A method for quantitatively adding multi-strain-volcanic rock carrier compound and application thereof

Abstract

This invention belongs to the field of aquaculture wastewater treatment technology, specifically relating to a quantitative addition of a multi-strain volcanic rock carrier composite, its preparation method, and its application. The floating volcanic rock of this invention can be stably suspended in the upper high-oxygen zone, suitable for aerobic nitrifying strains such as *Nitrobacterium velutipes* and *Nitrosomonas cerevisiae*; the suspended volcanic rock can remain in the middle medium-oxygen zone, carrying facultative strains such as *Pseudomonas fluorescens*; and the submerged volcanic rock can sink to the bottom low-oxygen / anaerobic zone, fixing denitrifying strains such as *Rhodopseudomonas palustris*. A vertical space of at least 2 meters in water depth is sufficient to construct a clear oxygen gradient, meeting the metabolic needs of different strains, thus achieving compatibility between the carrier and water depth and oxygen layer. This invention can precisely control the dissolved oxygen concentration in each layer, effectively avoiding oxygen layer homogenization caused by single aeration, helping to ensure the synergistic effect of aerobic, facultative, and anaerobic strains, and adapting to the growth needs of various functional bacterial communities.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture wastewater treatment technology, specifically relating to a quantitative addition of a multi-strain-volcanic rock carrier complex, its preparation method, and its application. Background Technology

[0002] With the rapid development of intensive aquaculture, the large amounts of high-concentration organic wastewater generated during the aquaculture process have become a key bottleneck restricting the industry's green and sustainable development. This type of wastewater typically contains high concentrations of ammonia nitrogen, nitrite, nitrate, phosphate, and high concentrations of chemical oxygen demand (COD) substances formed from a large amount of residual feed and excrement from aquaculture organisms. Its composition is complex and its pollution load fluctuates greatly. Direct discharge without effective treatment can easily lead to eutrophication of receiving water bodies, causing red tides, water quality deterioration, and a series of other environmental problems. Therefore, finding an efficient, stable, and cost-effective purification technology has become an urgent need for the industry.

[0003] Currently, the industry has explored various technological approaches for the purification and treatment of aquaculture wastewater. Physicochemical methods, such as flocculation sedimentation, adsorption, and advanced oxidation, while rapid and able to quickly remove some pollutants, often suffer from high treatment costs, potential secondary pollution, and incomplete removal of dissolved nutrients. These methods are ill-suited to the long-term treatment needs of continuously generated wastewater from aquaculture farms and are more appropriate for emergency or intensive treatment in specific situations. In contrast, biological purification technology, due to its environmental friendliness, relatively low treatment costs, and ability to completely transform pollutants, is considered a more promising mainstream approach. One common practice is to directly add microbial agents with specific degradation functions to the water body, such as adding nitrifying bacteria to reduce ammonia nitrogen or denitrifying bacteria to reduce nitrates. However, this "free-state" addition method has increasingly revealed limitations in practical applications: the bacteria have poor dispersibility in water, are easily lost with water flow, and it is difficult to maintain a high biological concentration, resulting in unstable treatment effects and short durations of action. Repeated additions are often necessary to maintain the effect, and long-term operating costs are not low.

[0004] To overcome the shortcomings of free microbial agents, microbial immobilization technology has emerged. This involves loading functional microorganisms onto specific carrier materials using physical or chemical methods to improve cell retention, enhance their resilience, and facilitate recovery. Porous materials, such as activated carbon, ceramsite, and polymer gels, have been extensively studied as carriers. However, existing immobilization technology research often focuses on immobilizing single species or simple bacterial communities on single types of carriers, and their treatment objectives are often relatively singular. When dealing with complex systems like aquaculture wastewater that require simultaneous nitrogen and phosphorus removal as well as organic matter removal, simply mixing and immobilizing various microorganisms with different functions (such as aerobic nitrifying bacteria, facultative and anaerobic denitrifying bacteria, and phosphorus-removing bacteria) on the same carrier can lead to new challenges. These microorganisms have drastically different requirements for dissolved oxygen and nutrients. Confining them to the same microenvironment leads to ineffective competition for oxygen and substrate among the bacterial communities, ultimately inhibiting the function of some species (especially denitrifying bacteria and certain phosphorus-removing bacteria that are extremely sensitive to oxygen concentration), failing to achieve the theoretical synergistic effect, and causing the overall purification efficiency to encounter a bottleneck.

[0005] Furthermore, even with a wide range of functional compound microbial agents, how to efficiently apply them in actual aquaculture water bodies—open, complex, and often static or micro-flowing systems—remains a huge challenge.

[0006] Traditional aeration or mixing methods typically aim to achieve uniform mixing and maintain a high dissolved oxygen level throughout the water body. While this benefits aerobic processes, it completely inhibits anaerobic / anoxic processes such as denitrification, causing the nitrogen removal process to be interrupted at the nitrate stage. Reducing or stopping aeration to create an anoxic environment, on the other hand, hinders ammonia oxidation, creating a dilemma for the entire system. Therefore, within a treatment unit, how to simultaneously create a suitable microenvironment for the efficient metabolism of microbial communities with different physiological characteristics, especially a stable and controllable dissolved oxygen gradient, is a deep-seated problem that current technologies have generally failed to systematically solve. Many studies remain focused on the control of small-scale test water bodies under ideal laboratory conditions. Once scaled up to actual aquaculture ponds or solidification tanks, the greater depth and poorer flow of the water make it difficult to form and maintain an ideal dissolved oxygen stratification structure. This prevents functional microorganisms from functioning properly, significantly reducing treatment effectiveness and compromising the replicability and stability of the technology. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of existing technologies in the biological purification of aquatic wastewater, such as the easy dispersion of bacterial strains in the aquatic environment, resulting in unstable treatment effects, difficulty in fixing various functional bacterial strains on the same carrier, difficulty in engineering application to actual aquatic wastewater processes, and difficulty in achieving efficient, stable, and simultaneous removal of multiple pollutants. The invention provides a method for quantitatively adding a multi-strain-volcanic rock carrier complex and its preparation, and applies it to the biological purification of aquatic wastewater.

[0008] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: A method for quantitatively adding a multi-strain-volcanic rock carrier complex, wherein the multi-strain-volcanic rock carrier complex comprises a mixed bacteria and a carrier, wherein the mixed bacteria include at least two functional bacteria among nitrifying bacteria, denitrifying bacteria, organic matter degrading bacteria, and phosphorus-removing bacteria; the carrier is volcanic rock particles; and the total viable count of the multi-strain-volcanic rock carrier complex is ≥1×10⁻⁶. 11 CFU / g.

[0009] This invention selects functional microbial communities covering key biochemical pathways from ammonia oxidation to nitrate reduction, from organic matter decomposition to phosphorus absorption and transformation, as a mixed microbial population. These are then immobilized on volcanic rock granular carriers. This process helps extend the residence time of the functional microbial communities in the water, effectively preventing their rapid loss with the water flow and providing a stable and durable "microreactor" for microbial colonization and functional expression. Simultaneously, the physical protection provided by the volcanic rock granular carrier enhances the community's tolerance to external environmental fluctuations. The diverse combination of functional microbial communities helps to promote the simultaneous and parallel degradation or transformation of major pollutants in wastewater, such as ammonia nitrogen, nitrite, nitrate, organic pollutants, and phosphate. Furthermore, this invention achieves a total viable count ≥1×10⁻⁶ by quantitatively adding and limiting the total viable count of the multi-strain-volcanic rock carrier complex. 11 CFU / g helps ensure that each addition introduces a sufficiently high concentration and high activity of functional biomass into the system, fundamentally and effectively avoiding the problems of slow start-up and poor effect caused by insufficient biomass or low activity of traditional microbial agents.

