Method for regulating seawater carbon cycle and carbon storage based on oyster culture

Abstract

The application relates to the technical field of aquaculture regulation, and discloses a method for regulating seawater carbon cycle and carbon storage based on oyster culture, which comprises the following steps: demarcating an oyster culture area and a control area in a target water area, arranging a floating raft in the culture area, and culturing Hong Kong oysters; after a set culture period, water samples are collected from the two areas and are filtered to separate filtrate and retentate; the dissolved organic carbon and dissolved inorganic carbon content values of the filtrate are measured, deoxyribonucleic acid of the retentate is extracted to perform metagenomic sequencing, and the abundance values of microorganism species, metabolic function and carbon cycle function genes are output; when the above carbon content values and the specified microorganism and function gene abundance values of the culture area are all greater than those of the control area, a command is triggered to maintain the culture state. The present application expands the determination dimension to water phase dissolved carbon and microecological sequence, establishes a multi-dimensional biochemical data comparison threshold, and realizes closed-loop feedback control of the seawater carbon storage regulation execution steps.

Description

Technical Field

[0001] This invention relates to the field of aquaculture regulation technology, specifically a method for regulating seawater carbon cycle and carbon storage based on oyster farming. Background Technology

[0002] Bivalve aquaculture is widely deployed in nearshore bays and estuaries. Oysters, representing bivalve mollusks, create localized water currents during their culture cycle by opening their shells and beating their cilia, filtering phytoplankton and suspended particulate matter from the water. This process assimilates and binds carbon into the calcium carbonate shell and soft tissues. Simultaneously, undigested material is expelled through excretory pores, settling to the bottom as biomass feces and pseudofeces, which are denser than seawater, and forming sedimentary carbon deposits.

[0003] Current carbon storage measurement schemes for the aforementioned aquaculture systems primarily rely on mechanically weighing harvested oysters and combining this with chemical analysis to determine the carbon sequestration quality of shells and soft tissues, or on calculating the content of deposited carbon through sediment column samples. This conventional approach only covers the macroscopic dimension of solid carbon acquisition. In real-world environmental interactions, oysters' filter-feeding and excretory metabolic activities continuously release water-soluble products into the surrounding water, directly altering the concentrations of dissolved organic and inorganic carbon in the local water. Simultaneously, the microbial community within the aquatic body is the fundamental driving force behind carbon state transfer and biochemical degradation. Existing monitoring and measurement systems lack procedures for in-situ filtration and DNA extraction in localized water bodies, thus failing to incorporate aquatic microecological data into their analysis.

[0004] Due to the lack of chemical instruments for detecting carbon concentration in filtrate and metagenomic sequencing of retained samples, current technologies cannot obtain abundance values ​​for specific microbial species, nor can they quantify changes in the number of sequences of metabolic pathway and carbon cycling functional genes caused by excrement substrate input. This lack of measurement dimensions prevents the system from objectively recording the shift in local water bodies from macromolecular carbon retention mechanisms to rapid degradation metabolic mechanisms. Incomplete input variables prevent existing regulatory systems from establishing comparison thresholds based on multidimensional biochemical measurement data and from outputting clear Boolean logic signals through differences in control data. Without these feedback instructions, the system cannot perform scientific judgment and closed-loop control of the raft coverage area and oyster retention behavior within the aquaculture area. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for regulating seawater carbon cycling and carbon storage based on oyster farming. This method solves the problem that existing shellfish farming carbon measurement is limited to macroscopic solid phase carbon measurement and lacks extraction and analysis of dissolved carbon concentration in water and gene sequences of micro-ecological carbon cycling functions. As a result, the system cannot establish logical judgment thresholds based on multi-dimensional aquatic biochemical comparison data, and thus cannot perform closed-loop feedback regulation on the layout of farming facilities.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for regulating seawater carbon cycling and carbon storage based on oyster farming, comprising the following steps: Oyster farming areas and control areas were delineated in the target waters. Floating rafts were set up in the oyster farming areas and Hong Kong oysters were farmed there, while no oysters were farmed in the control areas. After the culture cycle, water samples were collected from the oyster culture area and the control area, and the water samples were filtered to obtain filtrate and retentate, respectively. The dissolved organic carbon content and dissolved inorganic carbon content in the filtrate were measured, and the deoxyribonucleic acid of the retentate was extracted for metagenomic sequencing to obtain the abundance of microbial species, relative abundance of metabolic functions, and abundance of carbon cycling genes in the oyster farming area and the control area, respectively. When the dissolved organic carbon content, dissolved inorganic carbon content, abundance of microbial species of methane consumers, organic matter degraders, and nitrogen-carbon cyclers in the oyster farming area, relative abundance of metabolic functions such as amino acid metabolism, energy metabolism, and carbohydrate metabolism, and abundance of carbon cycling functional genes such as acyl-CoA dehydrogenase and mannosidase in the oyster farming area are all greater than those in the control area, the farming state is maintained.

