Slow-release cultivation granules for in-situ selective cultivation of photosynthetic bacteria in contaminated water bodies, and preparation method and application thereof

Abstract

The application discloses a slow-release and expansion granule for in-situ selective culture of photosynthetic bacteria in a polluted water body and a preparation method and application thereof, and belongs to the technical field of biological remediation of the polluted water body, and solves the problems that the bacteria amount is determined when the bacteria agent adding route is used, the nutrients are rapidly released, the planting is unstable, and the non-sterilized selective culture cannot be used in the open water body. The granule is prepared by integrally granulating plastic extrusion of raw materials, and does not contain immobilized bacteria bodies. The true density of the granule is 1.05-1.30 g / cm3, and the particle size is 3-10 mm. The granule is used for in-situ selective culture of photosynthetic bacteria in the non-sterilized open polluted water body, and realizes in-situ remediation of the polluted water body.

Description

Technical Field

[0001] This invention relates to the field of bioremediation technology for polluted water bodies, specifically to a slow-release expansion granule for in-situ selective culture of photosynthetic bacteria in polluted water bodies, its preparation method, and its application. Background Technology

[0002] Photosynthetic bacteria (PSB) are a type of prokaryotic microorganism that can perform anoxic photosynthesis under anaerobic light conditions, using organic matter, sulfides, and ammonia as hydrogen donors and carbon sources. PSB possesses various metabolic capabilities, including degrading organic pollutants, removing nitrogen and phosphorus, fixing nitrogen, and producing hydrogen. They are widely used in environmental protection fields such as biological treatment of organic wastewater, aquatic ecosystem restoration, aquaculture water purification, and soil remediation. The core prerequisite for these applications is the large-scale, low-cost cultivation of PSB.

[0003] In the actual operation of PSB cultivation, the general practice of existing technologies is as follows: In a laboratory or culture workshop, the culture medium is prepared and then sterilized by high-temperature and high-pressure sterilization (121℃, autoclave) or membrane filtration (0.22μm microporous membrane). After cooling, PSB inoculum is inoculated in a clean bench and cultured in pure culture under controlled light and temperature conditions. After cultivation, the PSB culture is transported to the application site for use, or, if conditions permit, it is sterilized and cultured on-site before direct inoculation. After each cultivation cycle, the culture medium must be prepared again and the above sterilization-inoculation-cultivation process must be repeated.

[0004] However, the above approach faces a core contradiction in engineering applications: In practical applications, such as wastewater treatment plants requiring on-site cultivation of PSB bacterial solution and direct addition to the reaction tank, aquaculture ponds needing on-site expansion of PSB bacterial solution in open-air culture tanks, and water remediation projects requiring on-site preparation of PSB bacterial solution in mobile culture devices, these scenarios lack the operational conditions for autoclaving equipment such as autoclaves, as well as the equipment conditions for membrane filtration. In open environments where sterilization is not possible, rapidly growing heterotrophic bacteria such as Pseudomonas and Bacillus, as well as facultative anaerobic bacteria such as Escherichia coli, will compete with PSB for nutrients in the culture medium, leading to inhibited PSB growth, bacterial solution collapse (turning black, green, or gray, producing an odor, and a sharp increase in ammonia nitrogen concentration), and culture failure.

[0005] Existing solutions all rely on external equipment or operations to control contamination, failing to achieve selective inhibition through the culture medium formulation itself. While anaerobic light conditions do exhibit some selectivity for aerobic bacteria, they are insufficient to inhibit facultative anaerobes, which can still multiply using the organic nutrients in the culture medium under anaerobic conditions. Furthermore, existing culture media are all single-use, requiring re-preparation and addition for each culture, resulting in cumbersome operations and high labor costs.

[0006] In the application of photosynthetic bacteria in water pollution control (such as black and odorous water treatment, river and lake ecological restoration, aquaculture wastewater purification, and control of endogenous pollution in bottom sediments), existing technologies generally adopt the bacterial agent addition route. That is, the bacteria are first cultured in the laboratory or factory, and then the live bacteria are immobilized into bacterial agents through adsorption, encapsulation, etc., before being added to the polluted water body. For example, CN109609406A discloses a slow-release microbial agent for black and odorous water treatment, which is prepared into slow-release granules by adsorption, freeze-drying, and cross-linking with PVA-sodium alginate-chitosan; CN113683277A discloses a slow-release disintegrating microbial preparation for treating bottom sediments of black and odorous water bodies, which disintegrates and releases bacteria within 15 to 60 minutes after the granules are added to the water.

[0007] CN121380051A discloses a submersible slow-release aquaculture water quality improvement composite bacterial tablet. The core material contains immobilized live bacteria such as Bacillus subtilis, Bacillus licheniformis freeze-dried powder and photosynthetic bacteria composite preparation. The nutrient matrix is ​​water-soluble nutrients such as molasses and yeast extract. The outer layer is covered with a sodium alginate-calcium chloride microcapsule slow-release layer.