[0010] Preferably, the nitrifying bacteria include at least one of nitrite-oxidizing bacteria and nitrifying bacteria; the denitrifying bacteria include at least one of heterotrophic denitrifying bacteria and photosynthetic denitrifying bacteria; the organic matter degrading bacteria include at least one of Bacillus spp. and Pseudomonas spp.; and the phosphorus-removing bacteria include at least one of polyphosphate-accumulating bacteria and denitrifying phosphorus-removing bacteria.

[0011] Preferably, the mixed bacteria include at least three of the following: Nitrobacter winogradskyi, Nitrosomonas europaea, Pseudomonas fluorescens, Alcaligenes faecalis, Bacillus subtilis, Bacillus licheniformis, Paracoccus denitrificans, Rhodopseudomonas palustris, Rhodobacter sphaeroides, Citrobacter koseri, and Lysinibacillus sphaericus.

[0012] The diverse combination of functional microbial communities in mixed bacteria enables the simultaneous and parallel degradation or transformation of major pollutants in wastewater, such as ammonia nitrogen, nitrite, nitrate, organic pollutants, and phosphate, covering the entire chain of pollutant removal and overcoming the limitations of single-functional microbial agents in terms of treatment targets.

[0013] Preferably, the volcanic rock particles include at least one of floating, suspended, and sinking types.

[0014] As a further preferred embodiment, the floating volcanic rock particles have a particle size of 4-6 mm; the suspended volcanic rock particles have a particle size of 6-10 mm; the submerged volcanic rock particles have a particle size of 8-12 mm; and the specific surface area of ​​the volcanic rock particles is 12-18 m². 2 / g, with a porosity of 45~55%.

[0015] The volcanic rock particle carrier material of the present invention can increase air permeability and compatibility, and the surface is easy to form a biofilm, which is beneficial to bacterial attachment and metabolic activities.

[0016] Volcanic rock particles have a naturally porous structure, providing a broad attachment surface and a stable habitat for microorganisms. The characteristics of the raw materials allow for the production of particles with different densities through process adjustments, which in turn allow them to float, suspend, or settle in water, laying the physical foundation for subsequent spatial positioning and deployment in three-dimensional water bodies.

[0017] The method for preparing a quantitatively added multi-strain-volcanic rock carrier complex, as described above, includes the following steps: A mixed bacterial strain containing at least two functional bacteria from nitrifying bacteria, denitrifying bacteria, organic matter degrading bacteria, and phosphorus-removing bacteria was immobilized on a volcanic rock particle carrier to obtain a multi-strain-volcanic rock carrier complex.

[0018] As a further preferred option, the volume-to-mass ratio of the mixed bacterial strain suspension to the volcanic rock particle carrier is 2.5:1 (mL / g).

[0019] The application of a quantitative dosing of multi-strain-volcanic rock carrier complex in the purification of aquaculture wastewater, as described above.

[0020] Preferably, the method of application is as follows: The multi-strain-volcanic rock carrier complex was added to the aquaculture wastewater to be treated in layers according to the differences in dissolved oxygen requirements of the microorganisms therein, and then aerated in layers.

[0021] This invention provides operators with a means for precise spatial control in the vertical direction by leveraging the designability of the carrier (e.g., density) and the functional orientation of the microbial community (e.g., aerobic / anaerobic characteristics). It allows technicians to deploy complexes adapted to aerobic processes in the upper layer and complexes adapted to anoxic / anaerobic processes in the middle and lower layers, based on the natural or artificially formed gradient of dissolved oxygen in the water. This physically isolates parallel aerobic and anoxic / anaerobic zones within the same water body, enabling faster and more synchronized removal of various pollutants from wastewater and significantly improving the system's purification efficiency. Furthermore, because the functional microbial community is fixed and positioned within its suitable survival range, the system's resistance to shock loads and operational stability are significantly enhanced, eliminating the need for frequent addition of microbial agents. Simultaneously, this method reduces reliance on complex mechanical equipment and large-scale civil engineering, relying primarily on the biological processes themselves, making overall operation simpler and long-term operational controllability stronger.

[0022] Preferably, the stratified addition is as follows: a multi-strain-volcanic rock carrier complex adapted to high dissolved oxygen conditions is added to the upper layer of wastewater and fixed; a multi-strain-volcanic rock carrier complex adapted to medium dissolved oxygen conditions is added to the middle layer of wastewater and fixed; and a multi-strain-volcanic rock carrier complex adapted to low dissolved oxygen or anaerobic conditions is added to the bottom layer of wastewater and fixed.

[0023] Preferably, the carrier used in the multi-strain-volcanic rock carrier complex added to the upper layer of sewage is floating volcanic rock particles; the carrier used in the multi-strain-volcanic rock carrier complex added to the middle layer of sewage is suspended volcanic rock particles; and the carrier used in the multi-strain-volcanic rock carrier complex added to the bottom layer of sewage is submerged volcanic rock particles.

[0024] Preferably, the mixed bacteria are divided into an upper high-oxygen group, a middle medium-oxygen group, and a bottom low-oxygen or anaerobic group, and are respectively fixed on volcanic rock particles to form corresponding upper multi-strain-volcanic rock carrier complex, middle multi-strain-volcanic rock carrier complex, and bottom multi-strain-volcanic rock carrier complex.

[0025] As a further preferred option, the upper high-oxygen group contains at least *Nitrobacterium velutipes*, *Nitrosomonas cerevisiae*, *Bacillus subtilis*, and *Bacillus licheniformis*; the middle medium-oxygen group contains at least *Pseudomonas fluorescens*, *Alcaligenes faecalis*, *Bacillus spheroides*, and *Paracococcus denitrificans*; and the bottom low-oxygen or anaerobic group contains at least *Rhodopseudomonas palustris*, *Rhodopseudomonas spheroides*, *Paracococcus denitrificans*, and *Citrobacter korshinskii*.

[0026] As a further optimization, the strains contained in the upper hyperoxia group were inoculated into a nitrifying-Bacillus composite culture medium. Seed cultures of *Nitrobacterium velutipes*, *Nitrosomonas cerevisiae*, *Bacillus subtilis*, and *Bacillus licheniformis* were mixed in a volume ratio of 3:3:2:2, with a total inoculum of 10% (v / v). The temperature was 28–30°C, the dissolved oxygen (DO) concentration was ≥5 mg / L, and continuous aeration was maintained at a rate of 0.8–1.0 L / (L·min). The culture time was 48–72 h. 600 =0.8~1.0, viable count ≥1.2×10 10 CFU / mL.

[0027] As a further optimization, the strains contained in the middle layer of the oxygen-rich medium were inoculated into a facultative denitrification-phosphorus accumulation composite medium. Seed cultures of *Pseudomonas fluorescens*, *Alcaligenes faecalis*, *Bacillus spheroides*, and *Paracoccus denitrifyingus* were mixed at a volume ratio of 3:2:2:1, with a total inoculum of 8% (v / v). The temperature was 30–32°C, the dissolved oxygen (DO) concentration was 0.5–1.0 mg / L, and continuous static aeration was maintained at an aeration rate of 0.3–0.5 L / (L·min). The culture was conducted under low light conditions for 36–48 h. OD... 600 =0.7~0.9, viable count ≥1.2×10 10 CFU / mL.

