidota (76.19%→94.04%) in Example 1 increased significantly; the dominant phylum Bacteroidota (66.78%→92.31%) in Example 2 increased significantly; and the dominant phylum Bacteroidota (70.34%→92.35%) in Example 3 increased significantly. It was found that the bacterial cultivation carrier of the present invention is beneficial to the growth and development of microorganisms, and the electrospinning of cellulose and hemicellulose has a good promoting effect on the bacterial flora inside the embedded particles.

[0085] In summary, the bacterial cultivation carrier prepared by the present invention has an inner and outer two-layer structure, the inner layer is a cross-linked coating layer obtained by ionic cross-linking, and the outer layer is a seaweed hemicellulose / cellulose porous membrane prepared by electrospinning. The prepared bacterial cultivation carrier has excellent biocompatibility and mechanical properties, a large specific surface area, is conducive to the growth and breeding of internal microorganisms, creates excellent conditions for substrate transport, and the preparation process is controllable. The electrospinning embedded particles can maintain good microbial activity inside, so that the microorganisms can be aggregated and cultured at high concentrations; at the same time, the electrospinning embedded particles have excellent acid and alkali resistance, and have important application prospects in sewage and sludge treatment.

[0086] Specifically, the bacterial cultivation carrier of the present invention can be placed in the early operation start-up stage of the sewage sludge treatment process, and it can be directly added to the SBR reactor, without adding activated sludge and other substances in the reactor for experiments. The bacterial cultivation carrier of the present invention enriches the key bacterial flora at a high concentration, which can shorten the time for culturing microbial flora in the early process, and more quickly carry out biological denitrification and phosphorus removal, aerobic anaerobic fermentation and other processes. At the same time, if the bacterial cultivation carrier of the present invention is placed in the early start-up stage of acidic and alkaline sewage sludge, the high activity of the dominant bacterial flora can be protected, so that the microorganisms can efficiently carry out the biological treatment process.

[0087] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a bacterial culture carrier, characterized in that: The preparation method of the bacterial culture carrier comprises the following steps: Step (i): preparing electrospinning solution and embedded particles; and Step (ii): electrospinning the embedded particles using the electrospinning solution, Wherein, in the step (i), the electrospinning solution is prepared by extracting cellulose and / or hemicellulose from seaweed; the embedded particles include a core containing denitrifying bacteria and a shell layer formed by cross-linking with a cross-linking agent and covering the core.

2. The method for preparing a bacteria culture carrier according to claim 1, characterized in that: In the step (i), the cross-linking agent is any one of polyvinyl alcohol-sodium alginate, polyethylene glycol, polyvinyl alcohol, and waterborne polyurethane, or a combination of several of them.

3. The method for preparing a bacteria culture carrier according to claim 2, characterized in that: The embedded particles are prepared by the following method: polyvinyl alcohol and sodium alginate are used as the crosslinking agent in a mass ratio of 3:1 to 5:1, the crosslinking agent is dissolved in water at a concentration of 50 g / L to 150 g / L as a crosslinking agent solution, denitrification sludge is added to the crosslinking agent solution at a volume ratio of the crosslinking agent solution to denitrification sludge of 1:2 to 3:2, activated carbon is added and mixed, and the obtained mixed solution is added to a CaCl2 aqueous solution to perform a crosslinking immobilization reaction to obtain the embedded particles. Among them, the added amount of the activated carbon is 0.5g~2g of the activated carbon per 100mL of the denitrification sludge, the mass concentration of the CaCl2 aqueous solution is 20g / L~80g / L, and the volume ratio of the CaCl2 aqueous solution to the denitrification sludge is 4:1~10:

1.

4. The method for preparing a bacteria culture carrier according to claim 2, characterized in that: The embedded particles are prepared by the following method: polyethylene glycol is used as the cross-linking agent, the cross-linking agent is dissolved in water at a concentration of 90 g / L to 120 g / L as a cross-linking agent solution, denitrification sludge is added to the cross-linking agent solution at a volume ratio of the cross-linking agent solution to denitrification sludge of 1:2 to 3:2, activated carbon, N,N,N,N-tetramethylenediamine and potassium persulfate are added, and a cross-linking reaction is performed to obtain the embedded particles. The amount of activated carbon added is 0.5 to 5 g per 100 mL of the denitrification sludge, and the mass ratio of polyethylene glycol, N,N,N,N-tetramethylenediamine and potassium persulfate is 15:1:1 to 30:1:

1.

5. The method for preparing a bacteria culture carrier according to any one of claims 1 to 4, characterized in that: The electrospinning solution is prepared by the following method: 10 to 20 parts by weight of hemicellulose and / or cellulose extracted from the seaweed is dissolved in water at a concentration of 400 g / L to 800 g / L, 0.15 to 0.25 parts by weight of ammonium persulfate and 0.15 to 0.25 parts by weight of anhydrous sodium sulfite are added to react, and then 2 to 3 parts by weight of acrylic acid are added to react to obtain a seaweed spinning solution, and the viscosity of the seaweed spinning solution is adjusted to 7 to 10 Pa·s with anhydrous ethanol to obtain the electrospinning solution.

6. The method for preparing a bacteria culture carrier according to claim 5, characterized in that: In the step (i), the hemicellulose is prepared by the following method: the seaweed is mechanically crushed to obtain seaweed powder, the seaweed powder is dissolved in an aqueous solution containing 4.8 g / L NaOH and 1 volume % H2O2 at a concentration of 40 g / L to 60 g / L, the extract is stirred and centrifuged to obtain an extract, the protein is removed from the extract, anhydrous ethanol is added to precipitate solids, and the hemicellulose is obtained by drying.

7. The method for preparing a bacteria culture carrier according to claim 5, characterized in that: In the step (i), the cellulose is prepared by the following method: the seaweed is mechanically crushed to obtain seaweed powder, the seaweed powder is defatted in methanol, bleached, alkali treated, washed to neutrality, filtered and dried to obtain the cellulose.

8. The method for preparing a bacteria culture carrier according to any one of claims 1 to 4, characterized in that: In the step (ii), the electrospinning liquid is sprayed onto the outside of the embedded particles using an electrospinning machine, wherein the ejection rate of the electrospinning liquid is 0.4 mL / h to 0.6 mL / h and the spinning temperature is 20° C. to 25° C.

9. A bacterial culture carrier, characterized in that: The bacteria culture carrier is prepared by the method for preparing a bacteria culture carrier according to any one of claims 1 to 8.

10. Use of a bacterial culture carrier in sewage and / or sludge treatment, characterized in that: The bacteria culture carrier is prepared by the method for preparing a bacteria culture carrier according to any one of claims 1 to 8.

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