[0007] Preferably, the target water area specifically includes: The target waters are semi-enclosed inner bays or estuaries; The water temperature of the target water area is between 20°C and 22°C. The salinity of the target water area is limited to between 18 ppt and 33 ppt; The pH of the target water body is limited to between 8.0 and 8.4; The dissolved oxygen concentration in the target water area is limited to between 6.8 mg / L and 7.1 mg / L; The chlorophyll a concentration in the target water area is limited to between 1.69 μg / L and 2.75 μg / L.

[0008] Preferably, the raft is 6 meters long and 6 meters wide, and the area covered by the rafts in the oyster farming area accounts for more than 30% of the total area of ​​the target water area.

[0009] Preferably, the breeding cycle specifically includes: The Hong Kong oysters filter-feed on phytoplankton and organic particles in the water within the oyster farming area; The Hong Kong oyster assimilates carbon into its soft tissue and shell; The Hong Kong oysters enrich their sediments through excretion and biodeposition.

[0010] Preferably, the collection of water samples from the oyster farming area and the control area specifically includes: Three sampling points were set up within the oyster farming area; Three control area sampling points were set within the control area; Surface seawater was collected from the sampling points in the three aquaculture areas and the three control areas, respectively. The collected surface seawater was stored away from light and used as the water sample.

[0011] Preferably, determining the dissolved organic carbon content and dissolved inorganic carbon content in the filtrate specifically includes: The filtrate was chemically analyzed using a total organic carbon analyzer, and the dissolved organic carbon content and dissolved inorganic carbon content were output. The dissolved organic carbon content in the oyster farming area is limited to the range of 3.56 mg / L to 3.82 mg / L; The dissolved inorganic carbon content in the oyster farming area is limited to the range of 0.84 mg / L to 0.96 mg / L.

[0012] Preferably, extracting the deoxyribonucleic acid of the retained material for metagenomic sequencing specifically includes: Separate the microbial community attached to the surface of the retrieval material; The microbiome was subjected to cell disruption and deoxyribonucleic acid (DNA) release. The purity of the released deoxyribonucleic acid was tested. A sequencing library was constructed from deoxyribonucleic acid that passed purity testing. High-throughput metagenomic sequencing is performed on the sequencing library and the metagenomic sequencing sequence is output.

[0013] Preferably, the abundance values ​​of microbial species, relative abundance values ​​of metabolic functions, and abundance values ​​of carbon cycling function genes in the oyster farming area and the control area specifically include: The abundance values ​​of microbial species are expressed as percentages of the absolute sequence counts of methane consumers, organic matter degraders, and nitrogen-carbon cyclers, respectively, divided by the total sequence count. The relative abundance of metabolic functions is expressed as a percentage of the absolute number of amino acid metabolism sequences, the absolute number of energy metabolism sequences, and the absolute number of carbohydrate metabolism sequences, divided by the total number of sequences. The abundance values ​​of carbon cycle functional genes are expressed as percentages of the absolute sequence number of the acyl-CoA dehydrogenase gene and the absolute sequence number of the mannosidase gene, respectively, divided by the total sequence number.

[0014] Preferably, filtering the water sample to obtain filtrate and retentate specifically includes: The water sample was separated using a glass fiber filter membrane with a pore size of 0.45 μm. The liquid passing through the glass fiber filter membrane with a pore size of 0.45 μm is collected as the filtrate; The material adhering to the surface of the glass fiber filter membrane with a pore size of 0.45 μm is collected as the retentate.