[0028] As a further optimization, strains contained in the bottom hypoxic or anaerobic group were inoculated into a photosynthetic denitrification-anaerobic phosphorus release composite medium. Seed cultures of *Rhodopseudomonas palustris*, *Rhodophyton floccosum*, *Paradiabetic*, and *Citrobacter korshinskii* were mixed at a volume ratio of 3:3:2:2, with a total inoculum of 12% (v / v). The temperature was 25–28°C, dissolved oxygen (DO) concentration ≤0.5 mg / L, and the medium was sealed and cultured under light (white light source, 20–30 cm away from the culture flask, continuous illumination) for 72–96 hours. 600=0.9~1.1, viable count ≥1.2×10 10 CFU / mL.

[0029] As a further optimization, when the strains contained in the bottom hypoxic or anaerobic group are cultured in the same layer, photosynthetic bacteria are cultured first. After 24 hours of photosynthetic bacteria culture, denitrifying paracocci and Citrobacter korshinskii are then introduced to reduce carbon source competition.

[0030] Preferably, the stratified aeration is performed as follows: the upper layer of wastewater is aerated at a first intensity to maintain a high dissolved oxygen concentration, the middle layer of wastewater is aerated at a second intensity to maintain a medium dissolved oxygen concentration, wherein the first intensity is greater than the second intensity; and the bottom layer of wastewater is not aerated or is aerated at a lower intensity than the second intensity to maintain a low dissolved oxygen or anaerobic environment.

[0031] Preferably, the first aeration intensity is 0.8~1.0 L / (L·min), and the second intensity is 0.3~0.5 L / (L·min).

[0032] Preferably, the high dissolved oxygen concentration is ≥5 mg / L, the medium dissolved oxygen concentration is 0.5~1.0 mg / L, and the dissolved oxygen concentration in the low dissolved oxygen or anaerobic environment is ≤0.5 mg / L.

[0033] Preferably, the order of layered application is from the bottom layer to the top layer.

[0034] In the aforementioned application, before adding the multi-strain-volcanic rock carrier complex in layers, the aquaculture wastewater is pretreated to ensure that the chemical oxygen demand (COD) of the wastewater is ≤500mg / L and the pH value is adjusted to 7.0~8.5.

[0035] Preferably, the application is suitable for static or micro-flow aquaculture wastewater ponds with a water depth of ≥2 meters that can form a dissolved oxygen gradient.

[0036] As a further preferred option, the application is suitable for static or micro-flow aquaculture wastewater ponds with a water depth ≥2 meters, a diameter ≥2 meters, and capable of forming a dissolved oxygen gradient.

[0037] As a further preferred option, when the water depth is 2 meters, the stratification is divided into an upper layer of sewage with a water depth of 0~0.5m, a middle layer of sewage with a water depth of 0.5~1.5m, and a bottom layer of sewage with a water depth of 1.5~2.0m.

[0038] As a further optimization, when the water depth is 2 meters, the total water volume V in the aquaculture wastewater pond is... 总 =πr 2 h=π×1 2 ×2≈6.28m 3 The volume of the upper layer of sewage is V1 = π × 1 2 ×0.5≈1.57m3 The volume of the middle layer of wastewater is V² = π × 1. 2 ×1≈3.14m 3 The volume of the bottom layer of sewage is V3 = π × 1 2 ×0.5≈1.57m 3 .

[0039] Therefore, the present invention has the following beneficial effects: (1) When the water depth is 2 meters, the floating volcanic rock of the present invention can be stably suspended in the upper layer (0~0.5m) high oxygen zone, which is suitable for aerobic nitrifying strains such as *Nitrosporium vesiculosus* and *Nitrosporium cerevisiae*; the suspended volcanic rock can stay in the middle layer (0.5~1.5m) medium oxygen zone, which can carry facultative strains such as *Pseudomonas fluorescens*; the submerged volcanic rock can sink to the bottom layer (1.5~2.0m) low oxygen / anaerobic zone, which can fix denitrification strains such as *Rhodopseudomonas swampus*. The vertical space of 2 meters water depth is sufficient to construct a clear oxygen layer gradient, which can meet the metabolic needs of different strains, thereby achieving the compatibility of the carrier with water depth and oxygen layer. (2) Total viable count ≥ 1 × 10 11 The high-concentration multi-strain-volcanic rock carrier complex (CFU / g) can rapidly form a dominant bacterial community in static wastewater, making up for the slow diffusion rate of strains in static systems. Combined with the immobilization effect of the volcanic rock carrier, it can significantly prolong the duration of strain effectiveness and improve nitrogen and phosphorus removal efficiency. (3) A sewage tank with a water depth of at least 2 meters is convenient for the installation of a layered aeration device (upper layer microporous aeration, middle layer intermittent aeration, and bottom layer no aeration), which can accurately control the dissolved oxygen concentration of each layer, effectively avoid the homogenization of the oxygen layer caused by single aeration, and help ensure the synergistic effect of aerobic, facultative and anaerobic strains, and adapt to the growth needs of each functional bacterial group. (4) The present invention uses a small vortex blower and a disc microporous aerator for aeration, which reduces energy consumption by 40% to 50% compared with traditional surface aeration; the present invention uses solar charging, and the volcanic rock carrier is widely available and inexpensive, with low energy consumption and low cost, making it suitable for small-scale rural scenarios. (5) This invention achieves a low-carbon, circular governance effect by applying near-zero-energy biological denitrification technology and renewable volcanic rock particle carriers. It reduces aquaculture emissions, a major cause of non-point source pollution, at the source and is a key practice to promote agriculture to break away from the "high-pollution, high-consumption" model and shift to green and ecological development. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0041] The strains used in the following examples are from the following sources: Nitrobacter winogradskyi (ATCC 25391) was purchased from Guangzhou Mianyuan Environmental Technology Co., Ltd.; Nitrosomonas europaea (ATCC 25978) was purchased from Guangzhou Mianyuan Environmental Technology Co., Ltd.; Pseudomonas fluorescens (ATCC 13525) was purchased from Shandong Beijia Biotechnology Co., Ltd.; Alcaligenes faecalis (ATCC 8750) was purchased from Shandong Beijia Biotechnology Co., Ltd.; Bacillus subtilis (ATCC 6633) was purchased from Shandong Beijia Biotechnology Co., Ltd.; Bacillus licheniformis (ATCC 14580) was purchased from Shandong Beijia Biotechnology Co., Ltd.; Paracoccus denitrificans (ATCC 14580) was purchased from Shandong Beijia Biotechnology Co., Ltd.; 17741 was purchased from Shandong Beijia Biotechnology Co., Ltd.; Rhodopseudomonas palustris (ATCC 33872) was purchased from Shandong Beijia Biotechnology Co., Ltd.; Rhodobacter sphaeroides (ATCC 17023) was purchased from Guangzhou Mianyuan Environmental Technology Co., Ltd.; Citrobacter koseri (ATCC 25408) was purchased from Guangzhou Mianyuan Environmental Technology Co., Ltd.; LysiniLysinibacillus sphaericus (ATCC 14577) was purchased from Guangzhou Mianyuan Environmental Technology Co., Ltd.