[0008] The above-mentioned bacterial agent addition routes share the following shortcomings: First, the bacterial quantity is fixed at the factory. The number of live bacteria in the immobilized bacterial agent is fixed during production. After being added to polluted water, the bacterial count only decreases and does not multiply, requiring periodic re-addition, resulting in high long-term operating costs. Second, nutrients are rapidly released. The nutrients in this type of bacterial agent are mostly water-soluble sugars such as brown sugar and black sugar, which are depleted within a few days after being added to the water, making it difficult to support long-term bacterial colonization. Third, colonization is unstable. The proportion of photosynthetic bacteria in the compound bacterial agent is limited. After being added to open water, there is a lack of selectivity when competing with a large number of indigenous microorganisms. The bacteria are easily diluted by the water flow or eliminated by competition, resulting in unstable colonization efficiency and purification effect.

[0009] On the other hand, CN110373355A discloses a long-lasting culture medium for the selective cultivation of photosynthetic bacteria. Although it enables the selective cultivation of photosynthetic bacteria under non-sterilized conditions, the cultivation process must be carried out in a culture container isolated from the outside environment, and it cannot be directly applied to open water bodies. Therefore, how to achieve the selective cultivation of photosynthetic bacteria in non-sterilized open polluted water bodies, and enable them to proliferate in situ and become the dominant bacterial group, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0010] To address the shortcomings of existing technologies, such as fixed bacterial count at the factory, rapid nutrient release, and unstable colonization, as well as the limitation that non-sterile selective culture can only be achieved in culture containers and cannot be applied to open water bodies, the present invention aims to provide a slow-release amplification granule for in-situ selective culture of photosynthetic bacteria in polluted water bodies, its preparation method, and its application. In open, non-sterile polluted water environments containing various indigenous bacteria, the slow-release amplification granule itself enables the selective culture of photosynthetic bacteria, allowing in-situ proliferation of inoculated or inherent photosynthetic bacteria in the water body to become the dominant bacterial population.

[0011] To achieve the above objectives, the present invention provides the following technical solution: A slow-release amplification granule for in-situ selective cultivation of photosynthetic bacteria in polluted water is made by plasticizing and extruding a raw material containing the following components in parts by weight: 40-90 parts protein powder, 5-30 parts biodegradable aliphatic polyester, 1-5 parts gelling agent, 0-5 parts inorganic salt, 5-30 parts density regulator, 1-5 parts plasticizer, 0.1-2 parts dispersant, and 0.1-1 parts lubricant. The granules do not contain immobilized bacteria, have a true density of 1.05-1.30 g / cm³, and a particle size of 3-10 mm. After being added to polluted water, the granules settle at the sediment-water interface or remain suspended in the water. Nutrients are released gradually by the gradual hydrolysis of the protein, and the nutrient release rate is controlled by the biodegradable aliphatic polyester skeleton, allowing inoculated or water-native photosynthetic bacteria to attach to the granule surface and proliferate into a dominant bacterial population.

[0012] Furthermore, the protein powder is one or more of soybean protein powder, corn protein powder, wheat protein powder, cottonseed protein powder, and peanut protein powder, and the dosage is 55 to 90 parts.

[0013] Furthermore, the biodegradable aliphatic polyester is one or more of polyhydroxyalkanoates, polylactic acid, polycaprolactone, and polybutylene succinate, and is used in an amount of 8 to 20 parts.

[0014] Further, the density regulator is one or more of heavy calcium carbonate, silica powder and barium sulfate, and the dosage is 5 to 20 parts; the true density of the particles is 1.10 to 1.30 g / cm³, and after being added to the polluted water body, they settle to the sediment-water interface; or the true density of the particles is 1.05 to 1.10 g / cm³, and after being added to the polluted water body, they slowly sink or are approximately suspended.

[0015] Furthermore, the particles do not disintegrate when immersed in water, the mass loss rate is ≤50% after soaking in still water at 25℃ for 30 days, and the nutrient release cycle is ≥30 days.

[0016] Further, the gelling agent is one or more of methylcellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, sodium alginate, and chitosan; the plasticizer is one or more of glycerol, ethylene glycol, polyethylene glycol, urea, epoxidized soybean oil, and maleic anhydride; the lubricant is one or more of magnesium stearate, paraffin wax, and beeswax; the dispersant is one or more of light calcium carbonate and glyceryl monostearate; and the inorganic salt is one or more of calcium phosphate, potassium sulfate, and magnesium sulfate.

[0017] The present invention also provides a method for preparing the above-mentioned sustained-release cultured granules, comprising the following steps: S1. Grind the protein powder, pass it through a 150-200 mesh sieve, and dry it until the moisture content is below 1%. S2. Add the dried protein powder and plasticizer to a high-speed mixer, heat to 80-100℃ at 500-1000 rpm for 15-45 min, and cool to room temperature to obtain plasticized protein powder. S3. Add biodegradable aliphatic polyester, gelling agent, inorganic salt, dispersant, lubricant and density regulator to the plasticized protein powder, and mix at high speed for 15-30 min to obtain a mixture; S4. The mixture is extruded and granulated using a screw extruder, with the three temperature stages being 80-90℃, 100-110℃, and 110-120℃ respectively, the screw speed being 10-20 rpm, and the die head temperature being 110-120℃. S5. Perform cold or hot granulation, and sieve to obtain the slow-release expanded granules in the 3-10 mm particle size range.

