Preparation method of nitrobacteria inoculant block

Abstract

The invention relates to a preparation method of a nitrifying bacterium agent block, which comprises the following steps: mixing and grinding a mineral carrier to a specified particle size, and respectively preparing a PVA (Polyvinyl Alcohol) solution and a composite adhesive; mixing and kneading the pretreated mineral carrier with a PVA solution, then adding a composite adhesive, kneading, and finally adding the bacterial suspension for embedding to prepare a dough-shaped material; filling a double-layer nested mold with the dough-shaped material in batches, and respectively applying different pressures to form a gradient structure fungicide block which is dense outside and sparse inside; soaking the formed microbial agent block in a calcium chloride solution for cross-linking reaction; and sequentially carrying out normal-temperature pre-curing and low-temperature activation on the cured microbial agent block to obtain a finished product. The problems that in the prior art, the survival rate of flora is low and the slow release period is short are solved by combining a composite mineral carrier with a specific formula and a multi-component organic adhesive with a unique gradient compaction mold forming and calcium ion cross-linking curing process.

Description

Technical Field

[0001] This application relates to the field of microbial preparations and water treatment technology, and in particular to a method for preparing nitrifying bacteria inoculum blocks. Background Technology

[0002] In aquaculture and wastewater treatment, nitrifying bacteria are key microorganisms for degrading ammonia nitrogen and nitrite. However, traditional liquid bacterial agents are difficult to preserve and easily lost after addition; conventional solid bacterial agents often face two main contradictions: firstly, the high temperature or high pressure during the molding process can easily lead to the death of the bacteria; secondly, if the carrier is too dense, it will be difficult to release, or if it is too loose, it will disintegrate too quickly, making it difficult to achieve long-term slow release (e.g., 60-90 days). Therefore, developing a method for preparing bacterial agent blocks that can maintain high bacterial activity and achieve controllable slow release through structural design is a technical problem that urgently needs to be solved in the industry. Summary of the Invention

[0003] This application provides a method for preparing nitrifying bacteria inoculant blocks. By using a composite mineral carrier with a specific formulation and a multi-component organic binder, combined with a unique gradient compaction mold forming and calcium ion cross-linking curing process, the method solves the problems of low bacterial survival rate and short slow-release cycle in the prior art.

[0004] This application provides a method for preparing nitrifying bacteria inoculum blocks, comprising the following steps:

[0005] S1: Raw material pretreatment: Mix and grind the mineral carrier to the specified particle size to prepare PVA solution and composite adhesive respectively;

[0006] S2: Preparation of dough-like material: The pretreated mineral carrier is mixed and kneaded with PVA solution, then a composite adhesive is added and kneaded again, and finally a bacterial suspension is added for encapsulation to obtain dough-like material;

[0007] S3: Gradient compaction mold forming: The dough-like material is filled into a double-layer nested mold in batches, and different pressures are applied to form a gradient structure of bacterial agent blocks with a dense outer layer and a loose inner layer.

[0008] S4: Calcium ion cross-linking and curing: The molded bacterial agent block is immersed in calcium chloride solution to carry out the cross-linking reaction;

[0009] S5: Gradient drying and activation: The cured bacterial agent blocks are sequentially pre-cured at room temperature and activated at low temperature to obtain the finished product.

[0010] By adopting the above technical solution, the overall process route of "raw materials-kneading-molding-curing-activation" breaks the traditional single-mixing molding mode. Through step-by-step processing, it resolves the contradiction between maintaining microbial activity and sustained-release performance. S2's "layered kneading" ensures the uniform dispersion of all components, especially the microbial community, and reduces mechanical damage. S3's "gradient compaction" achieves physical regulation of the release rate through physical structure design. S4's "chemical cross-linking" constructs a semi-permeable membrane on the surface, further chemically regulating the release rate. S5's "gradient drying" removes moisture while avoiding cracking caused by rapid drying and utilizes a CO2 environment to promote microbial colonization. The overall process achieves a long-lasting sustained release of 60-90 days.

[0011] In one specific implementation, in step S1, the mineral carrier is composed of the following raw materials in parts by weight: 25%-28% bentonite, 20%-22% diatomite, 15%-17% zeolite powder, 7%-9% calcium carbonate, and the balance being an inert mineral filler.

[0012] By adopting the above technical solution, a specific ratio of bentonite, diatomite, zeolite powder and calcium carbonate is selected and compounded. The aim is to use the high porosity of diatomite and zeolite powder to provide microbial attachment sites, use the adhesiveness and water absorption and swelling properties of bentonite to enhance the molding effect, and use calcium carbonate to provide pH buffering.

[0013] In one specific implementation scheme, the mineral carrier mixing and grinding specifically involves: mixing and stirring all the mineral carrier raw materials for 10-15 minutes, and then grinding them to a particle size of 25-40 μm.

[0014] By adopting the above technical solution, strictly controlling the particle size to ≤40μm (passing through a 325-mesh sieve) is the key process parameter. Fine particles can significantly increase the specific surface area, which is conducive to the formation of a dense micro-network with the binder in the subsequent process, preventing structural defects and stress concentration caused by large particles, thereby improving the mechanical strength of the finished product.

