Use of a hydrogen substrate bacteria-algal symbiotic membrane system in improving the yield and / or quality of tilapia

Abstract

This invention provides an application of a hydrogen-based microbial-algae symbiotic membrane system in improving tilapia yield and / or quality, relating to the field of aquatic ecological aquaculture technology. This invention utilizes a hydrogen-based microbial-algae symbiotic membrane system to promote tilapia farming. The system employs a hydrogen-releasing membrane to slowly release hydrogen, achieving stable transport of high-purity hydrogen in the water without affecting tilapia growth. The hydrogen-based microbial-algae biofilm is rich in beneficial bacteria and algae, promoting fish growth performance, improving fish survival rate and yield, and enhancing fish meat quality.

Description

Technical Field

[0001] This invention belongs to the field of aquatic ecological aquaculture technology, specifically relating to the application of a hydrogen-based microbial-algae symbiotic membrane system in improving tilapia yield and / or quality. Background Technology

[0002] Under intensive aquaculture conditions, high stocking densities can cause oxidative stress, further damaging intestinal and muscle tissue, thus affecting yield and muscle quality. Hydrogen has antioxidant, anti-inflammatory, and anti-apoptotic effects, which can improve the gut microbiota of farmed animals, promote their health, and improve their muscle quality. However, hydrogen has low solubility in water and escapes rapidly. Producing hydrogen-rich water for aquaculture not only requires significant energy consumption but also has low hydrogen utilization, necessitating frequent replacement of the hydrogen-rich water. This process is complex and costly.

[0003] Patent CN 117902736 A discloses a hydrogen-based algae-bacterial symbiotic membrane system and its application in nitrogen and phosphorus removal and suppression of golden apple snails. Utilizing the characteristic of eutrophic water bodies prone to algal blooms, it attaches algae grown under natural light to a hollow fiber membrane to form an algae-bacterial symbiotic membrane, thereby alleviating the problem of algal blooms and suppressing the growth of golden apple snails in the water. However, this patented technology inserts the algae-bacterial symbiotic membrane into a wetland system. The types of algae and bacteria in the bio-symbiotic membrane are inevitably affected by the wetland sediment. What impact would it have if it were used directly for tilapia aquaculture without being placed in a wetland? There are no relevant reports in the existing technology. Summary of the Invention

[0004] The main objective of this invention is to provide an application of a hydrogen-based bacterial-algae symbiotic membrane system in improving tilapia yield and / or quality.

[0005] Another objective of this invention is to provide an application of a hydrogen-based bacterial-algae symbiotic membrane system in increasing the abundance of beneficial bacteria in the gut microbiota of tilapia and / or inhibiting pathogenic bacteria in the gut microbiota of tilapia.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides an application of a hydrogen-based bacterial-algae symbiotic membrane system in improving tilapia yield and / or quality.

[0008] The present invention also provides an application of a hydrogen-based algal-bacterial symbiotic membrane system in the following a and / or b:

[0009] a. Increase the abundance of beneficial bacteria in the gut microbiota of tilapia;

[0010] b. Inhibit pathogenic bacteria in the gut microbiota of tilapia.

[0011] Preferably, the hydrogen-based algae-bacterial symbiotic membrane system includes a hydrogen slow-release membrane and an algae-bacterial biofilm.

[0012] Preferably, the hydrogen slow-release membrane includes a membrane module and a hydrogen source; the membrane module contains a strip-shaped hollow fiber membrane, and the top of the membrane module is connected to the hydrogen source through a gas pipeline; the hydrogen source is a gas bag or cylinder containing high-purity hydrogen, or a hydrogen gas generator, or a hydrogen-producing material, or hydrogen produced by electrolysis of water; the purity of the high-purity hydrogen is >99%.

[0013] Preferably, at least one membrane module is provided per cubic meter of aquaculture pond, and each membrane module contains 30 to 50 hollow fiber membranes; the hydrogen consumption is 1.8 to 2.2 L / day.

[0014] Preferably, the hollow fiber membrane is made of polyvinylidene fluoride (PVDF) or polypropylene (PP); the pore size of the hollow fiber membrane is 0.2 μm.