[0015] Preferably, maintaining the aquaculture state specifically includes: The Hong Kong oysters cultivated in the oyster farming area are retained; The cultivated Hong Kong oysters continue to perform excretion and biodeposition processes; Dissolved organic carbon and dissolved inorganic carbon are continuously introduced into the water body of the oyster farming area through the aforementioned excretion and biological sedimentation processes; The dissolved organic carbon content and dissolved inorganic carbon content in the oyster farming area are maintained to be higher than those in the control area.

[0016] This invention provides a method for regulating seawater carbon cycling and carbon storage based on oyster farming. It has the following beneficial effects: 1. This invention expands the assessment of seawater carbon storage to include dissolved carbon components in water bodies by determining the dissolved organic carbon and dissolved inorganic carbon content in the filtrate of water samples from a designated control area and an oyster farming area within the target water body. This detection method overcomes the shortcomings of existing technologies that only calculate carbon content from oyster excretion and biological deposition, providing quantifiable and direct data to verify the process by which oyster farming promotes the enrichment of dissolved carbon in seawater.

[0017] 2. This invention utilizes metagenomic sequencing to extract deoxyribonucleic acid from water samples and determine the abundance of microbial species involved in methane consumption, organic matter degradation, and nitrogen-carbon cycling. By using the succession of aquatic microbial community structure as a monitoring indicator for carbon storage regulation, the specific microbial groups involved in aquatic carbon conversion processes are identified, providing objective microscopic evidence for analyzing seawater carbon retention capacity.

[0018] 3. This invention uses the relative abundance values ​​of metabolic functions such as amino acid metabolism, energy metabolism, and carbohydrate metabolism, as well as the abundance values ​​of carbon cycle functional genes such as acyl-CoA dehydrogenase and mannosidase, as criteria for evaluating state maintenance. A correlation is established between macroscopic oyster farming behavior and microscopic deoxyribonucleic acid sequence distribution, quantifying the metabolic alteration pathways of aquatic microorganisms under organic substrate input at the molecular level.

[0019] 4. This invention establishes a multi-dimensional index comparison process that includes physicochemical concentration measurement, community abundance measurement, and functional gene abundance measurement, and sets a rule for maintaining the aquaculture state when all measured values ​​are greater than the control area. It constructs a closed-loop workflow between index monitoring and aquaculture behavior intervention, eliminating potential analytical biases associated with single evaluation indicators and improving the repeatability of carbon storage regulation and accounting in aquaculture. Attached Figure Description

[0020] Figure 1 This is a distribution map of sampling points in the Maoweihai research area of ​​this invention; Figure 2 This is a graph showing the results of non-metric multidimensional scale (NMDS) analysis of the aquatic microbial community at the species level in the oyster farming area and the control area of ​​this invention. Figure 3 This is a comparison of the relative abundance of aquatic microorganisms at the species level between the oyster farming area and the control area in this invention; Figure 4 This is a diagram showing the NMDS analysis results of functional genes in the water bodies of the oyster farming area and the control area in this invention. Figure 5 These are the Bray-Curtis analysis results of functional genes in the water bodies of the oyster farming area and the control area in this invention; Figure 6 This is a graph showing the results of multidimensional scale (MDS) analysis of carbon cycling-related genes in the oyster farming area and control area of ​​this invention. Figure 7 This is a relative abundance diagram of the top 30 carbon cycle-related functional genes in the water of the oyster farming area and the control area of ​​this invention. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Figure 1The distribution of sampling points in the Maoweihai research area in this embodiment of the invention is shown. The locations of six sampling points are marked in the figure, where B1, B2, and B3 are sampling points in the Hong Kong oyster farming area, and C1, C2, and C3 are sampling points in the control area. Figure 1 The spatial layout of the oyster farming area and the control area was clearly defined, providing a basis for assessing the impact of oyster farming on seawater carbon storage. Maoling River; Qinjiang River; Maowei Sea; Qinzhou Bay.

[0023] Figure 2 The results of non-metric multidimensional scale (NMDS) analysis of the aquatic microbial communities in the oyster farming area and the control area in this embodiment of the invention are shown. Figure 2 The study showed that microbial diversity formed three spatially based clusters. The microbial diversity of samples from the control area was more clustered than that of samples from the oyster farming area, indicating that in addition to environmental parameters, oyster farming in Hong Kong also altered the microbial diversity of the water body.