[0015] In one specific implementation scheme, in step S1, the PVA solution is prepared by: maintaining the temperature of deionized water at 75-85°C, then adding polyvinyl alcohol to the deionized water, wherein the solid-liquid ratio of polyvinyl alcohol to deionized water is 1:10-17, stirring for 30-35 minutes, and cooling to 25-30°C after dissolution to obtain the PVA solution.

[0016] By adopting the above technical solution, cooling the PVA solution to 25-30℃ is a key measure to prevent high-temperature inactivation of subsequently added microorganisms.

[0017] In one specific implementation scheme, the composite adhesive is prepared by sequentially adding sodium carboxymethyl cellulose solution, gelatinized starch, and sodium alginate solution, stirring for 15-20 minutes, and then adding glycerin to form a paste-like adhesive. The mass ratio of each component in the composite adhesive is as follows: 33%-39% sodium carboxymethyl cellulose solution, 27%-33% gelatinized starch, 26%-32% sodium alginate solution, and 1.5%-2.5% glycerin, and the sum of the mass fractions of each component is 100%.

[0018] By adopting the above technical solution, the composite adhesive uses a multi-component system of "CMC + gelatinized starch + sodium alginate + glycerin": CMC provides thickening and water retention; gelatinized starch (gelatinized at 85℃ for 40 minutes) enhances adhesion and serves as a carbon source; sodium alginate is the matrix for subsequent calcium ion cross-linking reaction; and glycerin acts as a plasticizer to regulate extensibility. This "paste-like" form (with a flow similar to yogurt) ensures that it can quickly penetrate into the micropores of the mineral carrier.

[0019] In one specific implementation scheme, in step S2, the preparation of the dough-like material is divided into three stages: basic dough preparation, binder fusion, and microbial encapsulation. In the basic dough preparation stage, PVA solution is added in 3-5 batches, with each batch kneaded for 3-5 minutes, and the kneading temperature is controlled at 25-30℃ and the ambient humidity at 50-60%. In the binder fusion stage, after adding the composite binder, the dough is kneaded for 8-10 minutes using a "press-fold" method, and the dough moisture content is controlled at 38-40%.

[0020] By adopting the above technical solutions, the "phased addition" and "press-fold" techniques originate from improvements in pasta processing technology. The aim is to form a gluten-like network structure, allowing inorganic mineral powders to form a continuous phase under the connection of organic polymers. Controlling the moisture content at 38-40% is the critical point of the process. At this point, the dough is in a state where it is "not sticky to the touch and does not deform when squeezed," ensuring plasticity during shaping while avoiding shrinkage and cracking during drying due to excessive moisture or insufficient compaction due to insufficient moisture.

[0021] In one specific implementation, during the microbial colony encapsulation stage, the number of viable bacteria pre-cooled to 4°C is ≥5 × 10⁻⁶. 9 The bacterial suspension (CFU / mL) is added to the dough after it has been blended with the binder in 5-7 portions. Each addition is followed by a "spiral kneading" method. The total amount of bacterial suspension added is 15% of the total dry mass of the dough. The kneading time is controlled within 25 minutes.

[0022] By employing the above technical solution, pre-cooling the bacterial solution to 4°C is intended to induce a dormant state in the bacteria, reducing their metabolic level and thus increasing their tolerance to mechanical shear forces. The "spiral kneading" method is gentler than vigorous mixing, effectively dispersing the bacteria evenly deep within the carrier pores without damaging the bacterial cell walls. Strictly limiting the kneading time to within 25 minutes minimizes the time the bacteria are exposed to unsuitable environments, preventing frictional heat from causing localized temperature increases that could kill the bacteria.

[0023] In one specific implementation scheme, in S3, the double-layer nested mold includes an outer mold and an inner mold; the molding operation is as follows: first, 50-60% of the dough-like material is filled into the outer mold, and 0.3-0.5MPa pressure is applied to compact it to form a dense outer layer; then, the remaining dough-like material is filled into the inner mold, and 0.1-0.15MPa pressure is applied to compact it to form a loose central layer.

[0024] By employing the above technical solution and controlling the pressure differentially at 0.3-0.5 MPa (high pressure) and 0.1-0.15 MPa (low pressure), a density gradient structure with a dense outer layer and a loose inner layer was constructed. The outer dense layer has low porosity and high water flow resistance, primarily serving as a protective framework and controlling initial release to prevent the bacterial agent blocks from dispersing upon entering the water. The inner loose layer has high porosity, which facilitates the proliferation of internal bacterial communities and the excretion of metabolic products. This structural design simulates the structure of soil aggregates in nature and directly determines the length of the slow-release period. Insufficient pressure in the outer layer will lead to an excessively high initial release.

[0025] In one specific implementation scheme, in step S4, the concentration of the calcium chloride solution is 1-2%, the liquid-solid ratio of the calcium chloride solution and the gradient structure bacterial agent block is 3:1, and the solution is soaked at 25-30℃ for 30-40 minutes, turning it 2-3 times during the soaking period. The soaking is ended when the pH value of the system is stabilized in the range of 7.3-7.7.