[0018] The present invention also provides a method for in-situ remediation of polluted water, comprising the following steps: adding the above-mentioned slow-release propagation granules to the polluted water at a dosage of 0.05–0.5 kg / m³ of water; simultaneously inoculating photosynthetic bacteria solution at a dosage of 0.1%–5% (v / v) of the polluted water, or without inoculating photosynthetic bacteria solution, relying on the nutrients slowly released by the slow-release propagation granules to naturally enrich the photosynthetic bacteria inherent in the polluted water; under anaerobic light conditions, the photosynthetic bacteria proliferate into a dominant bacterial community on the surface of the granules and in the surrounding water, degrading organic matter in the water, converting ammonia nitrogen, and oxidizing sulfides; the nutrient release cycle is ≥30 days, and the granules are replenished after their nutrients are exhausted.

[0019] Furthermore, the polluted water body is a black and odorous water body. The slow-release propagation particles settle to the sediment-water interface and form a photosynthetic bacterial microdomain at the sediment-water interface, which intercepts and purifies the release of endogenous pollutants from the sediment.

[0020] Furthermore, the slow-release propagation granules are added according to the pollution load of the polluted water body, with localized and intensified addition in heavily polluted areas downstream of the sewage outlet, in the sediment enrichment area, and in the stagnant water area.

[0021] The beneficial effects of this invention are as follows: (1) The particles of this invention do not contain immobilized bacteria, and the bacterial count is not fixed upon addition. The photosynthetic bacteria proliferate in a self-regulating manner within the biofilm on the particle surface according to the pollution load. A single addition can provide a continuous supply of bacteria, overcoming the shortcomings of existing immobilized bacterial agents where the bacterial count is fixed at the factory and only decreases without proliferating after addition. Under in-bottle culture conditions, the concentration of photosynthetic bacteria was maintained at over 1 billion CFU / mL for 10 consecutive culture cycles (see Examples 1 and 2).

[0022] (2) The main nutrient component of this invention is plasticized protein powder. The release rate is controlled by the coupling of degradation of the biodegradable aliphatic polyester skeleton and gradual hydrolysis of the protein. The nutrient release cycle is ≥30 days. Compared with the rapid release method of water-soluble sugar nutrients, which is exhausted in a few days, the selective pressure can be maintained for several weeks, so that the time window of selective culture matches the establishment cycle of the dominant photosynthetic bacterial community. Under in-bottle culture conditions, a single addition of granules can continuously supply nutrients for 10 culture cycles (more than 40 days) (see Examples 1 and 2); the particle-level release behavior is measured in Example 3.

[0023] (3) The slow-release protein hydrolysis products (peptides, amino acids) in the particles, together with the organic acids, sulfides and ammonia in the water, constitute a substrate spectrum that photosynthetic bacteria can utilize efficiently, while other bacteria have difficulty utilizing it under anaerobic light. Together with the anaerobic light conditions, they form a dual selection pressure on photosynthetic bacteria, enabling photosynthetic bacteria to attach to the particle surface in open water, form a biofilm and proliferate in situ to become the dominant bacterial group, thus overcoming the shortcomings of existing bacterial agents that cause unstable colonization after addition. The ability to maintain the dominance of photosynthetic bacteria under non-sterilized conditions has been verified under in-bottle culture conditions (see Examples 1 and 2). In the in situ remediation system, the total relative abundance of photosynthetic bacteria (Heterochromobacterium, Thiobaca, and Rhodopseudomonas) reached 52.84%, making them the absolutely dominant group in the community. In contrast, photosynthetic bacteria did not form a dominance in the control system, which was directly granulated without the addition of plasticizers. More importantly, this control system did not form a dominance even when inoculated with the same amount of photosynthetic bacteria, indicating that the above-mentioned selective culture effect did not come from the simple addition of direct protein granulation, but from the continuous maintenance of selective pressure by the granule slow-release system (see Example 5).

[0024] (4) The selective inhibition ability of the present invention originates from the granule formulation itself, eliminating the need for external equipment and operational procedures such as sterilizers, membrane filters, chemical disinfectants, or closed photobioreactors. This allows the cultivation of photosynthetic bacteria to move from a sterile laboratory environment to an open environment at the engineering site, significantly reducing cultivation costs and equipment investment. Data from Example 5 show that the relative abundance of Acinetobacter spp., Desulfuric microorganisms, and other miscellaneous bacteria in the granule system of the present invention is only 0.35% and 0.03%, respectively, which is significantly lower than that of the control system directly granulated without plasticizer (15.11% and 2.98%, respectively), demonstrating a clear selective inhibition effect.