[0015] Preferably, the hydrogen slow-release membrane is immersed in the aquaculture water. After 3 to 7 days, the algal biofilm begins to form and attach to the surface of the hollow fiber membrane. The algal biofilm includes algae and bacteria. The aquaculture water is river water.

[0016] Preferably, the bacterial-algal biofilm is calculated based on the gene abundance ratio as follows: the algae include more than 10% of cyanobacteria, the bacteria include more than 10% of Proteobacteria, more than 1% of Bacteroidetes, more than 1% of Chlorobacteria, more than 1% of Acidobacteria, more than 1% of Actinobacteria, more than 1% of Verrucomicrobial and more than 1% of Bacillus, and the remainder are miscellaneous bacteria not intended for culture.

[0017] Preferably, the stocking density of tilapia is 10-15 fish / m². 3 During the rearing of tilapia, the water temperature should be controlled at 25-30℃, dissolved oxygen >5mg / L, and pH 7.5-8.0. Feed three times a day, with the amount of food given each time initially based on 5% of the fish's body weight, and then ensuring that there is no uneaten food in the water 10 minutes after feeding.

[0018] Preferably, the tilapia are cultured for 60 days, and the tilapia species include one or more of Nile tilapia, Nile tilapia, Oreochromis aureus, Mozambique tilapia, and red tilapia.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention utilizes a hydrogen-based algae-microbe symbiotic membrane system for tilapia farming. The system employs a hydrogen-releasing membrane to slowly release hydrogen, achieving stable transport of high-purity hydrogen in the water without affecting tilapia growth. The hydrogen-based algae-microbe biofilm is rich in beneficial bacteria and algae, promoting tilapia growth, increasing survival rate and yield, and improving fish meat quality. The algae-microbe biofilm formed on the hollow fiber membrane surface of the system helps improve water quality, further increasing the abundance of beneficial bacteria in the tilapia's gut microbiota, inhibiting pathogenic bacteria, and promoting intestinal health. Attached Figure Description

[0021] Figure 1 The results show the water quality comparison under different aquaculture methods in Experiment Example 1 of this invention, where * indicates p<0.05.

[0022] Figure 2 The results show the water quality and microbial diversity and abundance of the bacterial and algal biofilm produced in Example 1 of this invention under different aquaculture methods in Example 1 and Comparative Example 1.

[0023] Figure 3 The results show the comparison of total tilapia weight under different farming methods in Experiment 1 of this invention, where * indicates p<0.05.

[0024] Figure 4 The results of the detection of essential amino acid content in tilapia flesh tissue under different farming methods in Experiment Example 1 of this invention are shown. * indicates p<0.05.

[0025] Figure 5 The results of crude fat content detection in tilapia flesh tissue under different farming methods in Experiment Example 1 of this invention are shown. * indicates p<0.05.

[0026] Figure 6 The results show the microbial diversity and abundance of tilapia gut microbiota under different farming methods in Experiment Example 1 of this invention. Detailed Implementation

[0027] This invention provides an application of a hydrogen-based bacterial-algae symbiotic membrane system in improving tilapia yield and / or quality.

[0028] This invention also provides the application of a hydrogen-based bacterial-algae symbiotic membrane system in increasing the abundance of beneficial bacteria in the gut microbiota of tilapia and / or inhibiting pathogenic bacteria in the gut microbiota of tilapia.

[0029] In this invention, the hydrogen-based algae-bacterial symbiotic membrane system includes a hydrogen slow-release membrane and an algae-bacterial biofilm. Preferably, the hydrogen slow-release membrane includes a membrane module and a hydrogen source; the membrane module contains a strip-shaped hollow fiber membrane, and the top of the membrane module is connected to the hydrogen source via a gas pipeline; the hydrogen source is a gas bag or cylinder containing high-purity hydrogen, a hydrogen gas generator, a hydrogen-producing material, or hydrogen produced by electrolysis of water. As an optional embodiment, the hydrogen source is an aluminum foil gas bag containing high-purity hydrogen with a purity >99%.