[0026] By adopting the above technical solution, the -COO in sodium alginate is utilized - Group and Ca 2+ A cross-linking reaction occurs, forming an "egg-box" gel structure. This reaction generates a tough, semi-permeable membrane on the surface of the bacterial agent block. This membrane, like skin, protects the internal structure and strictly controls the permeation rate of water and nutrients. The reaction endpoint is determined by ensuring that the cross-linking reaction is complete and that excessive soaking does not damage the bacterial cells or cause structural brittleness.

[0027] In one specific implementation scheme, in step S5, the room temperature pre-curing conditions are: placing in static air at a temperature of 23-27℃ and a relative humidity of 55-60% for 6-7 hours; the low temperature activation conditions are: a temperature of 29-31℃, introducing humidified air containing 5-6% CO2 at a flow rate of 0.5-0.6L / min, and processing for 12-14 hours.

[0028] By adopting the above technical solution, the pre-curing stage uses "static air" to slowly evaporate moisture, avoiding the capillary tension difference caused by inconsistent drying rates inside and outside, thus preventing surface cracking. The low-temperature activation stage introduces 5% CO2 to simulate the natural growth environment of nitrifying bacteria (autotrophic bacteria), providing them with an inorganic carbon source and promoting the "colonization" and recovery of the bacterial community within the micropores of the carrier, enabling them to maintain a very high survival rate after drying (≥1×10⁻⁶ on the carrier surface). 9 CFU / g).

[0029] In summary, this application includes the following beneficial technical effects:

[0030] 1. A sustained-release cycle of 60-90 days is achieved through a gradient structure of "dense on the outside and sparse on the inside" and a semi-permeable membrane formed by surface calcium ion cross-linking.

[0031] 2. The entire process was carried out at low temperatures (PVA cooling, 4℃ bacterial solution, low-temperature activation) and activated with CO2-containing humidified air, ensuring that the number of viable bacteria on the carrier surface was ≥1×10⁻⁶. 9 CFU / g;

[0032] 3. The composite adhesive and gradient compaction process endow the product with excellent mechanical strength, with a surface compressive strength ≥2.0MPa. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0034] The following disclosure provides numerous different embodiments or examples for implementing various structures of this application. To simplify the disclosure, specific examples of components and arrangements are described below. These are merely examples and are not intended to limit the scope of this application. Furthermore, reference numerals and / or letters may be repeated in different examples. Such repetition is for simplification and clarity and does not in itself indicate a relationship between the various embodiments and / or arrangements discussed.

[0035] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0036] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of the application. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0037] It should also be further understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0038] As used in this specification and the appended claims, the term "if" may be interpreted, depending on the context, as "when," "once," "in response to determination," or "in response to detection." Similarly, the phrases "if determined" or "if [described condition or event] is detected" may be interpreted, depending on the context, as "once determined," "in response to determination," "once [described condition or event] is detected," or "in response to detection of [described condition or event]."

[0039] The present application will be further described in detail below with reference to the embodiments.

[0040] This application addresses the industry pain points of existing nitrifying bacteria agents in water treatment applications, namely "excessively rapid initial release (burst release)," "weak release in the later stage," and "inactivation of bacterial communities due to the molding process," and proposes a method for preparing nitrifying bacteria agent blocks.

[0041] Its core technological principle lies in constructing a three-tiered sustained-release barrier and a full-process activity protection system:

[0042] The first level of barrier (physical gradient): Through a double-layered nested mold and differentiated pressure (0.3MPa vs 0.1MPa), a heterogeneous structure with a "dense outer layer and loose inner layer" is constructed inside the bacterial agent block. The outer dense zone has low porosity, significantly increasing the mass transfer resistance for water molecule entry and nutrient dissolution, mainly serving as mechanical support and initial controlled release; the central loose zone retains high porosity, providing ample oxygen transport channels and proliferation space for aerobic nitrifying bacteria, ensuring continuous release in the middle and later stages.

[0043] The second level of barrier (chemical cross-linking): Utilizing the ion exchange reaction between sodium alginate and calcium ions, a dense "egg-box" semi-permeable gel membrane is formed on the outermost layer of the bacterial agent block. This membrane layer has selective permeability, effectively locking in large molecular nutrients and bacterial flocs while allowing the permeation of water molecules and small molecular matrices (such as ammonia nitrogen).

[0044] The third level of barrier (matrix network): an interpenetrating network structure formed by PVA, gelatinized starch and mineral powder is used to physically anchor the bacteria in a micron-sized inorganic framework, preventing them from being quickly washed away by water flow.

[0045] Active protection system: The "low temperature operation" strategy (PVA cooling, bacterial pre-cooling, low temperature activation) throughout the entire process and the CO-assisted colonization technology that simulates the natural environment solve the problem of large-scale death of microbial communities caused by traditional hot pressing or sintering processes.