[0025] (5) All components of the particles of the present invention are conventional industrial raw materials, which can be produced in large-scale continuous production by screw extruder. The preparation process is mature, the equipment is versatile, and the processing cost is low, which is conducive to industrialization and promotion.

[0026] (6) Particles settle to the bottom sediment-water interface and continuously release nutrients, supporting photosynthetic bacteria to proliferate in situ in the polluted water body and become the dominant bacterial group, simultaneously degrading organic matter, converting ammonia nitrogen and oxidizing sulfides, thus achieving in situ remediation of polluted water bodies (see Example 4 for in situ remediation verification).

[0027] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 This is a schematic diagram showing the slow-release propagation particles of the present invention settling at the sediment-water interface to form a nutrient slow-release microdomain and a photosynthetic bacterial biofilm. Figure 2 This is a diagram showing the relative abundance composition of the microbial community at the genus level in the in-situ remediation system of this invention; Figure 3 A graph showing the relative abundance composition of the microbial community at the genus level in the control system of direct protein granulation without the addition of plasticizers; Figure 4 This is a comparison diagram of the relative abundance of microbial communities at the genus level in the in-situ remediation system and the control system of this invention. Detailed Implementation

[0029] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0030] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0031] The slow-release culture granules of the present invention are, by weight, made from raw materials comprising the following components through plasticizing extrusion granulation: 40-90 parts protein powder, preferably 55-90 parts; 5-30 parts biodegradable aliphatic polyester, preferably 8-20 parts; 1-5 parts gelling agent; 0-5 parts inorganic salt; 5-30 parts density regulator, preferably 5-20 parts; 1-5 parts plasticizer; 0.1-2 parts dispersant; and 0.1-1 parts lubricant. The granules do not contain immobilized bacterial cells and have a particle size of 3-10 mm (5-8 mm for sedimentation type).

[0032] The functions and interrelationships of each component during the in-situ amplification process are as follows: (1) Protein powder as the main nutrient: After the granules are immersed in water, the protein is gradually hydrolyzed into peptides and amino acids by microorganisms (including extracellular proteases secreted by photosynthetic bacteria) on the surface and inside of the granules and released slowly, serving as a source of nitrogen, carbon and electron donors for photosynthetic bacteria. The slow hydrolysis process of protein allows the nutrient release cycle to last for several weeks, which is different from the rapid release of water-soluble carbohydrates. Protein powder can be selected from one or more of soybean protein powder, corn protein powder, wheat protein powder, cottonseed protein powder and peanut protein powder. Different protein sources have different hydrolysis rates and product profiles, which can be used to adjust the release rate and bacterial selectivity.

[0033] (2) Degradable aliphatic polyester as the sustained-release backbone: one or more of polyhydroxyalkanoates (PHA), polylactic acid (PLA), polycaprolactone (PCL), and polybutylene succinate (PBS) can be selected. Hydrophobic polyesters swell and degrade at a low rate in water, forming an interpenetrating matrix with plasticized proteins, controlling the rate of diffusion of protein hydrolysis products out of the particles, which is the main controlling factor of sustained-release behavior; the final degradation products are carbon dioxide and water, with no secondary pollution.

[0034] (3) After absorbing water and swelling, the gelling agent (methylcellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, sodium alginate, chitosan, etc.) forms a water conduction channel inside the particle, so that the protein inside the particle can also participate in hydrolysis, avoiding the loss of effective capacity caused by only the surface layer being consumed.

[0035] (4) Density regulators (heavy calcium carbonate, silica powder, barium sulfate, etc.) adjust the true density of particles to 1.05-1.30 g / cm³: the sediment-type particles with a true density of 1.10-1.30 g / cm³ settle to the bottom sediment-water interface after entering the water. This interface is the main pollution accumulation area and endogenous release area of ​​black and odorous water bodies and high-load organic wastewater. It can make the nutrient slow-release micro-domain and the pollution release area spatially overlap; the near-suspended particles with a true density of 1.05-1.10 g / cm³ slowly sink or are nearly suspended in the water body, which is suitable for the purification needs of surface water bodies.

[0036] (5) Plasticizers (glycerol, ethylene glycol, polyethylene glycol, urea, epoxidized soybean oil, maleic anhydride, etc.) are used to plasticize proteins and improve extrusion processability. Maleic anhydride undergoes a grafting reaction with proteins during plasticization and also has the effect of protein modification. Lubricants (magnesium stearate, white wax, beeswax, etc.) and dispersants (light calcium carbonate, glyceryl monostearate, etc.) are processing aids. Inorganic salts (calcium phosphate, potassium sulfate, magnesium sulfate) supplement phosphorus, potassium and magnesium. Among them, sulfur and magnesium elements have a direct supporting effect on sulfur metabolism of photosynthetic bacteria and photosynthetic pigment synthesis.