[0030] In this invention, the hydrogen slow-release membrane is preferably prepared by the following method:

[0031] (1) Fix 30 to 50 hollow fiber membranes, each 30 cm long, with glue to form a bundle of hollow fiber membranes, and place it in a separate aquaculture water body for 3 days to eliminate its biological toxicity.

[0032] (2) Inject high-purity hydrogen gas with a purity specification of >99% into the gas bag to make a hydrogen gas bag;

[0033] (3) Connect the hydrogen bag in step (2) and the hollow fiber membrane bundle in step (1) using an explosion-proof gas tube to make a hydrogen slow-release membrane assembly.

[0034] As one possible implementation, a bundle of hollow fiber membranes contains 30 hollow fiber membranes, each 30 cm long.

[0035] In this invention, the hollow fiber membrane can be made of polyvinylidene fluoride (PVDF) or polypropylene (PP); preferably polyvinylidene fluoride (PVDF), and more preferably with a pore size of 0.2 μm.

[0036] In this invention, at least one membrane module is installed per cubic meter of aquaculture pond. As an optional implementation, six membrane modules are installed in a 4 cubic meter aquaculture pond. In this invention, each membrane module is a bundle of solid hollow fiber membranes; after hydrogen is passed through the membrane module, it becomes a complete hydrogen slow-release membrane module. The preferred hydrogen consumption is 1.8–2.2 L / day, more preferably 2 L / day. As an optional implementation, the total hydrogen consumption during the entire aquaculture period is 30 L.

[0037] In this invention, the hydrogen slow-release membrane is immersed in the aquaculture water. After 3-7 days, the algal biofilm begins to form and adheres to the surface of the hollow fiber membrane. The algal biofilm includes algae and bacteria. Preferably, the algal biofilm, calculated by gene abundance ratio, comprises: algae greater than 10% of cyanobacteria; bacteria greater than 10% of Proteobacteria, greater than 1% of Bacteroidetes, greater than 1% of Chlorobacteria, greater than 1% of Acidobacteria, greater than 1% of Actinobacteria, greater than 1% of Verrucomicrobial, and greater than 1% of Bacillus; the remainder are miscellaneous bacteria not intended for culture. More preferably, the algae, calculated by gene abundance ratio, include 33.9% cyanobacteria, and the bacteria, calculated by gene abundance ratio, include 33.9% Proteobacteria, 11.1% Bacteroidetes, 5.3% Chlorobacteria, 3.2% Acidobacteria, 3.0% Actinobacteria, 1.3% Verrucous Microbes, and 1.2% Bacillus, with the remainder being miscellaneous bacteria not intended for culture.

[0038] In this invention, the stocking density of tilapia is 10-15 fish / m². 3 The preferred number is 11-14 fish / m 3 A further preferred ratio is 12-13 fish / m 3 The optimal value is 12.5 tails / m 3 During tilapia rearing, the water temperature should be controlled at 25–30℃, dissolved oxygen >5 mg / L, and pH 7.5–8.0. Feed three times a day, with each feeding initially based on 5% of the fish's body weight, followed by feeding until no uneaten food remains in the water 10 minutes later. The tilapia species include one or more of Nile tilapia, Nile tilapia, Oreochromis aureus, Mozambique tilapia, and red tilapia, with Nile tilapia being more preferred. The Nile tilapia is a hybrid of Oreochromis aureus and Nile tilapia. As an optional implementation method, the tilapia are reared for 60 days; the total hydrogen consumption during the entire rearing period is 30L.

[0039] In this invention, the bacterial and algal biofilm and the hydrogen slow-release membrane work synergistically to improve the yield, survival rate, quality, and abundance of beneficial bacteria in the tilapia gut microbiota. Specifically, the improvement in quality refers to increasing the content of essential amino acids and reducing the crude fat content of tilapia.