[0046] In this embodiment, the mineral carrier is not only a habitat for microorganisms, but also the skeleton that constitutes the mechanical strength of the bacterial agent block.

[0047] Bentonite (25%-28%, preferably 25%): Sodium-based bentonite is selected, taking advantage of its high expansion ratio and cation exchange capacity (CEC). Upon contact with water, the bentonite layers absorb water and swell, which can block some of the excessively large pores and prevent the bacterial agent block from disintegrating. At the same time, its interlayer charge helps to adsorb ammonia nitrogen and increase the local concentration of the substrate.

[0048] Diatomaceous earth (20%-22%, preferably 20%): Calcinated diatomaceous earth is selected, whose main component is amorphous silica. It has a unique microporous structure (pore size 0.1-1μm). This pore size range is highly matched with the size of nitrifying bacteria, providing the bacteria with a "micro-refuge" to protect them from predation by protozoa.

[0049] Zeolite powder (15%-17%, preferably 15%): Clinoptilolite is selected, which uses the cavities in its skeleton structure to screen molecules and has a strong selective adsorption capacity for ammonia nitrogen. It can create a "nitrogen-rich environment" around the bacteria and remove the limitation of low substrate concentration on the nitrification reaction.

[0050] Calcium carbonate (7%-9%, preferably 7%): serves as a pH buffer and inorganic carbon source. Nitrification consumes alkalinity and produces hydrogen ions, leading to microenvironment acidification and inhibiting bacterial activity. The slow dissolution of calcium carbonate can sustainably neutralize acidity, maintaining the microenvironment pH within the optimal range of 7.5-8.0.

[0051] Detailed explanation of pretreatment process parameters: In order to ensure that the above four mineral powders of different densities are mixed evenly and form a dense packing, a "premixing + fine grinding" process must be carried out.

[0052] Mixing: Use a three-dimensional motion mixer, set the speed to 150 rpm, and run for 10-15 minutes. The multi-directional motion of the three-dimensional mixer effectively eliminates dead zones and prevents the heavier calcium carbonate from settling to the bottom.

[0053] Grinding: The mixture is fed into a colloid mill, and the grinding disc gap is adjusted to 0.05 mm. The purpose of grinding is to break up the agglomerates of mineral particles and force mechanochemical activation to increase the surface energy of the particles.

[0054] Screening: The ground material must pass through a 325-mesh sieve (approximately 45μm aperture). Strictly controlling the particle size to ≤40μm is crucial. If the particles are too large, macroscopic defects will exist inside the formed bacterial agent blocks, becoming a breakthrough point for water erosion and causing structural collapse.

[0055] PVA solution (polyvinyl alcohol):

[0056] Specifications: High degree of polymerization PVA with a degree of alcoholysis of 99% (such as type 1799) is selected to ensure water resistance after film formation.

[0057] Preparation: Add 6% (by weight) PVA to deionized water (solid-liquid ratio 1:10-17). Due to the difficulty in dissolving highly polymerized PVA, it needs to be stirred in a constant temperature water bath at 75-85℃ (optimal 80℃) for 30-35 minutes (optimal 30 minutes) until completely transparent.

[0058] Critical control point: After dissolving, the solution must be cooled to below 30°C (25-30°C) before use. This is because the subsequent kneading step will directly introduce the bacteria, and if the temperature is above 40°C, the enzymes of nitrifying bacteria (especially Nitrosomonas) will denature and become inactive.

[0059] Composite adhesive (slurry):

[0060] 33%-39% CMC (sodium carboxymethyl cellulose): 6% solution. CMC acts as a thickener, providing wet binding force and giving the dough good water retention.

[0061] 27%-33% gelatinized starch: made from 10% corn starch and 20% water. It must be gelatinized at 85°C for 40 minutes. At this point, the starch granules completely break down, amylose dissolves, and a high-viscosity, translucent paste is formed. Incompletely gelatinized starch has poor binding force and is easily consumed by bacteria, leading to structural collapse.

[0062] 26%-32% sodium alginate: 4% solution. As a backbone molecule for chemical cross-linking, the content of its G block directly affects the gel strength.

[0063] Glycerin: 1.5%-2.5%. As a plasticizer, it inserts between polymer chains to increase free volume and prevent the bacterial agent block from becoming brittle due to excessive shrinkage stress during the drying process.

[0064] Mixing process: CMC solution, gelatinized starch, and sodium alginate solution are added sequentially to a stainless steel tank. After stirring for 15 minutes, glycerin is added last. The resulting adhesive is a "yogurt-like" fluid with shear-thinning pseudoplastic fluid characteristics, which is beneficial for pumping and mixing.

[0065] Bacterial composition: a mixed bacterial suspension of Nitrosomonas and Nitrobacterium, in a ratio of approximately 1:1.

[0066] Indicator requirement: viable bacteria count ≥ 5 × 10⁹ CFU / mL.

[0067] Temperature control: The cells must be pre-cooled in a refrigerator at 4°C before use. Low temperature puts the cells into a dormant state, increases cell wall toughness, and minimizes metabolic activity, thereby significantly improving their tolerance to subsequent mechanical kneading and shearing forces.