[0042] Example 1: Experiment on the ratio of bacterial strains in the upper hyperoxia group (1) Consumables preparation: The sources of livestock wastewater include, but are not limited to, pig farms, employing either soaking or dry manure cleaning processes. This wastewater contains urine, feces, and pen flushing water, contains no drugs detrimental to bacterial growth, and is wastewater discharged within the past week. The wastewater undergoes pretreatment with a two-stage sedimentation + filtration + dilution process. Sedimentation includes, but is not limited to, using quicklime and polyaluminum chloride (PAC) to remove heavy metals from the water. Before adding the multi-strain-volcanic rock carrier complex to the wastewater, baseline water quality measurements are performed to determine the ammonia nitrogen (NH4) content. + The concentrations of total nitrogen (TN), total phosphorus (TP), and chemical oxygen demand (COD) were measured. Under rural conditions, wastewater was diluted as follows: when the initial wastewater COD was approximately 800-1000 mg / L, a wastewater:clean water ratio of 1:1 was used; when the initial wastewater COD was approximately 1000-1500 mg / L, a wastewater:clean water ratio of 1:2 was used, aiming to achieve a COD ≤ 500 mg / L after dilution for optimal purification. In mountainous rural areas without testing facilities, the dilution ratio was controlled based on the standard that "the mixed wastewater has no obvious fecal odor and the bottom of the container is clearly visible." The pH was adjusted to 7.0-8.5. The wastewater quality for this experiment was: TN 120 mg / L, TP 12 mg / L, NH4+... + -N 90mg / L, COD 350mg / L, PH 7.5.

[0043] Device: A 1000mL stoppered graduated cylinder was used, and the wastewater was divided into three layers according to its height: upper layer (0~22.3cm), middle layer (22.3~44.7cm), and lower layer (44.7~66.7cm). The volume of a single wastewater layer is 1000 / 3 = 0.001m³. 3 .

[0044] Parameters of bacterial culture and multi-strain-volcanic rock carrier complex: The concentration of bacterial solution in the upper hyperoxia group was 1×10⁻⁶. 8 CFU / mL; the dosage of the multi-strain-volcanic rock carrier complex was 50 g / L; the volume-to-mass ratio of the bacterial culture to the volcanic rock carrier in the upper hyperoxia group was bacterial culture: volcanic rock carrier = 2.5:1 (mL / g).

[0045] The experimental conditions are now met: floating-type upper-layer hyperoxia multi-strain-volcanic rock carrier complexes with different strain ratios have been prepared. The preparation method for the floating-type upper-layer hyperoxia multi-strain-volcanic rock carrier complex is as follows: After the upper layer of high-oxygen group strains were mixed and cultured, they were mixed with pretreated floating volcanic rock carrier at a ratio of strain liquid to volcanic rock carrier = 2.5:1 (mL / g), adsorbed at room temperature for 2 hours, separated into solid and liquid, and dried to prepare the product.

[0046] The pretreatment method for floating volcanic rock carriers is as follows: Take floating volcanic rock particles and rinse them with running water 3-5 times. Then soak them in 5% dilute hydrochloric acid (HCl) for 2-4 hours. After rinsing, rinse them repeatedly with distilled water / purified water until the rinse water pH=6.5-7.5 (neutral), without acidic taste or turbidity. Then put them in an oven and dry them at 105℃ to constant weight. Cool them for later use.

[0047] The test strains were A (Nitrobacterium velutipes), B (Nitrosomonas cerevisiae), C (Bacillus subtilis), and D (Bacillus licheniformis). The experiment was divided into four groups: AA1, AA2, AA3, and AA4. The mixing volume ratio of pure culture solutions in each group (A:B:C:D) was set as follows: AA1: 3:3:2:2; AA2: 4:2:2:2; AA3: 2:4:2:2; AA4: 3:2:3:2.

[0048] (2) Preparatory work before the experiment: Take a sufficient amount of homogenized aquaculture wastewater, stir it evenly, and dispense it into 1000mL graduated cylinders, 1000mL per group, and label the corresponding group. Set up 4 experimental groups (AA1~AA4 groups) and 1 blank control group (sterile carrier only), with 3 replicates per group. The bacterial strain composition, pure bacterial solution mixing volume ratio, and dosage of multi-strain-volcanic rock carrier complex in the upper high-oxygen group are shown in Table 1 below.

[0049] Table 1: Composition of bacterial strains in the upper hyperoxia group, volume ratio of pure bacterial suspension, and dosage of multi-strain-volcanic rock carrier complex. .

[0050] Initial index determination: TN, TP, and NH4 were measured in wastewater from each treatment group. + Record raw data for N, COD, pH, and dissolved oxygen (DO) to ensure that there are no significant differences in initial indicators among treatment groups.

[0051] (3) Experimental operation steps: Dosing and mixing of multi-strain-volcanic rock carrier complex: According to the group number, accurately add the corresponding proportion of multi-strain-volcanic rock carrier complex to the appropriate position in each graduated cylinder for wastewater, ensuring that the multi-strain-volcanic rock carrier complex is on the surface of the wastewater. Add an equal amount of sterile volcanic rock to the blank control group. Gently stir along the cylinder wall with a sterile glass rod for 1 minute to ensure the multi-strain-volcanic rock carrier complex is uniformly suspended, avoiding the generation of excessive air bubbles.

[0052] Culture environment: All graduated cylinders were placed in a constant-temperature water bath, with the temperature stabilized at 28–30°C. Each graduated cylinder was connected to a micro aerator, with the aeration intensity adjusted to 0.8–1.0 L / (L·min) to maintain DO ≥ 5 mg / L. Dissolved oxygen was monitored every 2 hours using a dissolved oxygen meter. Continuous aeration was maintained throughout the entire process, operating statically. The graduated cylinders were completely covered with a black light-shielding cover for complete light protection during cultivation to prevent light from inhibiting nitrifying bacteria activity. The wastewater pH was measured every 6 hours. If the wastewater pH deviated from the range of 7.5–8.0, it was fine-tuned with 1 mol / L HCl or NaOH.

[0053] (4) Sampling and testing: Sampling time points: 0 (initial), 24h, 48h, 72h, 96h.

[0054] A 50 mL sample was taken from the middle layer of wastewater (halfway below the surface) for testing. The levels of TN, TP, and NH4 in the wastewater were measured. + -N, COD, DO (dissolved oxygen), calculate the removal rate of each indicator.

[0055] The detection method uses reagent kits. The kits include TN detection kit, TP detection kit, and NH4 detection kit. + The -N detection kit, COD detection kit, and dissolved oxygen detection kit were all purchased from Zhejiang Luheng Environmental Technology Co., Ltd., and provided by Nanning Yuanheng Veterinary Service Center. The test results are shown in Table 2 below.

[0056] Table 2: Results of the strain ratio test in the upper hyperoxia group Group AA1 20 60 42.1 90 65 Mild reunion Group AA2 20 30 31.5 65 40 Good dispersion Group AA3 20 45 36.5 80 55 Good dispersion Group AA4 20 63 45.5 93 72 Clear reunion Blank control group Sterile volcanic rock 20 12.4 12.2 5.1 18.1 -

[0057] As shown in Table 2, when the volume ratio of the pure culture bacterial solution in the upper high-oxygen group (A:B:C:D) is 3:3:2:2, the ammonia nitrogen removal rate is greater than or equal to 90%, the carrier is in good condition, and the cost performance is optimal.