[0040] This invention utilizes a hydrogen-based algae-bacterial symbiotic membrane system for tilapia culture, which increases the total nitrogen and chlorophyll concentrations in the culture water. The total nitrogen concentration is 16.1 mg / L, and the chlorophyll concentration is 3.4 μg / L. Figure 1The chlorophyll concentration in the water reflects the amount of phytoplankton. Therefore, the addition of a hydrogen film can increase the concentration of nutrients and phytoplankton in the aquaculture water, thereby achieving the purpose of fertilization. Simultaneously, the beneficial environmental bacteria in the aquaculture water include Comamonadaceae (5.1%), Limnohabitans (4.1%), Novosphingobium (3.5%), and Sediminibacterium (2.2%); all of which are higher than those in the control group (Comamonadaceae 2.1%, Limnohabitans 3.6%, Novosphingobium 0.2%, Sediminibacterium 1.5%). Therefore, the hydrogen-based microbial-algae symbiotic film system has the function of regulating the microbial community and improving the aquaculture ecosystem.

[0041] This invention uses a hydrogen-based bacterial-algae symbiotic membrane system to cultivate tilapia, increasing tilapia yield by 30%.

[0042] The tilapia cultured using the hydrogen-based microbial-algae symbiotic membrane system of this invention exhibited significant improvements in nutritional quality: the essential amino acid content increased by 4.16 mg / g; this increase in essential amino acid content may be attributed to the synergistic metabolic effects of microorganisms and algae within the microbial-algae membrane. The microbial-algae membrane enhances the fish's nitrogen utilization efficiency and promotes muscle protein deposition through nitrogen fixation, enzyme production, or amino acid synthesis (such as glutamate secretion by cyanobacteria). The crude fat content decreased slightly by 0.0075% (fresh weight), suggesting that the microbial-algae membrane may reduce fat accumulation by regulating energy distribution in the fish (such as activating fatty acid oxidation pathways) or providing low-fat biological feed (algae). The hydrogen-based microbial-algae symbiotic membrane system of this invention has the potential to optimize the metabolic balance of tilapia: it can both enhance the nutritional value of fish meat through biosynthesis and moderately inhibit fat deposition, thus improving the texture and mouthfeel.

[0043] In this invention, a hydrogen-based algae-bacterial symbiotic membrane system was used to culture tilapia. Beneficial bacteria detected in the tilapia's intestinal flora included Bacillus, Cetobacterium, Gemmataceae, Hyphomicrobium, Exiguobacterium, Saccharomyces, and Propionibacteriaceae. Potential pathogenic bacteria included Aura ntimicrobium, Mycobacterium, Acinetobacter, Legionella, Staphylococcus, and Neochlamydia. Among them, the abundance of beneficial bacteria genus *Cetobacterium* increased significantly, while the abundance of potential pathogenic bacteria genus *Aurantimicrobium* decreased significantly; specifically, the abundance ratio of beneficial bacteria genus *Cetobacterium* increased from 0.1% to 23.7%, while the abundance ratio of potential pathogenic bacteria genus *Aurantimicrobium* decreased from 3.1% to 0.3%.

[0044] In the following embodiments of the present invention, the aquaculture water is river water, taken from the inland waterway of the Zhuangxing Experimental Station of the Shanghai Academy of Agricultural Sciences.

[0045] In the following embodiments of the present invention, the specific species of tilapia is Nile tilapia.

[0046] In the following embodiments of the present invention, the hollow fiber membrane is PVDF with a pore size of 0.2 μm, and was purchased from Guangzhou Haike Filter Membrane Technology Co., Ltd.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0048] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] This embodiment provides a method for tilapia farming based on a hydrogen-based microbial-algae symbiotic membrane system. The hydrogen-based microbial-algae symbiotic membrane system includes a hydrogen slow-release membrane and a microbial-algae biofilm. The hydrogen slow-release membrane is placed in a farming pond for tilapia farming. The microbial-algae biofilm is generated during the farming process and attaches to the hydrogen slow-release membrane. The microbial-algae biofilm includes bacteria and algae. The hydrogen slow-release membrane and the microbial-algae biofilm work synergistically to promote tilapia growth.

[0051] The experiment was conducted in a cement aquaculture pond at the Zhuangxing Experimental Station of the Shanghai Academy of Agricultural Sciences.