[0068] The preparation method of this application includes the following five steps, each with key process control parameters.

[0069] S1: Raw material pretreatment and functional modification (basic construction)

[0070] This step is the starting point of quality control, and its main task is to transform raw materials with vastly different physical properties into homogeneous and stable intermediate materials.

[0071] Composite and ultrafine mineral carriers:

[0072] Operating steps: Add bentonite, diatomaceous earth, zeolite powder and calcium carbonate into the three-dimensional mixer.

[0073] Parameter control: Set the rotation speed to 150 rpm and the mixing time to 10-15 minutes. After mixing, the material must be ground using a colloid mill or air jet mill.

[0074] Key indicator: The particle size after grinding must be ≤40μm (passing through a 325-mesh sieve).

[0075] Explanation of the principle: This step is not just about mixing, but also about eliminating stress concentration points that may be caused by large mineral particles. Particle size smaller than 40μm can significantly increase the specific surface area, allowing the organic binder to more fully encapsulate each inorganic particle and prevent structural loosening caused by subsequent "dry mixing".

[0076] Preparation of rheology adhesives:

[0077] PVA solution preparation: Add 6% PVA powder to hot water at 75-85℃ (optimal 80℃) (solid-liquid ratio 1:10-17) and dissolve completely. Then, cool to below 30℃ (25-30℃) before use. Excessive temperature will directly kill any subsequent microorganisms.

[0078] Preparation of composite paste: CMC solution (thickening), gelatinized starch (binding), and sodium alginate solution (cross-linking backbone) are added sequentially to a stainless steel bucket. After stirring for 15 minutes, glycerol is added.

[0079] Explanation of the principle: Gelatinized starch needs to be treated at 85℃ for 40 minutes in advance to completely destroy the starch granules and release the amylopectin; otherwise, it lacks sufficient binding force at room temperature.

[0080] S2: Preparation of dough-like materials (step-by-step kneading process)

[0081] Traditional processes often involve a single mixing step, which can easily lead to localized over-wetting or clumping of dry flour. This application employs a three-step kneading process to simulate the gluten formation process in pasta processing.

[0082] Basic dough preparation (solid-liquid wetting):

[0083] The pretreated mineral carrier is fed into a stainless steel double-helix conical mixer or a special kneading tank.

[0084] The prepared PVA solution is sprayed in 3-5 batches. Each batch is 1 / 3 to 1 / 5 of the total volume. After spraying, the mixture is kneaded at low speed for 3-5 minutes.

[0085] Mechanism: The phased addition of liquid ensures that PVA molecules can be uniformly adsorbed on the surface of each mineral particle, forming a preliminary "organic-inorganic" binding layer, thus avoiding the "dry-on-wet" phenomenon.

[0086] Adhesive fusion (rheological modification):

[0087] Add a composite adhesive (CMC + starch + sodium alginate + glycerin).

[0088] The kneading process uses a mechanical method of "pressing and folding" (simulating hand kneading) and takes 8-10 minutes.

[0089] Endpoint determination: Continue until the material reaches a "three-smooth" state (smooth container walls, smooth mixing paddle, and smooth dough surface). At this point, the dough moisture content is precisely controlled between 38-40%.

[0090] Parameter significance: A moisture content of 38-40% is the rheological critical point. Below 38%, the material is too dry, the compaction density is insufficient, and it is easy to crumble; above 40%, the material has fluidity, cannot maintain the gradient structure, and the porosity is too large after drying.

[0091] Microbial encapsulation (bioaugmentation):

[0092] Add the bacterial suspension pre-cooled to 4°C. The total amount of bacterial suspension should be 15% of the dry mass (referring to the solid components in the mineral carrier + composite binder, such as CMC, corn starch, sodium alginate, PVA, etc.).

[0093] Adding method: Do not pour all at once. Add in 5-7 portions, mixing with a "spiral kneading" method after each addition.

[0094] Time control: The total operation time for this stage is strictly limited to 25 minutes (including liquid addition and mixing time).

[0095] Explanation of the reason: Nitrifying bacteria are sensitive to shear forces, and prolonged exposure to non-aqueous environments can lead to cell dehydration. The shear heat generated by spiral mixing is low, and combined with the low temperature of the bacterial solution, the temperature rise during the mixing process can be controlled within 2°C.

[0096] S3: Gradient compaction mold forming (structural construction)

[0097] This is the most critical step in achieving long-term sustained release in this application.

[0098] Mold Equipment:

[0099] It uses a custom-designed double-layer nested silicone mold.

[0100] Outer mold: inner diameter 80mm, height 50mm.

[0101] Inner mold: inner diameter 40mm, height 45mm.

[0102] The guide channel design: The inner wall of the mold is engraved with a spiral groove that is 2mm wide and 1mm deep, which forms a guide channel on the surface of the bacterial agent block after demolding.

[0103] Operating procedures (secondary packing method):

[0104] Step 1 (Outer Wall Construction): Weigh 50-60% of the total dough material and fill it into the outer mold (i.e., the annular space or bottom of the mold should be filled first). Apply pressure of 0.3-0.5 MPa using a pneumatic pressure rod with a pressure sensor or a screw-on mold cap.