[0058] Example 2: Experiment on the ratio of strains in the mid-layer oxygen group (1) Consumables preparation: The water quality of the aquaculture wastewater to be tested, the preparation of the equipment, and the parameters of the bacterial solution and the multi-strain-volcanic rock carrier complex were operated in accordance with the procedures in Example 1.

[0059] The experimental conditions are now met: suspension-type meso-aerobic multi-strain-volcanic rock carrier complexes with different strain ratios have been prepared. The preparation method for the suspension-type meso-aerobic multi-strain-volcanic rock carrier complex is as follows: After being cultured together with the meso-aerobic strains in the middle layer, the mixture was combined with pretreated suspended volcanic rock carrier at a volume-to-mass ratio of strain culture to volcanic rock carrier of 2.5:1 (mL / g). The mixture was adsorbed at room temperature for 2 hours, followed by solid-liquid separation and drying. The pretreatment method for the suspended volcanic rock carrier was as described in Example 1.

[0060] The test strains were E (Pseudomonas fluorescens), F (Alcaligenes faecalis), G (Bacillus spheroidans), and H (Paracoccus denitrificans). The experiment included groups BB1, BB2, BB3, and BB4, with the following pure culture volume ratios (E:F:G:H): BB1: 3:2:2:1; BB2: 4:1:2:1; BB3: 2:4:1:1; BB4: 2:1:4:1.

[0061] (2) Preparatory work before the experiment Take a sufficient amount of homogenized aquaculture wastewater, stir it evenly, and dispense it into 1000mL graduated cylinders, 1000mL per group, and label the corresponding group. Set up 4 experimental groups (BB1~BB4 groups) and 1 blank control group, with 3 replicates per group. The bacterial strain composition of the mid-layer medium-oxygen group, the mixed volume ratio of pure bacterial solutions (E:F:G:H) of each group, and the dosage of multi-strain-volcanic rock carrier complex are shown in Table 3 below.

[0062] Table 3: Composition of strains in the meso-aerobic group of the middle layer, mixing volume ratio of pure bacterial cultures in each group (E:F:G:H), and dosage of multi-strain-volcanic rock carrier complex. .

[0063] The initial index determination was performed in accordance with the procedure in Example 1.

[0064] (3) Experimental operation steps: Dosing and mixing of multi-strain-volcanic rock carrier complex: According to the group number, accurately add the corresponding proportion of multi-strain-volcanic rock carrier complex to each graduated cylinder of wastewater, ensuring that the multi-strain-volcanic rock carrier complex is in the middle layer of the wastewater. Add an equal amount of sterile volcanic rock to the control group. Gently stir along the wall of the graduated cylinder with a sterile glass rod for 1 minute to ensure that the multi-strain-volcanic rock carrier complex is uniformly suspended, avoiding the generation of a large number of air bubbles.

[0065] Culture environment: All graduated cylinders were placed in a constant-temperature water bath, with the temperature stabilized at 30-32℃. Each graduated cylinder was connected to a micro aerator, and the aeration intensity was adjusted to 0.3-0.5 L / (L·min) to maintain a dissolved oxygen (DO) concentration of 0.5-1.0 mg / L. The DO value was monitored every 4 hours using a dissolved oxygen meter, and the aeration parameters were adjusted accordingly. All graduated cylinders were covered with a light-shielding net to create a low-light environment of 500-1000 lux, avoiding strong light that could inhibit denitrifying enzyme activity. The wastewater pH was measured every 6 hours. If the wastewater pH deviated from the range of 7.0-7.5, it was fine-tuned with 1 mol / L HCl or NaOH.

[0066] (4) Sampling and testing: The sampling time, sampling location, detection indicators, and detection methods were performed in accordance with Example 1. The test results are shown in Table 4 below.

[0067] Table 4: Results of the strain ratio test in the middle layer of the oxygen-rich group BB1 Group 15 80 60 85 75 Mild reunion BB2 Group 15 70 45 75 65 Good dispersion BB3 Group 15 83 60 88 80 Clear reunion BB4 group 15 85 63 90 80 serious family reunification Blank control group Sterile volcanic rock 15 12.0 12.1 5.1 18.0 -

[0068] As shown in Table 4, when the volume ratio of pure bacterial culture in the middle layer of the oxygen-rich group (E:F:G:H) is 3:2:2:1, the ammonia nitrogen removal rate is greater than or equal to 85%, the carrier is in good condition, and the cost performance is optimal.

[0069] Example 3: Experiment on the ratio of strains in the bottom hypoxia / anaerobic group (1) Consumables preparation: The water quality of the aquaculture wastewater to be tested, the equipment preparation, and the parameters of the bacterial solution and the multi-strain-volcanic rock carrier complex are the same as in Example 1.

[0070] The experimental conditions are now met: submersible bottom-layer hypoxic / anaerobic multi-strain-volcanic rock carrier complexes with different strain ratios have been prepared. The preparation method for the submersible bottom-layer hypoxic / anaerobic multi-strain-volcanic rock carrier complex is as follows: After mixed culture of the low-oxygen / anaerobic strains at the bottom layer, they were mixed with pretreated submerged volcanic rock carrier at a volume-to-mass ratio of strain culture to volcanic rock carrier = 2.5:1 (mL / g). The mixture was adsorbed at room temperature for 2 hours, followed by solid-liquid separation and drying. The pretreatment method for the submerged volcanic rock carrier was as described in Example 1.

[0071] The test strains were I (Rhodopseudomonas palustris), J (Rhodopseudomonas aeruginosa), K (Paradiabetic paracocci), and L (Citrobacter korshinskii). The experiment was divided into DD1, DD2, DD3, and DD4 groups, with the following pure culture volume ratios (I:J:K:L): DD1: 3:3:2:2; DD2: 4:2:2:2; DD3: 2:2:3:3; DD4: 2:3:2:3.

[0072] (2) Preparatory work before the experiment: Take a sufficient amount of homogenized aquaculture wastewater, stir it evenly, and then dispense it into 1000mL graduated cylinders, 1000mL per group, and label the corresponding group. Set up 4 experimental groups (DD1~DD4 groups) and 1 blank control group, with 3 replicates per group. The bacterial strain composition of the bottom hypoxia / anaerobic group, the mixing volume ratio of pure bacterial solutions in each group, and the dosage of multi-strain-volcanic rock carrier complex are shown in Table 5 below.

[0073] Table 5: Composition of strains in the bottom hypoxic / anaerobic group, mixing volume ratio of pure bacterial cultures in each group, and dosage of multi-strain-volcanic rock carrier complex.

[0074] Initial index determination: Refer to the procedure in Example 1.

[0075] (3) Experimental operation steps: Dosing and mixing of multi-strain-volcanic rock carrier complex: According to the group number, the corresponding proportion of multi-strain-volcanic rock carrier complex was precisely added to each graduated cylinder of wastewater, ensuring that the multi-strain-volcanic rock carrier complex was at the bottom of the wastewater. An equal amount of sterile volcanic rock was added to the blank control group. The mixture was gently stirred along the cylinder wall with a sterile glass rod for 1 minute to ensure uniform suspension of the multi-strain-volcanic rock carrier complex, avoiding the generation of excessive air bubbles. Nitrogen gas was then introduced into each graduated cylinder for 10 minutes to drive oxygenation. The cylinder openings were then sealed with sterile plastic wrap and reinforced with rubber stoppers to ensure an effective anaerobic seal.