[0052] Specifically, the steps include the following:

[0053] 1. Preparation of hydrogen slow-release membrane

[0054] (1) Fix 30 hollow fiber membranes, each 30cm long, with glue to form a solid hollow fiber membrane bundle, and place it in a separate aquaculture water body to soak for 3 days to eliminate its biological toxicity.

[0055] (2) Inject 99.99% high-purity hydrogen into three 10L aluminum foil gas bags to make hydrogen bags; ensure that each hollow fiber membrane module can provide 30L of hydrogen (1 bar, i.e., 1 atmosphere of hydrogen volume is 30L).

[0056] (3) Connect the hydrogen bag in step (2) to the hollow fiber membrane bundle in step (1) using an explosion-proof gas tube; to make a hydrogen slow-release membrane assembly.

[0057] Following the steps above, a total of 6 hydrogen slow-release membrane modules were manufactured. These 6 hydrogen membrane modules were placed in aquaculture ponds, and tilapia were cultured after hydrogen was introduced.

[0058] 2. Tilapia farming steps:

[0059] Aquaculture water source: taken from surrounding rivers.

[0060] Culture pond setup: Outdoor cement pond culture, with a pond volume of 6m³. 3 Water surface area 4m 2 The water depth is 1m, which means the volume of the aquaculture water is 4m³. 3 .

[0061] Culture conditions: During the rearing period, the water temperature was controlled at 25-30℃, dissolved oxygen >5mg / L, and pH 7.5-8.0. Rearing cycle: 60 days; oxygen was aerated 24 hours a day during the experiment, and wastewater was removed and the water changed every 5 days. Hydrogen aeration: The six hydrogen slow-release membranes prepared above were placed in the culture pond and submerged in the culture water. The gas bags were not replaced during the experiment, and the total amount of hydrogen introduced during the entire rearing period was 30L.

[0062] Fish fry stocking: On the first day of the experiment, 50 Nile tilapia fry weighing 3g each were randomly stocked into each breeding pond.

[0063] Feeding: Feed three times a day at 7:00, 12:00, and 17:00. Each feeding should initially be based on 5% of the fish's body weight, and then ensure no uneaten feed remains in the water 10 minutes after feeding. Record the daily feed amount, maintaining a consistent feeding rate across all ponds. The entire rearing period is 60 days.

[0064] On the third day after the hydrogen slow-release membrane was immersed in the aquaculture water, bacterial and algal biofilms began to attach to the surface of the hollow fiber membrane bundles and remained stable until the seventh day.

[0065] Comparative Example 1

[0066] Tilapia were cultured using the method described in Example 1, with the following differences: no slow-release hydrogen membrane was used during hydrogen aeration, and no bacterial or algal biofilm was generated; a hydrogen cylinder equipped with a flow meter was used, and the hydrogen purity was 99.99%. Hydrogen was aerated in the culture pond every 6 days at a flow rate of 0.1 L / min for 30 minutes, for a total of 10 days during the experiment, with a total hydrogen volume of 30 L (30 L in total, with a 5-day interval).

[0067] Experimental Example 1

[0068] Experiments showed that in Example 1, on the third day after the hydrogen slow-release membrane was immersed in the aquaculture water, bacterial and algal biofilms began to attach to the surface of the hollow fiber membrane bundles and remained stable until the seventh day.

[0069] Aquaculture water quality testing:

[0070] The water was not changed for one week before sampling. Water quality tests were conducted on the aquaculture water of Example 1 and Comparative Example 1, and the results are as follows: Figure 1 As shown, the total nitrogen concentration in the water of Comparative Example 1 was measured to be 7.8 mg / L, and the chlorophyll concentration was 2.1 μg / L; Example 1 increased the total nitrogen and chlorophyll concentrations in the aquaculture water, with the total nitrogen concentration reaching 16.1 mg / L and the chlorophyll concentration reaching 3.4 μg / L. Figure 1 The chlorophyll concentration in water reflects the amount of phytoplankton in the water. Therefore, the addition of hydrogen film can increase the concentration of nutrients and phytoplankton in the aquaculture water, thereby achieving the purpose of fertilizing the water.