[0024] Figure 3 The figure shows the relative abundance of aquatic microorganisms at the species level in the oyster farming area and the control area in this embodiment of the invention. The figure shows the top 50 species in relative abundance. The control area was enriched with decomposers such as Pseudomonas spp., Streptomyces spp., Chloroflexi spp., and Mesotoga spp., while the oyster farming area was enriched with methane consumers, organic matter degraders, and nitrogen and carbon cyclers such as Methylocapsa spp., Methanohalophilus spp., Cycloclasticus spp., Vibrio spp., and Nitrospirae spp.

[0025] Figure 4 The NMDS analysis results of functional genes in the water bodies of the oyster farming area and the control area in this embodiment of the invention are shown. Similar to the microbial community structure, the relative abundance of functional genes also showed spatial structure, with the homogeneity among samples in the control area being higher than that among samples in the oyster farming area.

[0026] Figure 5 The Bray-Curtis analysis results of functional genes in the oyster farming area and control area in this embodiment of the invention are shown. The figure shows that the oyster farming area is enriched in amino acid metabolism, energy metabolism, and carbohydrate metabolism functions, while the control area shows a wider range of functional categories, including glycan synthesis and cell community function.

[0027] Figure 6The results of multidimensional scale (MDS) analysis of carbon cycle-related genes in the oyster farming area and control area in this embodiment of the invention are shown. The figure shows that the relative abundance of carbon cycle-related genes exhibits spatial structure, and the similarity between samples from the oyster farming area is higher than that from the control area.

[0028] Figure 7 The figure shows the relative abundance of the top 30 carbon cycle-related functional genes in the oyster farming area and the control area in this embodiment of the invention. The figure shows that the abundance of genes related to carbon metabolism (carB), carbohydrate utilization (gmd), and biofilm-mediated carbon persistence strategies is higher in the control area than in the oyster farming area, while the abundance of genes related to rapid carbon degradation is higher in the oyster farming area.

[0029] Figure 2 , Figure 4 , Figure 6 The English explanation of NMDS / MDS dimensionality reduction scatter plot: MDS1 / MDS2: Two reduced-dimensional coordinate axes for multidimensional scaling analysis. In the legend, blue dot B and dark dot C: B represents the culture area (oyster group), and C represents the control area.

[0030] Figure 3 Comparison of relative abundance of microbial communities: The horizontal axis represents abundance (numerical value).

[0031] Group on the right side of the figure: (B is the breeding area, C is the control area).

[0032] Figure 5 Relative abundance of metabolic functions: Relative Abundance: relative abundance.

[0033] Amino acid metabolism.

[0034] Carbohydrate metabolism.

[0035] Energy metabolism.

[0036] Metabolism of cofactors and vitamins.

[0037] Translation: The process of translating.

[0038] Nucleotide metabolism.

[0039] Replication and repair: Replication and repair.

[0040] Membrane transport: Membrane transport.

[0041] Signal transduction: Signal transduction.

[0042] Glycan biosynthesis and metabolism: Glycan biosynthesis and metabolism.

[0043] Lipid metabolism: Lipid metabolism.

[0044] Cellular community - prokaryotes: Cellular community - prokaryotes.

[0045] Folding, sorting and degradation: Folding, sorting and degradation.

[0046] Metabolism of other amino acids: Metabolism of other amino acids.

[0047] Xenobiotics biodegradation and metabolism: Xenobiotics biodegradation and metabolism.

[0048] Biosynthesis of other secondary metabolites: Biosynthesis of other secondary metabolites.

[0049] Figure 7 : Relative abundance diagram of functional genes related to carbon cycle.