[0076] Culture environment: All graduated cylinders were placed in a constant-temperature water bath, with the temperature stabilized at 25-28℃. An incandescent lamp was placed directly above the graduated cylinders, at a distance of 20-30cm, with the light intensity adjusted to 3000-5000 lux to maintain continuous illumination (essential for photosynthetic bacteria growth). The dissolved oxygen (DO) concentration was rapidly monitored every 24 hours, ensuring it was ≤0.5 mg / L. After monitoring, the cylinders were immediately resealed and nitrogen was introduced for 5 minutes. The wastewater pH was rapidly measured every 12 hours. If the wastewater pH deviated from the range of 6.8-7.2, it was fine-tuned using 1 mol / L HCl or NaOH.

[0077] (4) Sampling and testing: The sampling time, sampling location, detection indicators, and detection methods were performed in accordance with Example 1. The test results are shown in Table 6 below.

[0078] Table 6: Results of the strain ratio test in the bottom hypoxic / anaerobic group DD1 Group 15 85 70 93 80 Mild reunion DD2 group 15 75 55 91.1 70 Good dispersion DD3 group 15 88 75 95.5 83 Clear reunion DD4 group 15 90 78 96.1 85 serious family reunification Blank control group Sterile volcanic rock 15 12.5 18.3 5.3 12.1 -

[0079] As shown in Table 6, when the volume ratio of the pure culture liquid in the bottom hypoxia / anaerobic group (I:J:K:L) is 3:3:2:2, the ammonia nitrogen removal rate is greater than or equal to 93%, the carrier is in good condition, and the cost performance is optimal.

[0080] Example 4: Optimal ratio test of strains in the same layer (1) Consumables preparation: The wastewater from the aquaculture experiment had the following parameters: TN 100 mg / L, TP 15 mg / L, and NH4+. +-N 60mg / L, COD 350mg / L, adjust pH to 7.5±0.1. The equipment preparation and parameters for the bacterial culture and multi-strain-volcanic rock carrier complex are as described in Example 1.

[0081] The experimental conditions are now met: the optimal ratio of the three-layer multi-strain-volcanic rock carrier complex (top, middle, and bottom layers) has been prepared. The preparation method for each layer of the multi-strain-volcanic rock carrier complex is as described in Examples 1-3.

[0082] (2) Preparatory work before the experiment: Take 1000 mL of homogenized aquaculture wastewater and place it into a 1000 mL graduated cylinder. Label the stratified experimental group and the blank control group, with 3 replicates for each group. Add an equal volume of sterile volcanic rock to the blank control group. The bacterial strain composition, pure bacterial culture mixing volume ratio, and dosage of multi-strain-volcanic rock carrier complex in each layer of the stratified experimental group and the blank control group are shown in Table 7 below.

[0083] Table 7: Strain composition of each layer, volume ratio of pure culture solutions, and dosage of multi-strain-volcanic rock carrier complex. .

[0084] Initial parameters of wastewater in all treatment groups were measured: total nitrogen (TN), total phosphorus (TP), and nitrous oxide (NH4). + -N, COD, pH, DO, record the raw data to ensure that there is no significant difference between the two initial indicators.

[0085] (3) Operating steps: Stratified test group: The multi-strain-volcanic rock carrier complex was added in the order of bottom layer, middle layer and top layer in the stratified test group.

[0086] The addition of the multi-strain-volcanic rock carrier complex to the bottom layer and the construction of the culture environment were carried out in accordance with the procedures in Example 3; the addition of the multi-strain-volcanic rock carrier complex to the middle layer and the construction of the medium-oxygen environment were carried out in accordance with the procedures in Example 2; and the addition of the multi-strain-volcanic rock carrier complex to the top layer and the construction of the high-oxygen environment were carried out in accordance with the procedures in Example 1.

[0087] Procedure for blank control group: Add 50g of sterile volcanic rock (consistent with the total amount added in the stratified test group) and stir for 1 minute.

[0088] Environmental parameters were adjusted synchronously with those of the experimental group: 0~24h anaerobic + strong light + 25~28℃; 24~48h medium oxygen + weak light + 30~32℃; 48~72h high oxygen + dark + 28~30℃.

[0089] (4) Sampling and testing: The sampling time, sampling location, detection indicators, and detection methods were performed in accordance with Example 1. The results of the optimal ratio test of strains in the same layer are shown in Table 8 below.

[0090] Table 8: Results of the optimal ratio of strains in the same layer Stratified experimental group 0 average value 100.0 0.0 15.0 0.0 60.0 0.0 350.0 0.0 - Blank control group 0 average value 100.0 0.0 15.0 0.0 60.0 0.0 350.0 0.0 No carrier, only sewage Stratified experimental group 24 average value 78.5 21.5 10.2 31.8 38.5 35.8 265.0 24.3 The upper layer is stable when floating, and the middle layer is stable when suspended. Blank control group 24 average value 95.2 4.8 13.8 8.2 55.1 8.1 327.7 6.1 No significant changes in the wastewater Stratified experimental group 48 average value 56.5 43.5 6.5 56.5 22.4 62.7 203.3 41.6 The carrier separates into layers without migration, and a preliminary bacterial film is formed. Blank control group 48 average value 90.5 9.5 12.5 16.7 50.3 16.2 305.0 12.9 Slightly turbid sewage Stratified experimental group 72 average value 35.8 64.2 3.2 78.5 10.6 82.3 148.3 57.6 The bacterial film is thick and the carrier is stable. Blank control group 72 average value 85.6 14.4 11.3 24.7 45.6 24.0 280.0 20.0 Increased turbidity of sewage Stratified experimental group 96 average value 26.5 73.5 2.1 85.6 7.3 87.8 102.0 70.6 The carrier was intact and the bacterial film did not detach. Blank control group 96 average value 80.8 19.2 10.2 32.0 40.5 32.5 255.0 27.1 The wastewater was river-brown in color with a small amount of sediment.

[0091] As shown in Table 8, the blank control group contained only aquaculture wastewater (without sterile carriers), and its natural purification efficiency within 96 hours was extremely low (TN removal rate 19.2%, TP 32.0%, NH4+ removal rate 32.0%). + -N 32.5%, COD 27.1%); the removal rates of all indicators in the stratified experimental group at 96 h were significantly higher than those in the blank control group: TN 73.5%, TP 85.8%, NH4+ 32.5%, COD 27.1%; + -N 87.8%, COD 70.6%.

[0092] The results of the optimal ratio of strains in the same stratum were analyzed using an independent samples t-test to compare the differences in indicators between the stratified experimental group (multiple strains-volcanic rock carrier complex) and the blank control group (sterile carrier only) at different time points. The significance level was α=0.05 (P<0.05 was considered significant). The statistical significance analysis results of the optimal ratio of strains in the same stratum are shown in Table 9 below. The statistical significance analysis results of the nitrogen and phosphorus removal experiment in aquaculture wastewater are shown in Table 10 below.