[0037] The release behavior of the particles of this invention is characterized by: non-disintegration upon immersion in water, a mass loss rate of ≤50% after 30 days of static water immersion at 25°C, and a nutrient release cycle of ≥30 days. This release behavior is controlled by the coupled rate of degradation of the biodegradable aliphatic polyester backbone and gradual hydrolysis of the protein, which is different from the disintegration time limit control of disintegrating formulations and the dissolution and diffusion control of water-soluble nutrient balls.

[0038] The environmental conditions for the application and propagation of the particles of this invention are anaerobic light: the light intensity can be in the range of 500 to 4000 lux (which can be adjusted in stages, such as about 500 lux in the early stage of propagation and about 1500 lux in the later stage), and the pH should be 7.5 to 8.5.

[0039] like Figure 1 As shown, the process of the slow-release propagation particles of the present invention after being added to polluted water is as follows: the particles settle to the sediment-water interface → the protein is gradually hydrolyzed to release nutrients and form a nutrient slow-release microdomain around the particles → photosynthetic bacteria attach to the particle surface to form a biofilm → sulfides, ammonia nitrogen and organic matter released from the sediment are utilized and transformed by photosynthetic bacteria as they migrate upward through the microdomain, thus intercepting and purifying the release of endogenous pollutants. Figure 1 In the diagram, water is on top and sediment is below. Particles are distributed at the sediment-water interface. The halo around the particles represents the nutrient slow-release microdomain, the shaded layer on the particle surface represents the photosynthetic bacterial biofilm, the upward arrow indicates the migration direction of endogenous pollutants in the sediment, and the arrows from the particles outward indicate the slow-release diffusion direction of nutrients.

[0040] Example 1 Formula (by weight): 86.4 parts soybean protein powder, 10 parts polyhydroxyalkanoate, 1 part carboxymethyl cellulose, 0.1 parts calcium phosphate, 0.1 parts potassium sulfate, 0.1 parts magnesium sulfate, 1 part light calcium carbonate (dispersant), 10 parts heavy calcium carbonate (density regulator), 1 part glycerin, 0.4 parts epoxidized soybean oil, and 0.1 parts paraffin wax.

[0041] Preparation: Grind soybean protein and sieve it (sieve mesh range of 150-200 mesh is acceptable, 200 mesh is preferred in this embodiment), and dry it until the moisture content is below 1%; add the dried protein powder, glycerin, and epoxidized soybean oil to a high-speed mixer, heat to 90℃ at 750 rpm for 30 min (speed range of 500-1000 rpm, temperature range of 80-100℃, and plasticizing time range of 15-45 min is acceptable, 750 rpm, 90℃, and 30 min are preferred in this embodiment), cool to room temperature to obtain plasticized protein powder; add polyhydroxyalkanoates, carboxymethyl cellulose, calcium phosphate, potassium sulfate, magnesium sulfate, light calcium carbonate, heavy calcium carbonate, and paraffin wax to the plasticized protein powder, and mix at high speed for 15 min (mixing time range of 15-30 min). The mixture is obtained by extruding the mixture through a single-screw extruder. The three-stage temperatures are 90℃, 110℃, and 120℃ (the three-stage temperatures can be within the range of 80~90℃, 100~110℃, and 110~120℃ respectively), the screw speed is 12 rpm (the speed can be within the range of 10~20 rpm), and the die head temperature is 120℃. After drawing and cold cutting granulation, 5~8 mm particles are obtained by sieving (the particle size can be within the range of 3~10 mm, and 5~8 mm is preferred for the settling type), which yields soybean protein-based settling slow-release expanded granules. The true density of the particles is adjusted to the range of 1.10~1.30 g / cm³ by adjusting the amount of heavy calcium carbonate.

[0042] Cultivation Validation: The granules prepared in this example were added to an anaerobic bottle with an effective volume of 500 mL at a dosage of 50 g / L, and simultaneously inoculated with 10% (v / v) photosynthetic bacteria culture. The bottle was then incubated statically under 2000 lux light, with a cultivation cycle of 4 days. In each cycle, 90% of the supernatant was drained and the same volume of tap water was added. This process was repeated for 10 cycles. The results showed that the concentration of photosynthetic bacteria reached over 1 billion CFU / mL for all 10 cultivation cycles, indicating that the granules can sustain nutrient release for more than 40 days and maintain photosynthetic bacterial dominance even under non-sterilized conditions.

[0043] Example 2 Formula (by weight): 70 parts corn gluten, 20 parts polyhydroxyalkanoates, 4.3 parts polycaprolactone, 1 part sodium alginate, 1 part hydroxypropyl methylcellulose, 1 part calcium phosphate, 0.5 parts magnesium sulfate, 0.2 parts glyceryl monostearate, 2 parts maleic anhydride, 0.5 parts magnesium stearate, and 8 parts heavy calcium carbonate (density regulator).