[0105] Effect: Under this pressure, mineral particles are tightly packed together, and the porosity is compressed to a low level (<30%), forming a dense protective layer.

[0106] Step 2 (Core Filling): Fill the central cavity with the remaining 40-50% of the material. Apply a pressure of 0.1-0.15 MPa.

[0107] Effect: Low pressure allows the central area to retain a high porosity (>50%), forming a sponge-like structure that is conducive to the habitat of microorganisms.

[0108] Step 3 (Stationary Demolding): Maintain pressure and let stand for 5 minutes to eliminate elastic aftereffects, then demold. The resulting bacterial agent block is a single unit, but its internal density is distributed in a stepped pattern.

[0109] S4: Calcium ion crosslinking curing (surface sealing)

[0110] Although the bacterial culture blocks are formed after demolding, they are still prone to disintegration when exposed to water (both PVA and starch are water-soluble). Chemical curing is required.

[0111] Reaction system: 1% anhydrous calcium chloride (CaCl) aqueous solution.

[0112] Operating conditions: Completely immerse the inoculum block, liquid-to-solid ratio 3:1, temperature 25℃.

[0113] Reaction process:

[0114] guluronic acid (G unit) in sodium alginate reacts with Ca in the solution 2+ A chelation reaction occurs. Calcium ions bind between the polymer chains, forming an "eggbox" structure.

[0115] Kinetic control: The reaction penetrates from the surface to the interior. The soaking time is controlled at 30 minutes.

[0116] Endpoint control:

[0117] Touch: The surface of the fungal block changes from sticky and soft to a slightly elastic, rubbery feel.

[0118] pH testing: The pH of the soaking solution remained stable at 7.5 ± 0.2. A significant drop in pH indicates that calcium ions have displaced H+. + Too much pH indicates an overreaction; if the pH remains unchanged, it means the reaction has not proceeded.

[0119] Result: A calcium alginate gel film with a thickness of approximately 0.5 mm was eventually formed on the surface of the bacterial agent block. This film acts as a "valve" to control the sustained-release rate.

[0120] S5: Gradient drying activation (activity recovery)

[0121] Direct high-temperature drying can cause gel membrane rupture and bacterial death, so a two-step gradient drying method must be used.

[0122] Phase 1: Room temperature pre-curing (crack prevention):

[0123] Environment: Temperature 25±2℃, relative humidity 55-60%, still air (no wind).

[0124] Time: 6-7 hours.

[0125] Objective: To remove surface free water extremely slowly using a concentration gradient. If ventilation or heating is applied at this time, the surface moisture will evaporate too quickly, and the internal moisture will not have enough time to diffuse, resulting in huge capillary tension and causing surface cracking.

[0126] Second stage: Low temperature activation (colonization):

[0127] Environment: Temperature 30±1℃ (optimal growth temperature for nitrifying bacteria).

[0128] Atmosphere: Introduce humidified air containing 5-6% CO at a flow rate of 0.5-0.6 L / min.

[0129] Time: 12-14 hours.

[0130] Principle: During the drying process, the bacteria are gradually concentrated deep within the mineral pores. CO, as an inorganic carbon source, stimulates autotrophic nitrifying bacteria to undergo weak metabolism, causing them to firmly adsorb (colonize) onto the carrier wall. Humid air prevents excessive drying, ultimately controlling the moisture content at 25-28%.

[0131] Example 1:

[0132] Raw material ratio: Bentonite 250g, diatomaceous earth 200g, zeolite powder 150g, calcium carbonate 70g, kaolin 165g, talc powder 165g. PVA solution (6% concentration) 329g. Composite binder (containing 35% CMC solution, 33% gelatinized corn starch, 30% sodium alginate solution, 2% glycerol) 141g. Bacterial suspension (5×10⁻⁶) 9 188g (CFU / mL)

[0133] Mixing: The mineral powder was mixed in a three-dimensional mixer at 150 rpm for 10 minutes and then ground through a colloid mill through a 325-mesh sieve.

[0134] Kneading: At room temperature (25℃), first add PVA solution and knead for 9 minutes (in 3 batches); then add composite binder, press, fold, and knead for 9 minutes; finally, add pre-cooled bacterial solution in 5 batches and knead in a spiral motion for 20 minutes. At this point, the dough moisture content is measured to be 39%.

[0135] Molding: A double-layer mold is used. The outer layer contains 150g of material, compacted at 0.3MPa; the inner layer contains 100g of material, compacted at 0.1MPa. Demolding yields an 80mm cylinder.

[0136] Curing: Immerse in 1% CaCl2 solution for 30 minutes to form a gel layer on the surface.

[0137] Activation: Let stand at 25°C for 6 hours, then activate at 30°C by passing a 5% CO2 gas stream for 12 hours.

[0138] Product performance test results:

[0139] Appearance: Smooth surface, no cracks, obvious spiral guide grooves, and elastic when squeezed by hand.