[0071] Detection of microbial diversity in aquaculture water and algal biofilms:

[0072] DNA sample collection from bacterial and algal biofilms: After 60 days of culture, the hollow fiber membranes loaded with bacterial and algal biofilms were cut into 2-3 cm fragments using sterile scissors and then flash-frozen in liquid nitrogen.

[0073] DNA sample collection from aquaculture water:

[0074] A 200 mL water sample was filtered through a 0.22 μm filter membrane, and the microorganisms trapped on the filter membrane were then rapidly frozen in liquid nitrogen.

[0075] DNA was extracted from the aquaculture water and bacterial / algal biofilm samples to detect microbial diversity:

[0076] according to Following the instructions of the soil DNAkit (Omega Bio-tek, Norcross, GA, US), total genomic DNA was extracted from the microbial community of the above-mentioned aquaculture water and bacterial / algal biofilm DNA samples. The quality of the extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using a NanoDrop2000 (Thermo Scientific, USA).

[0077] Using the DNA from the qualified samples as templates, PCR amplification of the V3-V4 variable region of the 16S rRNA gene was performed using the upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3', SEQ ID NO.1) carrying the barcode sequence and the downstream primer 806R (5'-GGACTAC HVGGGTWTCTAAT-3', SEQ ID NO.2).

[0078] The PCR reaction mixture consisted of: 4 μL of 5×FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of forward primer (5 μM), 0.8 μL of reverse primer (5 μM), 0.4 μL of FastPfu polymerase, 0.2 μL of BSA, and 10 ng of template DNA, to a final volume of 20 μL. Each sample was tested in triplicate.

[0079] The amplification program was as follows: 95℃ pre-denaturation for 3 min, 27 cycles (95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s), followed by 72℃ stable extension for 10 min, and finally storage at 4℃ (PCR instrument: T100 Thermal Cycler PCR, USA).

[0080] The amplified products were sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing. The purified PCR products were used to construct a library using the RapidDN A-Seq Kit, and sequencing was performed using the Illumina Nextseq2000 platform to determine the microbial diversity and abundance of the tilapia gut microbiota.

[0081] Results: The detection results of microbial diversity and abundance in the water bodies of Example 1 and Comparative Example 1, as well as the bacterial and algal biofilm produced in Example 1, are as follows: Figure 2 As shown. Analysis Figure 2 The results show that:

[0082] The biofilm produced in Example 1, calculated by gene abundance ratio, included 33.9% cyanobacteria, 33.9% Proteobacteria, 11.1% Bacteroidetes, 5.3% Chlorobacteria, 3.2% Acidobacteria, 3.0% Actinobacteria, 1.3% Verrucomicrobium, and 1.2% Bacillus, with the remainder being miscellaneous bacteria not belonging to the culture.

[0083] Example 1 (Experimental Group): The aquaculture water contained, by gene abundance ratio, 17.7% Exiguobacterium, 5.1% Comamonadaceae, 4.7% hgcI_clade, 4.1% Limnohabitans, 3.5% Novosphingobium, 3.3% GKS98_freshwater_group, 3.0% Alsobacter, and 2.2% Sediminibacterium.

[0084] In contrast, the aquaculture water in Comparative Example 1 (control group) contained, by gene abundance ratio, 4.8% Exiguobacterium, 2.1% Comamonadaceae, 4.7% hgcI_clade, 3.6% Limnohabitans, 0.2% Novosphingobium, 0% GKS98_freshwater_group, 0.3% Alsobacter, and 1.5% Sediminibacterium.

[0085] As can be seen from the above, the microbial diversity and abundance in the experimental group's aquaculture water were higher than those in the control group. Among them, *Limnohabitans*, *Comamonadaceae*, *Novosphingobium*, and *Sediminibacterium* are beneficial environmental bacteria that support ecosystem functions. This indicates that the hydrogen-releasing membrane synergistic with microbial-algal biofilm (hydrogen-based microbial-algal symbiotic membrane system) has the function of improving the aquaculture environment ecosystem by regulating the microbial community.