[0044] Preparation: Grind corn protein through a 150-mesh sieve and dry it to a moisture content of less than 1%. Add the dried protein powder and maleic anhydride to a high-speed mixer and heat it to 80°C at 500 rpm for 20 min to plasticize it. Cool it to room temperature to obtain plasticized protein powder. Add the remaining materials and mix at high speed for 15 min to obtain a mixture. Extrude the mixture into granules using a single-screw extruder with three temperature stages of 80°C, 100°C, and 110°C, a screw speed of 15 rpm, and a die head temperature of 110°C. Cold-cut and granulate the mixture, and sieve to obtain 5-8 mm particles to obtain corn protein-based slow-release expanded granules.

[0045] Cultivation Validation: The granules prepared in this example were added to an anaerobic bottle with an effective volume of 500 mL at a dosage of 50 g / L, and simultaneously inoculated with 10% (v / v) photosynthetic bacteria culture. The mixture was then statically cultured under 2000 lux light, with a cultivation cycle of 4 days. In each cycle, 90% of the supernatant was drained and the same volume of tap water was added. This process was repeated for 10 cycles. The results showed that the concentration of photosynthetic bacteria reached over 2 billion CFU / mL for all 10 cultivation cycles, higher than the 1 billion CFU / mL of the soybean protein-based granules in Example 1, indicating that the slow-release nutrients from the corn protein-based granules were more abundant.

[0046] Example 3 Slow-release behavior determination: 20 g of the granules prepared in Example 1 were added to 1 L of tap water and allowed to stand at 25°C. Samples were taken on days 1, 7, 14, and 30, dried at 60°C to constant weight, and weighed to calculate the cumulative mass loss rate. Simultaneously, the soaking water sample was taken, and the total nitrogen content was determined using the Kjeldahl method or a nitrogen assay kit to calculate the cumulative total nitrogen release (as a percentage of the total nitrogen content of the granules). The results showed that the cumulative mass loss rate did not exceed 50% on day 30; the cumulative total nitrogen release continued to increase and did not reach a release plateau on day 30, indicating a nutrient release cycle ≥ 30 days.

[0047] The sustained-release performance was characterized by the sustained-release coefficient K = c / t1 / 2 (where c is the nutrient release concentration and t1 / 2 is the half-release period), or by the time series of the remaining nutrient rate on days 7, 14, and 30.

[0048] Example 4 In-situ remediation experiment of black and odorous water body: 20 L of black and odorous water body (background COD about 300 mg / L, ammonia nitrogen about 25 mg / L, sulfide about 2.5 mg / L, transparency about 15 cm) was taken into an open container, and the granules prepared in Example 1 were added. The amount added can be in the range of 0.05-0.5 kg / m³ of water body. In this example, 0.5 kg / m³ (i.e., 10 g added to 20 L of water body) was used. At the same time, Rhodopseudomonas palustris bacterial solution was inoculated. The inoculation amount can be in the range of 0.1%-5% (v / v). In this example, 1% (v / v) was used. The mixture was then incubated under natural light for 30 days. Samples were taken on days 4, 8, 15, and 30 to determine the concentrations of COD, ammonia nitrogen, sulfide, and transparency, and to count the concentration of photosynthetic bacteria. COD was determined using the potassium dichromate method, and ammonia nitrogen was determined using Nessler's reagent spectrophotometry. Microscopic examination (400×) was used to observe the formation of bacterial film on the particle surface and the proportion of photosynthetic bacteria to the total number of bacteria.

[0049] After the culture was completed, the particle samples were fixed, dried and observed by scanning electron microscopy (SEM). The pores on the particle surface and the morphology of bacterial attachment were used to characterize the entry and attachment of photosynthetic bacteria into the particles.

[0050] Microscopic examination on day 30 revealed a distinct bacterial film on the particle surface, with photosynthetic bacteria being the dominant bacterial group in the system. These results indicate that the slow-release nutrients from the particles can support the continuous proliferation and maintain the dominant position of photosynthetic bacteria in open, non-sterilized water bodies. COD, ammonia nitrogen, and sulfides decreased synchronously with the culture time, achieving in-situ remediation of polluted water bodies.