[0140] Viable bacteria count (plate count method): 1.2 × 10⁻⁵ bacteria in the surface layer of the carrier (0-5 mm depth). 9 CFU / g; central region: 6.5 × 10⁻⁶ 8 CFU / g.

[0141] Slow-release performance (dynamic leaching method): In flowing water at 25℃, the cumulative mass loss rate is 35% on day 30, 62% on day 60, and 88% on day 90.

[0142] Mechanical strength: Surface compressive strength 2.2MPa, central compressive strength 1.1MPa.

[0143] Example 2:

[0144] To address the cracking issue that may occur in production environments with low humidity (e.g., <40%), the formula was fine-tuned.

[0145] Adjustment points: Increase the PVA solution concentration to 7% and extend the room temperature pre-curing time to 8 hours. The remaining steps are the same as in Example 1.

[0146] Principle: Increasing the PVA concentration enhances the tensile strength of the polymer network, resisting drying shrinkage stress; extending the pre-curing time further reduces the steepness of the moisture evaporation gradient.

[0147] Test results: The finished product had no cracks and its mechanical strength was increased to 2.5MPa, but the initial release rate decreased slightly (30% release rate after 30 days), making it suitable for applications with rapid water flow.

[0148] Example 3:

[0149] For scenarios requiring rapid establishment of a nitration system, the solidification process is adjusted.

[0150] Adjustment points: shorten the calcium chloride soaking time to 15 minutes and adjust the outer mold pressure to 0.25 MPa.

[0151] Principle: Shortening the soaking time makes the surface gel film thinner (about 0.2 mm), reducing mass transfer resistance; reducing the outer layer pressure increases porosity.

[0152] Test results: 30-day release rate increased to 55%, 60-day release rate to 85%. Suitable for emergency water treatment.

[0153] To verify the effects of the "gradient structure" and "calcium ion crosslinking" in this application, the following comparative examples were set up.

[0154] Comparative Example 1:

[0155] Preparation difference: All materials are filled into the mold at one time and compacted by applying an average pressure of 0.2 MPa. The rest of the formula and post-processing steps are exactly the same as in Example 1.

[0156] Test results:

[0157] Initial release: The release rate is extremely high during days 1-10 (explosive release), which causes the water to become turbid.

[0158] Late release: By day 60, the inside of the bacterial agent block is basically hollowed out or collapsed, and it loses its function.

[0159] Conclusion: This study demonstrates that a gradient structure with a "dense outer layer and sparse inner layer" is key to achieving linear and stable sustained release.

[0160] Comparative Example 2:

[0161] Preparation difference: After molding, it does not undergo CaCl2 soaking and directly enters the drying process.

[0162] Test results:

[0163] Water resistance: After being immersed in water, the PVA and starch on the surface quickly absorb water and swell. Within 24 hours, the surface of the bacterial agent block begins to gelatinize and fall off.

[0164] Strength: The surface compressive strength is only 0.8 MPa.

[0165] Conclusion: This study demonstrates that the calcium alginate gel membrane plays a decisive role in maintaining the structural integrity of the bacterial agent block in water.

[0166] Comparative Example 3:

[0167] Preparation difference: The activation stage does not use a low-temperature CO2 process, but is directly placed in a 45℃ oven for rapid drying.

[0168] Test results:

[0169] Viable bacteria count: The number of viable bacteria on the surface of the carrier decreased sharply to 1×10⁻⁶. 6 CFU / g (decreased by 3 orders of magnitude).

[0170] Appearance: Obvious cracks appear on the surface.

[0171] Conclusion: This demonstrates the necessity of the "low temperature + CO2" activation process proposed in this application for protecting the activity of heat-sensitive nitrifying bacteria.

[0172] All data cited in the foregoing embodiments were obtained based on the following standard testing methods.

[0173] viable bacteria count detection:

[0174] Plate counting was used. 1g of bacterial agent block sample was ground, serially diluted, and spread onto PCA agar plates. The plates were incubated at 30℃ for 48 hours before counting.

[0175] Differential detection: Samples were taken from the surface (outer 5mm) and the center (core 10mm) to verify the distribution of bacteria in a gradient environment.

[0176] Sustained-release performance (cumulative release rate):

[0177] The dynamic leaching method was adopted. The weighed bacterial agent blocks were placed in a circulating water tank at 25℃, and the water flow rate was controlled at 0.1m / s (simulating general filtration water flow). Every 10 days, the bacterial agent blocks were removed, dried at 105℃ to constant weight, and the weight loss rate was calculated.

[0178] Calculation formula: R t =W0-W t / W0×100%, where W0 is the initial dry weight, W t Let t be the dry weight.

[0179] Mechanical strength (compressive strength):

[0180] Use a universal testing machine. Place the inoculum block between the pressure plates, apply a loading rate of 1 mm / min, and record the maximum load at the point of rupture.

[0181] Surface strength: Apply pressure directly to the intact fungal block.

[0182] Center strength: Cut out 1cm from the center 3 The cube is being tested.

[0183] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0184] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Since these modifications and variations fall within the scope of the claims and their equivalents, this application also intends to include these modifications and variations.