[0086] Tilapia sample testing:

[0087] 1. Tilapia quantity and yield detection: After 60 days of the experiment, all fish were harvested and weighed.

[0088] In Example 1, 48 fish remained, with a total weight of 927.4g and a juvenile survival rate of 96%; in Comparative Example 1, 35 fish remained, with a total weight of 711.5g and a juvenile survival rate of 70%. The results show that Example 1 yielded a 30% higher yield compared to Comparative Example 1. Figure 3 This increased the survival rate of juvenile fish by 26%.

[0089] 2. Detection of essential amino acid and crude fat content in tilapia flesh tissue

[0090] In Example 1 (experimental group) and Comparative Example 1 (control group), six fish were selected from each group, and their flesh tissue was collected. The content of essential amino acids in the tilapia flesh tissue was determined using an automated amino acid analyzer. The crude fat content in the tilapia flesh tissue was determined by Soxhlet extraction combined with gas chromatography.

[0091] Test results as follows Figure 4 and Figure 5 As shown, the essential amino acid content in the flesh of the experimental group tilapia increased by 4.16 mg / g compared to the control group, and the crude fat content decreased by 0.0075%. The experimental results indicate that the experimental group tilapia using the hydrogen-based algae-microbe film showed a significant improvement in nutritional quality compared to the control group simply exposed to hydrogen. The 4.16 mg / g increase in essential amino acid content compared to the control group may be attributed to the synergistic metabolic effects of microorganisms and algae within the algae-microbe film. The algae-microbe film enhances the fish's efficiency in utilizing nitrogen sources and promotes muscle protein deposition through nitrogen fixation, enzyme production, or amino acid synthesis (such as glutamate secreted by cyanobacteria).

[0092] The crude fat content decreased slightly by 0.0075% (fresh weight), suggesting that the bacterial and algal film may have reduced fat accumulation by regulating energy distribution in the fish (such as activating fatty acid oxidation pathways) or by providing low-fat biological feed (algae).

[0093] This result reveals the potential of hydrogen matrix bacteria and algae film in optimizing the metabolic balance of aquatic animals: it can both enhance the nutritional value of fish meat through biosynthesis (meeting the requirements of healthy food) and moderately inhibit fat deposition (improving the texture of the meat).

[0094] 3. Detection of microbial diversity and abundance in the gut microbiota of tilapia:

[0095] In Example 1 and Comparative Example 1, six fish were selected from each group for dissection, and samples from the terminal intestines were preserved in liquid nitrogen. According to... Total genomic DNA was extracted from the microbial community using the soil DNAkit (Omega Bio-tek, Norcross, GA, US) according to the instructions. The quality of the extracted genomic DNA was assessed using 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using a NanoDrop2000 (Thermo Scientific, USA).

[0096] Using quality-tested DNA as a template, PCR amplification of the V3-V4 variable region of the 16S rRNA gene was performed using upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3', SEQ ID NO.1) and downstream primer 806R (5'-GGACTACHVG GGTWTCTAAT-3', SEQ ID NO.2) carrying the barcode sequence.

[0097] The PCR reaction mixture consisted of: 4 μL of 5×FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of forward primer (5 μM), 0.8 μL of reverse primer (5 μM), 0.4 μL of FastPfu polymerase, 0.2 μL of BSA, and 10 ng of template DNA, to a final volume of 20 μL. Each sample was tested in triplicate.

[0098] The amplification program was as follows: 95℃ pre-denaturation for 3 min, 27 cycles (95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s), followed by 72℃ stable extension for 10 min, and finally storage at 4℃ (PCR instrument: T100 Thermal Cycler PCR, USA).

[0099] The amplified products were sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing. The purified PCR products were used to construct a library using the RapidDN A-Seq Kit, and sequencing was performed using the Illumina Nextseq2000 platform to determine the microbial diversity and abundance of the tilapia gut microbiota.

[0100] Results: Beneficial bacteria detected in the intestinal flora of tilapia cultured using the method of Example 1 (experimental group) included Bacillus, Cetobacterium, Gemmataceae, and Hyphomicrobium; potential pathogenic bacteria included Aurantimicrobium, Mycobacterium, Acinetobacter, and Staphylococcus.