[0051] Example 5 Microbial community composition analysis of the remediation system: Samples from the culture system were taken, total DNA was extracted, and 16S rRNA genes were sequenced using high-throughput sequencing. The relative abundance of each genus group was statistically analyzed at the genus level, and a genus-level relative abundance composition diagram of the microbial community was drawn (see the in-situ remediation system of this invention). Figure 2 See the comparison system. Figure 3 The two groups are compared. Figure 4 ). Figure 4In the figure, the vertical axis represents relative abundance (%), and the relative abundance of different genera within each system is represented by stacked segments. Sequencing results showed that photosynthetic bacteria were the dominant group in the system, with *Allochromatium* having a relative abundance of 47.47%, *Thiobaca* 3.03%, and *Rhodopseudomonas* 2.34%, all three being photosynthetic bacteria, with a combined relative abundance of 52.84%. Other groups included unnamed genera from the family Acidaminococcaceae (7.01%), unnamed genera from the family Rikenellaceae (4.74%), *Acetobacterium* (4.62%), and *Acetoanaerobium* (3.12%). As a control, the microbial community composition of the culture system of control granules prepared directly from protein powder without the addition of plasticizers was also analyzed (see also). Figure 3 , Figure 4The control experiment was identical to the in-situ remediation system of this invention, except for the different particles used, including the contaminated water, inoculum and inoculum size, culture conditions, and sampling time. Sequencing results from the control group showed that the most abundant taxa in the control system were *Acinetobacter* (15.11%), *Acetobacterium* (7.90%), unnamed genera from the family Anaerolineaceae (6.47%), unnamed genera from the family Rikenellaceae (6.15%), *Macellibacteroides* (5.28%), and unnamed genera from the family Acidaminococcaceae (4.94%). No photosynthetic bacteria were found among the top 20 most abundant taxa, indicating that photosynthetic bacteria did not form a dominant taxa; the community was dominated by miscellaneous bacteria such as *Acinetobacter*. The comparison of the two sets of data shows that the total relative abundance of photosynthetic bacteria in the particle system of the present invention reaches 52.84%, while the relative abundance of Acinetobacter spp. (15.11%), which is the highest in the control group, is only 0.35% in the particle system of the present invention. The relative abundance of desulfomicrobium (sulfate-reducing bacteria) in the control group is 2.98%, while it is only 0.03% in the particle system of the present invention. This indicates that the plasticized slow-release system of the particles of the present invention supports the selective proliferation of photosynthetic bacteria into the dominant bacterial group, while significantly inhibiting miscellaneous bacteria such as Acinetobacter spp. and sulfate-reducing bacteria that produce sulfur compounds. The above results indicate that in open, unsterilized polluted water environments, the inoculated photosynthetic bacteria proliferated into the dominant bacterial population after in situ amplification using slow-release granules, demonstrating the successful selective culture of photosynthetic bacteria by the granules. In contrast, although the control system was treated with the same amount of protein granules without plasticizers, the photosynthetic bacteria still failed to gain dominance in community competition. This suggests that simply adding protein granules without plasticizers cannot establish and maintain the dominant position of photosynthetic bacteria in open polluted water bodies. Maintaining selective pressure continuously through a slow-release system of plasticized protein powder is the key to achieving in situ selective culture of photosynthetic bacteria.

[0052] The application method of the slow-release amplification granules of this invention is as follows: by differentiating the amount of density regulator, two types are formed to match the pollution scenario, namely the sinking type (true density 1.10~1.30 g / cm³) and the near-suspended type (true density 1.05~1.10 g / cm³).

[0053] (1) In-situ remediation of black and odorous water bodies (sediment interface type): Select sediment-type particles and evenly spread them at a rate of 0.1 to 0.5 kg / m³ (or measure the amount per unit area in g / m² based on the sediment area). The particles can be added in zones according to the pollution load of the polluted water body, with localized and intensified addition in heavily polluted areas downstream of the sewage outlet, sediment enrichment areas, and dead water areas. After the particles settle to the sediment-water interface, a nutrient slow-release microdomain is formed at the interface. Inoculate with 0.5% to 2% (v / v) of purple non-sulfur bacteria such as Rhodopseudomonas palustris, or rely on the natural enrichment of photosynthetic bacteria inherent in the sediment. Photosynthetic bacteria use sulfides as electron donors and small molecule organic acids as carbon sources, which matches the substrate spectrum of black and odorous water bodies. The slow-release nutrients in the particle interface microdomain and the sulfides and ammonia nitrogen in the water constitute a dual substrate supply, enabling photosynthetic bacteria to occupy a nutrient site advantage in competition with sulfate-reducing bacteria and other miscellaneous bacteria, oxidizing hydrogen sulfide from the source and inhibiting the formation of black and odorous water. Supplement with the slow-release agent after 30-60 days; once transparency improves and light can reach the surface, the photosynthetic bacteria action layer moves downward, forming a self-reinforcing repair mechanism.

[0054] (2) Purification of polluted river / lake / reservoir water (suspended type): Near-suspended particles (3-5 mm in diameter) are selected and added at 0.05-0.2 kg / m³. The particles slowly release nutrients in the water, supporting photosynthetic bacteria to form bacterial-particle flocs. During the slow release period, the bacteria remain in the water with the particles, preventing them from being lost with the flow. In river sections with faster flow, slow-release bags or permeable enclosures can be used to fix the particles in net bags on the bank or riverbed. The timing of replenishment is determined according to the cross-sectional water quality monitoring data (COD, ammonia nitrogen, transparency).

[0055] (3) Purification of aquaculture wastewater and ponds: Select sinking or near-suspended granules and add them at 0.05-0.15 kg / m³; after in-situ propagation of photosynthetic bacteria, they degrade ammonia nitrogen and small molecule organic acids on the one hand, and enter the food chain as feed organisms on the other hand; the granules do not contain exogenous fixed bacteria, do not introduce the risk of contaminating bacteria, and are suitable for the microbial safety requirements of aquaculture scenarios. Replenish the granules every aquaculture cycle or when water quality indicators deteriorate.