[0093] Table 9: Statistical significance analysis of the results of the optimal ratio of strains in the same layer (independent samples t-test) TN (mg / L) 24 78.5±0.71 95.2±0.36 42.38 4 0.0001 Significant (P<0.05) yes TN (mg / L) 48 56.5±0.85 90.5±0.71 64.12 4 <0.0001 Significant (P<0.05) yes TN (mg / L) 72 35.8±0.65 85.6±0.70 98.73 4 <0.0001 Significant (P<0.05) yes TN (mg / L) 96 26.5±0.75 80.8±0.71 118.45 4 <0.0001 Significant (P<0.05) yes TP (mg / L) 24 10.2±0.25 13.8±0.15 37.95 4 0.0001 Significant (P<0.05) yes TP (mg / L) 48 6.5±0.25 12.5±0.20 89.44 4 <0.0001 Significant (P<0.05) yes TP (mg / L) 72 3.2±0.15 11.3±0.20 138.67 4 <0.0001 Significant (P<0.05) yes TP (mg / L) 96 2.1±0.15 10.2±0.20 165.29 4 <0.0001 Significant (P<0.05) yes <![CDATA[NH4 + -N(mg / L)]]> 24 38.5±0.70 55.1±0.40 0.0001 4 0.0001 Significant (P<0.05) yes <![CDATA[NH4 + -N(mg / L)]]> 48 22.4±0.65 50.3±0.45 87.69 4 <0.0001 Significant (P<0.05) yes <![CDATA[NH4 + -N(mg / L)]]> 72 10.6±0.55 45.6±0.50 108.36 4 <0.0001 Significant (P<0.05) yes <![CDATA[NH4 + -N(mg / L)]]> 96 7.3±0.45 40.5±0.50 123.81 4 <0.0001 Significant (P<0.05) yes COD (mg / L) 24 265.0±3.00 327.7±2.52 27.54 4 0.0002 Significant (P<0.05) yes COD (mg / L) 48 203.3±3.51 305.0±3.00 41.23 4 0.0001 Significant (P<0.05) yes COD (mg / L) 72 148.3±3.51 280.0±2.00 64.95 4 <0.0001 Significant (P<0.05) yes COD (mg / L) 96 102.0±3.00 255.0±3.00 78.10 4 <0.0001 Significant (P<0.05) yes pH 24 7.3±0.05 7.4±0.00 4.47 4 0.0102 Significant (P<0.05) yes pH 48 7.1±0.05 7.3±0.00 8.94 4 0.0013 Significant (P<0.05) yes

[0094] Table 10: Statistical significance analysis results of nitrogen and phosphorus removal experiments in aquaculture wastewater (independent samples t-test) TN (mg / L) 24 78.5±0.71 95.2±0.36 42.38 4 0.0001 Significant (P<0.05) yes TN (mg / L) 48 56.5±0.85 90.5±0.71 64.12 4 <0.0001 Significant (P<0.05) yes TN (mg / L) 72 35.8±0.65 85.6±0.70 98.73 4 <0.0001 Significant (P<0.05) yes TN (mg / L) 96 26.5±0.75 80.8±0.71 118.45 4 <0.0001 Significant (P<0.05) yes TP (mg / L) 24 10.2±0.25 13.8±0.15 37.95 4 0.0001 Significant (P<0.05) yes TP (mg / L) 48 6.5±0.25 12.5±0.20 89.44 4 <0.0001 Significant (P<0.05) yes TP (mg / L) 72 3.2±0.15 11.3±0.20 138.67 4 <0.0001 Significant (P<0.05) yes TP (mg / L) 96 2.1±0.15 10.2±0.20 165.29 4 <0.0001 Significant (P<0.05) yes <![CDATA[NH4 + -N(mg / L)]]> 24 38.5±0.70 55.1±0.40 0.0001 4 0.0001 Significant (P<0.05) yes <![CDATA[NH4 + -N(mg / L)]]> 48 22.4±0.65 50.3±0.45 87.69 4 <0.0001 Significant (P<0.05) yes <![CDATA[NH4 + -N(mg / L)]]> 72 10.6±0.55 45.6±0.50 108.36 4 <0.0001 Significant (P<0.05) yes <![CDATA[NH4 + -N(mg / L)]]> 96 7.3±0.45 40.5±0.50 123.81 4 <0.0001 Significant (P<0.05) yes COD (mg / L) 24 265.0±3.00 327.7±2.52 27.54 4 0.0002 Significant (P<0.05) yes COD (mg / L) 48 203.3±3.51 305.0±3.00 41.23 4 0.0001 Significant (P<0.05) yes COD (mg / L) 72 148.3±3.51 280.0±2.00 64.95 4 <0.0001 Significant (P<0.05) yes COD (mg / L) 96 102.0±3.00 255.0±3.00 78.10 4 <0.0001 Significant (P<0.05) yes pH 24 7.3±0.05 7.4±0.00 4.47 4 0.0102 Significant (P<0.05) yes pH 48 7.1±0.05 7.3±0.00 8.94 4 0.0013 Significant (P<0.05) yes pH 72 6.9±0.05 7.2±0.00 13.42 4 0.0003 Significant (P<0.05) yes pH 96 6.8±0.05 7.1±0.00 17.89 4 0.0001 Significant (P<0.05) yes DO (mg / L) 24 2.2±0.10 2.5±0.10 5.48 4 0.0063 Significant (P<0.05) yes DO (mg / L) 48 1.8±0.10 2.2±0.10 8.94 4 0.0013 Significant (P<0.05) yes DO (mg / L) 72 1.5±0.10 1.9±0.10 13.42 4 0.0003 Significant (P<0.05) yes DO (mg / L) 96 1.3±0.10 1.6±0.10 17.89 4 0.0001 Significant (P<0.05) yes

[0095] As shown in Tables 9 and 10, all detected indicators (TN, TP, NH4) + The P values ​​for nitrogen, COD, pH, and DO at 24h, 48h, 72h, and 96h were all <0.05, and most P values ​​were <0.0001, indicating that the stratified experimental group and the blank control group were highly significantly different, demonstrating that the multi-strain-volcanic rock carrier complex has a better effect on nitrogen and phosphorus removal and purification of aquaculture wastewater.

[0096] Example 5: Comparative Test of Wastewater Treatment with Compound Microbial Agents 1. Experimental Design: Experimental group: The compound microbial agent of this invention (i.e., the mixed bacteria consists of Nitrifying Bacillus vesiculosus, Nitrifying Monoclonalella Europeanis, Pseudomonas fluorescens, Alcaligenes faecalis, Bacillus subtilis, Bacillus licheniformis, Citrobacter korshinskii, Paracoccus denitrificans, Rhodopseudomonas palustris, Rhodopseudomonas coccidioides, and Bacillus spheroidae).

[0097] Control group 1: a single nitrifying bacteria group (composed of Nitrosomonas cerevisiae and Nitrosobacterium velutipes).

[0098] Control group 2: Commercially available Bacillus compound inoculant (mainly Bacillus subtilis and Bacillus licheniformis).

[0099] Blank control group: Only blank volcanic rock was added, without any bacteria.

[0100] The experiment consisted of three treatment groups (experimental group, control group 1, and control group 2) and one blank control group (volcanic rock only, without bacteria). Each group was replicated three times. Total nitrogen (TN), total phosphorus (TP), and ammonia nitrogen (NH4) were measured at 7 days. + The optimal ratio was determined by measuring the removal rates of nitrogen (N) and chemical oxygen demand (COD).