[0185] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing a nitrifying bacteria inoculant block, characterized in that, Includes the following steps: S1: Raw material pretreatment: Mix and grind the mineral carrier to the specified particle size to prepare PVA solution and composite adhesive respectively; S2: Preparation of dough-like material: The pretreated mineral carrier is mixed and kneaded with PVA solution, then a composite adhesive is added and kneaded again, and finally a bacterial suspension is added for encapsulation to obtain dough-like material; S3: Gradient compaction mold forming: The dough-like material is filled into a double-layer nested mold in batches, and different pressures are applied to form a gradient structure of bacterial agent blocks with a dense outer layer and a loose inner layer. S4: Calcium ion cross-linking and curing: The molded bacterial agent block is immersed in calcium chloride solution to carry out the cross-linking reaction; S5: Gradient drying and activation: The cured microbial agent blocks are sequentially pre-cured at room temperature and activated at low temperature to obtain the finished product.

2. The method for preparing nitrifying bacteria inoculant blocks as described in claim 1, characterized in that, In step S1, the mineral carrier is composed of the following raw materials in parts by weight: 25%-28% bentonite, 20%-22% diatomite, 15%-17% zeolite powder, 7%-9% calcium carbonate, and the balance being an inert mineral filler.

3. The method for preparing nitrifying bacteria inoculant blocks as described in claim 1, characterized in that, The specific process of mixing and grinding the mineral carrier is as follows: all the mineral carrier raw materials are mixed and stirred for 10-15 minutes, and then ground to a particle size of 25-40μm.

4. The method for preparing nitrifying bacteria inoculant blocks as described in claim 3, characterized in that, In step S1, the PVA solution is prepared by maintaining the temperature of deionized water at 75-85℃, then adding polyvinyl alcohol to the deionized water, wherein the solid-liquid ratio of polyvinyl alcohol to deionized water is 1:10-17, stirring for 30-35 minutes, and cooling to 25-30℃ after dissolution to obtain the PVA solution.

5. The method for preparing nitrifying bacteria inoculant blocks as described in claim 1, characterized in that, The composite adhesive is prepared by sequentially adding sodium carboxymethyl cellulose solution, gelatinized starch, and sodium alginate solution, stirring for 15-20 minutes, and then adding glycerin to form a paste-like adhesive. The mass ratio of each component in the composite adhesive is as follows: 33%-39% sodium carboxymethyl cellulose solution, 27%-33% gelatinized starch, 26%-32% sodium alginate solution, and 1.5%-2.5% glycerin, and the sum of the mass fractions of each component is 100%.

6. The method for preparing nitrifying bacteria inoculant blocks as described in claim 5, characterized in that, In step S2, the preparation of the dough-like material is divided into three stages: basic dough preparation, binder fusion, and microbial encapsulation. In the basic dough preparation stage, PVA solution is added in 3-5 batches, with each batch kneaded for 3-5 minutes, and the kneading temperature is controlled at 25-30℃ and the ambient humidity at 50-60%. In the binder fusion stage, after adding the composite binder, the dough is kneaded for 8-10 minutes using a "press-fold" method, and the moisture content of the dough is controlled at 38-40%.

7. The method for preparing nitrifying bacteria inoculant blocks as described in claim 1, characterized in that, During the bacterial colony encapsulation stage, the number of viable bacteria pre-cooled to 4°C is ≥5×10⁻⁶. 9 The bacterial suspension (CFU / mL) is added to the dough after it has been blended with the binder in 5-7 portions. Each addition is followed by a "spiral kneading" method. The total amount of bacterial suspension added is 15% of the total dry mass of the dough. The kneading time is controlled within 25 minutes.

8. The method for preparing nitrifying bacteria inoculant blocks as described in claim 7, characterized in that, In S3, the double-layer nested mold includes an outer mold and an inner mold; the molding operation is as follows: first, 50-60% of the dough-like material is filled into the outer mold, and 0.3-0.5MPa pressure is applied to compact it to form a dense outer layer; then, the remaining dough-like material is filled into the inner mold, and 0.1-0.15MPa pressure is applied to compact it to form a loose central layer.

9. The method for preparing nitrifying bacteria inoculant blocks as described in claim 1, characterized in that, In step S4, the concentration of the calcium chloride solution is 1-2%, and the liquid-solid ratio of the calcium chloride solution and the gradient structure bacterial agent block is 3:

1. The solution is soaked at 25-30℃ for 30-40 minutes, turning it 2-3 times during the soaking period. The soaking is ended when the pH value of the system is stabilized in the range of 7.3-7.

7.

10. The method for preparing nitrifying bacteria inoculant blocks as described in claim 1, characterized in that, In step S5, the room temperature pre-curing conditions are: placing in static air at a temperature of 23-27℃ and a relative humidity of 55-60% for 6-7 hours; the low temperature activation conditions are: a temperature of 29-31℃, passing in humid air containing 5-6% CO2 at a flow rate of 0.5-0.6L / min, and processing for 12-14 hours.