[0056] (4) Emergency treatment of high load impact (sewage outlet, stormwater and sewage mixed flow impact): After the impact occurs, add more sedimentary granules (0.3-0.5 kg / m³) to the polluted river section and inoculate with 1%-5% (v / v) photosynthetic bacteria solution. The slow-release nutrients of the granules support the rapid expansion of the bacteria within 24-72 hours to form a dominant bacterial group to cope with the impact load.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A slow-release amplification granule for in-situ selective culture of photosynthetic bacteria in polluted water, characterized in that, The granules are made from raw materials containing the following components by weight through plasticizing extrusion and granulation: 40-90 parts protein powder, 5-30 parts biodegradable aliphatic polyester, 1-5 parts gelling agent, 0-5 parts inorganic salt, 5-30 parts density regulator, 1-5 parts plasticizer, 0.1-2 parts dispersant, and 0.1-1 parts lubricant; the granules do not contain immobilized bacteria, have a true density of 1.05-1.30 g / cm³, and a particle size of 3-10 mm.

2. The sustained-release culture granules according to claim 1, characterized in that, The protein powder is one or more of soybean protein powder, corn protein powder, wheat protein powder, cottonseed protein powder, and peanut protein powder, and the dosage is 55 to 90 parts.

3. The sustained-release culture granules according to claim 1, characterized in that, The biodegradable aliphatic polyester is one or more of polyhydroxy fatty acid esters, polylactic acid, polycaprolactone, and polybutylene succinate, and is used in an amount of 8 to 20 parts.

4. The sustained-release culture granules according to claim 1, characterized in that, The density regulator is one or more of heavy calcium carbonate, silica powder and barium sulfate, and is used in an amount of 5 to 20 parts; the particles are settling type with a true density of 1.10 to 1.30 g / cm³; or the particles are near-suspended type with a true density of 1.05 to 1.10 g / cm³.

5. The sustained-release amplification granules according to claim 1, characterized in that, The particles do not disintegrate when immersed in water, and the mass loss rate after soaking in still water at 25°C for 30 days is ≤50%, with a nutrient release period of ≥30 days.

6. The sustained-release amplification granules according to claim 1, characterized in that, The gelling agent is one or more of methylcellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, sodium alginate, and chitosan; the plasticizer is one or more of glycerol, ethylene glycol, polyethylene glycol, urea, epoxidized soybean oil, and maleic anhydride; the lubricant is one or more of magnesium stearate, paraffin wax, and beeswax; the dispersant is one or more of light calcium carbonate and glyceryl monostearate; and the inorganic salt is one or more of calcium phosphate, potassium sulfate, and magnesium sulfate.

7. A method for preparing the sustained-release amplification granules according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1. Grind the protein powder, pass it through a 150-200 mesh sieve, and dry it until the moisture content is below 1%. S2. Add the dried protein powder and plasticizer to a high-speed mixer, heat to 80-100℃ at 500-1000 rpm for 15-45 min, and cool to room temperature to obtain plasticized protein powder. S3. Add biodegradable aliphatic polyester, gelling agent, inorganic salt, dispersant, lubricant and density regulator to the plasticized protein powder, and mix at high speed for 15-30 min to obtain a mixture; S4. The mixture is extruded and granulated using a screw extruder, with the three temperature stages being 80-90℃, 100-110℃, and 110-120℃ respectively, the screw speed being 10-20 rpm, and the die head temperature being 110-120℃. S5. Perform cold or hot granulation, and sieve to obtain the slow-release expanded granules in the 3-10 mm particle size range.

8. A method for in-situ remediation of polluted water bodies, characterized in that, Includes the following steps: Add the slow-release amplification granules according to any one of claims 1 to 6 to the polluted water body at a dosage of 0.05 to 0.5 kg / m³ of water body; inoculate with photosynthetic bacteria culture at a dosage of 0.1% to 5% (v / v) of the polluted water body, or do not inoculate with photosynthetic bacteria culture; culture under anaerobic light conditions, wherein the nutrient release cycle of the slow-release amplification granules is ≥30 days, and the slow-release amplification granules are replenished after the nutrients are exhausted.

9. The in-situ remediation method for polluted water according to claim 8, characterized in that, The polluted water body is a black and odorous water body. The slow-release propagation particles settle to the sediment-water interface and form a photosynthetic bacteria microdomain at the sediment-water interface, which intercepts and purifies the release of endogenous pollutants from the sediment.

10. The in-situ remediation method for polluted water according to claim 8, characterized in that, The slow-release propagation granules are added according to the pollution load of the polluted water body, with localized and intensified addition in heavily polluted areas downstream of the sewage outlet, in the sediment-rich area, and in the stagnant water area.

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