[0101] 2. Test conditions: The wastewater from the aquaculture experiment had the following parameters: TN 100 mg / L, TP 15 mg / L, and NH4+. + -N 60mg / L, COD 350mg / L, adjust pH to 7.5±0.1. Cylindrical graduated cylinder with an effective volume of 1000mL and a liquid capacity of 1000mL; submersible volcanic rock particles (particle size 8~12mm, specific surface area 12~18m²). 2 Using a carrier (with a porosity of 45-55%), each group of bacterial cultures had a concentration of 1×10⁻⁶ g. 8 The dosage of the multi-strain-volcanic rock carrier complex is 50 g / L (CFU / mL); the volume-to-mass ratio of bacterial culture to volcanic rock carrier is 2.5:1 (mL / g). Calculations show that 50 g of volcanic rock and 125 mL of bacterial culture are needed for a 1000 mL graduated cylinder.

[0102] All graduated cylinders were placed in a constant-temperature water bath, with the temperature stabilized at 28~30℃. Each graduated cylinder was connected to a micro aerator, and the aeration intensity was adjusted to 0.8~1.0 L / (L·min) to maintain a DO concentration ≥5mg / L. The dissolved oxygen concentration was monitored every 2 hours using a dissolved oxygen meter. Aeration was continuous throughout the process, and the system was operated statically. The wastewater pH was measured every 6 hours. If the wastewater pH deviated from the range of 7.5~8.0, it was finely adjusted with 1mol / L HCl or NaOH.

[0103] 3. Experimental Procedure: The bacterial agents used in the experimental group, control group 1, and control group 2 were activated, and the bacterial concentration was adjusted to 1×10⁻⁶ with sterile physiological saline. 8 CFU / mL.

[0104] Each bacterial agent was mixed with volcanic rock granular carrier for 24 hours to obtain a multi-strain-volcanic rock carrier complex containing each bacterial agent. A graduated cylinder was first filled with 1000 mL of the experimental aquaculture wastewater. Then, the multi-strain-volcanic rock carrier complex containing each bacterial agent was separately placed into an open mesh cage (its diameter is smaller than the treatment tank) inside the graduated cylinder, laid flat, and fixed to the bottom of the wastewater tank. The graduated cylinder was placed in a constant temperature environment of 25-30℃, connected to a small aeration pump, with an aeration intensity of 2-3 m³ / h. 3 / (m 2 • h), maintain dissolved oxygen at 2.5~4.0 mg / L; culture period 7 days.

[0105] 4. Sampling and testing: Sampling time points: 0 (initial), 24h, 48h, 72h, 96h.

[0106] A 50 mL sample was taken from the middle layer of wastewater (halfway below the surface) for testing. The levels of TN, TP, and NH4 in the wastewater were determined. + -N and COD values ​​are used to calculate the removal rate of each indicator.

[0107] The detection method uses reagent kits. The kits include TN detection kit, TP detection kit, and NH4 detection kit. + -N detection kit, COD detection kit and dissolved oxygen detection kit were all purchased from Zhejiang Luheng Environmental Technology Co., Ltd. and provided by Nanning Yuanheng Veterinary Service Center.

[0108] 5. The test results are shown in Table 11 below: Table 11: experimental group 63.5±0.8 75.8±0.5 77.8 60.6±0.15 Control Group 1 41.2±1.1 22.5±0.55 68.3 25.7±0.21 Control group 2 35.6±0.9 62.1±0.12 42.9 58.2±0.32 Blank control group 8.3±0.74 11.5±0.23 12.4 16.8±0.11

[0109] The multi-strain compound bacterial agent constructed in this invention exhibits significant advantages in the treatment of aquaculture wastewater. Compared with single nitrifying bacteria groups and ordinary commercially available Bacillus compound bacterial agents, it shows significant improvements in the removal of total nitrogen, ammonia nitrogen, total phosphorus, and organic matter, resulting in superior overall treatment performance.

[0110] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A method for quantitatively adding a multi-strain-volcanic rock carrier complex, characterized in that, The multi-strain-volcanic rock carrier complex comprises a mixed bacteria and a carrier. The mixed bacteria include at least two functional bacteria among nitrifying bacteria, denitrifying bacteria, organic matter degrading bacteria, and phosphorus-removing bacteria. The carrier is volcanic rock particles. The total viable count of the multi-strain-volcanic rock carrier complex is ≥1×10⁻⁶. 11 CFU / g.

2. The quantitative dosing multi-strain-volcanic rock carrier complex according to claim 1, characterized in that, The nitrifying bacteria include at least one of nitrite-oxidizing bacteria and nitrifying bacteria; the denitrifying bacteria include at least one of heterotrophic denitrifying bacteria and photosynthetic denitrifying bacteria; the organic matter degrading bacteria include at least one of Bacillus spp. and Pseudomonas spp.; and the phosphorus-removing bacteria include at least one of polyphosphate-accumulating bacteria and denitrifying phosphorus-removing bacteria.

3. The quantitative dosing of multi-strain-volcanic rock carrier complex according to claim 1, characterized in that, The mixed bacteria include at least three of the following: Nitrobacter winogradskyi, Nitrosomonaseuropaea, Pseudomonas fluorescens, Alcaligenes faecalis, Bacillus subtilis, Bacillus licheniformis, Paracoccus denitrificans, Rhodopseudomonas palustris, Rhodobacter sphaeroides, Citrobacter koseri, and Lysinibacillus sphaericus.

4. The quantitative dosing multi-strain-volcanic rock carrier complex according to claim 1, characterized in that, The volcanic rock particles include at least one of the following: floating, suspended, and sinking types.

5. A method for preparing a quantitatively added multi-strain-volcanic rock carrier complex as described in any one of claims 1 to 4, characterized in that, Includes the following steps: A mixed bacterial strain containing at least two functional bacteria from nitrifying bacteria, denitrifying bacteria, organic matter degrading bacteria, and phosphorus-removing bacteria was immobilized on a volcanic rock particle carrier to obtain a multi-strain-volcanic rock carrier complex.

6. The application of a quantitatively added multi-strain-volcanic rock carrier complex as described in any one of claims 1 to 4 in the purification of aquaculture wastewater.

7. The application according to claim 6, characterized in that, The method of application is as follows: The multi-strain-volcanic rock carrier complex was added to the aquaculture wastewater to be treated in layers according to the differences in dissolved oxygen requirements of the microorganisms therein, and then aerated in layers.

8. The application according to claim 7, characterized in that, The stratified addition is as follows: a multi-strain-volcanic rock carrier complex adapted to high dissolved oxygen conditions is added to the upper layer of wastewater and fixed; a multi-strain-volcanic rock carrier complex adapted to medium dissolved oxygen conditions is added to the middle layer of wastewater and fixed; and a multi-strain-volcanic rock carrier complex adapted to low dissolved oxygen or anaerobic conditions is added to the bottom layer of wastewater and fixed.

9. The application according to claim 8, characterized in that, The carrier used in the multi-strain-volcanic rock carrier complex added to the upper layer of sewage is floating volcanic rock particles; the carrier used in the multi-strain-volcanic rock carrier complex added to the middle layer of sewage is suspended volcanic rock particles; and the carrier used in the multi-strain-volcanic rock carrier complex added to the bottom layer of sewage is submerged volcanic rock particles.

10. The application according to claim 7, characterized in that, The stratified aeration is as follows: the upper layer of wastewater is aerated at a first intensity to maintain a high dissolved oxygen concentration, the middle layer of wastewater is aerated at a second intensity to maintain a medium dissolved oxygen concentration, and the first intensity is greater than the second intensity; the bottom layer of wastewater is not aerated or is aerated at a lower intensity than the second intensity to maintain a low dissolved oxygen or anaerobic environment.

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