[0101] Compared with Comparative Example 1, the beneficial bacteria in the tilapia gut microbiota of Example 1 increased from 2.2% to 8.0%, Cetobacterium from 0.1% to 23.7%, Gemmataceae from 0.4% to 3.5%, and Hyphomicrobium from 0.3% to 1.8%.

[0102] Compared with Comparative Example 1, the percentages of pathogenic bacteria in the tilapia gut microbiota of Example 1 decreased from 3.1% to 0.3%, Mycobacterium from 19.9% ​​to 6.4%, Acinetobacter from 7.7% to 0.1%, and Staphylococcus from 5.1% to 0%.

[0103] As shown above, in Example 1, the abundance of the beneficial bacterium *Cetobacterium* in the tilapia gut microbiota significantly increased from 0.1% to 23.7%, while the abundance of the pathogenic bacterium *Aurantimicrobium* significantly decreased from 3.1% to 0.3% (see Example 1). Figure 6 ).

[0104] In summary, the hydrogen-based algae-bacterial symbiotic membrane system of this invention can improve water quality, increase the abundance of beneficial bacteria in the gut microbiota of tilapia, inhibit pathogenic bacteria in the gut microbiota of tilapia, and promote the gut health of tilapia.

[0105] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a hydrogen-based microbial-algae symbiotic membrane system in improving tilapia yield and / or quality, characterized in that, The hydrogen-based algae-bacterial symbiotic membrane system increases the abundance of beneficial bacteria in the gut microbiota of tilapia and inhibits pathogenic bacteria in the gut microbiota of tilapia. The hydrogen-based algae-bacterial symbiotic membrane system includes a hydrogen slow-release membrane and an algae-bacterial biofilm. The biofilm contains the following algae and bacteria according to gene abundance ratio: algae include more than 10% cyanobacteria, bacteria include more than 10% Proteobacteria, more than 1% Bacteroidetes, more than 1% Chlorobacteria, more than 1% Acidobacteria, more than 1% Actinobacteria, more than 1% Verrucomicrobial and more than 1% Bacillus, and the remainder are miscellaneous bacteria not of the culture object.

2. The application according to claim 1, characterized in that, The hydrogen slow-release membrane includes a membrane module and a hydrogen source; the membrane module contains a hollow fiber membrane, and the top of the membrane module is connected to the hydrogen source through a gas pipeline; the hydrogen source is a gas bag or gas cylinder containing high-purity hydrogen, or a hydrogen gas generator, or a hydrogen-producing material, or hydrogen produced by electrolysis of water; the purity of the high-purity hydrogen is >99%.

3. The application according to claim 2, characterized in that, At least one membrane module is installed per cubic meter of aquaculture pond, and each membrane module contains 30 to 50 hollow fiber membranes; the hydrogen consumption is 1.8 to 2.2 L / day.

4. The application according to claim 2, characterized in that, The hollow fiber membrane is made of polyvinylidene fluoride (PVDF) or polypropylene (PP); the pore size of the hollow fiber membrane is 0.2 μm.

5. The application according to claim 1, characterized in that, The hydrogen slow-release membrane is immersed in the aquaculture water. After 3 to 7 days, the bacterial and algal biofilm begins to form and attach to the surface of the hollow fiber membrane. The bacterial and algal biofilm includes algae and bacteria. The aquaculture water is river water.

6. The application according to any one of claims 1 to 5, characterized in that, Tilapia stocking density is 10-15 fish / m² 3 During the rearing of tilapia, control the water temperature at 25~30℃, dissolved oxygen >5mg / L, and pH 7.5~8.0; feed three times a day, with the amount of food given each time being based on 5% of the fish's body weight, and then ensuring that there is no uneaten food in the water 10 minutes after feeding.

7. The application according to any one of claims 1 to 5, characterized in that, The tilapia are cultured for 60 days; the tilapia species include one or more of the following: Nile tilapia, Nile tilapia, Oreochromis aureus, Mozambique tilapia, and red tilapia.

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