[0050] With reference to Figures 1-7 , the present invention provides a method for regulating seawater carbon cycle and carbon storage based on oyster culture, the method comprising: Demarcating an oyster culture area and a control area in a target water area, arranging rafts and culturing Crassostrea hongkongensis in the oyster culture area, and culturing no oysters in the control area; After the culture cycle, collecting water samples from the oyster culture area and water samples from the control area respectively, and filtering the water samples to obtain filtrates and retentates respectively; Determining the value of dissolved organic carbon content and the value of dissolved inorganic carbon content in the filtrate, extracting deoxyribonucleic acid from the retentates for metagenomic sequencing, and respectively obtaining the value of microbial species abundance, the value of relative abundance of metabolic functions and the value of carbon cycle functional gene abundance of the oyster culture area and the control area; When the dissolved organic carbon content, dissolved inorganic carbon content, abundance of microbial species of methane consumers, organic matter degraders, and nitrogen-carbon cyclers in the oyster farming area, relative abundance of metabolic functions such as amino acid metabolism, energy metabolism, and carbohydrate metabolism, and abundance of carbon cycling functional genes such as acyl-CoA dehydrogenase and mannosidase in the oyster farming area are all greater than those in the control area, the farming status is maintained.

[0051] Existing carbon storage measurements for bivalve aquaculture typically only count the amount of carbon stored in soft tissue and inside the shell, as well as the amount of carbon deposited in the sediment. The technical solution provided in this embodiment extends the measurement dimensions to dissolved carbon components and micro-ecological sequence abundance in the water body, and outputs control commands by comparing and measuring the data.

[0052] The present invention is based on a semi-enclosed inner bay or estuary sea area, where the target water area has the conditions for freshwater input and forms a salinity gradient distribution; the above parameters and hydrochemical characteristics constitute the preconditions for Hong Kong oysters to perform filter feeding and excretion metabolic activities.

[0053] At the macro-implementation level, a floating raft system of specified size and specifications is deployed within a designated water area. This system provides attachment and suspension structures for the cultivation of Hong Kong oysters. During the cultivation cycle, Hong Kong oysters continuously filter-feed on water particles, excrete biomass, and promote particle deposition and biological processes. These processes directly alter the physicochemical composition of the local water in the target area, resulting in deviations in physicochemical values ​​between the oyster cultivation area and the control area.

[0054] At the detection and separation level, mechanical sampling and filtration are performed on water samples from localized areas. Through filtration, the water sample is separated into filtrate that has passed through the filter membrane and retained material adhering to the membrane surface. The filtrate serves as the input for chemical detection, and after being fed into the analytical instrument, it outputs measured concentration data of dissolved organic carbon and dissolved inorganic carbon. The retained material contains the total microbiome, and through cell disruption and nucleic acid extraction, deoxyribonucleic acid (DNA) fragments are extracted and fed into a high-throughput sequencing device to output metagenomic sequencing data.

[0055] At the data processing level, classification, alignment, and statistical operations are performed on the sequence data output by the sequencing equipment. This process outputs absolute sequence counts for microbial species, metabolic pathways, and functional genes. These absolute sequence counts are divided by the total sequence count of the sample to convert them into abundance values ​​expressed as percentages.

[0056] At the state execution level, a logical judgment threshold based on multi-dimensional measurement data is established. The detection mechanism extracts dissolved carbon concentration, microbial species abundance, relative abundance of metabolic functions, and abundance of carbon cycle functional genes, and compares them with the corresponding detection values ​​in the control area. When all specified detection items meet the condition of being greater than those in the control area, the system outputs a positive feedback command, triggering operations to maintain the deployment status of the floating rafts and Hong Kong oysters, forming a closed-loop control flow from water area delineation, aquaculture operations, sampling and sequencing to state determination.

[0057] Reference Figures 1-7 When implementing the method for regulating seawater carbon cycle and carbon storage based on oyster farming provided by the present invention, it includes the selection of the implementation water area, the confirmation of environmental parameter boundaries, and the mechanical deployment of artificial farming facilities.

[0058] The target waters are geographically defined as semi-enclosed bays or estuaries, characterized by the convergence of freshwater runoff from land and tidal saltwater, creating a salinity gradient within the water space. The purpose of selecting this type of water is to provide a suitable fluid environment and suspended particulate matter concentration for oyster attachment and feeding in Hong Kong.

[0059] Before delineating oyster farming and control areas, physical and chemical parameters of candidate water bodies were measured using multi-parameter water quality monitoring equipment deployed at a predetermined depth below the water surface. This monitoring process output multiple continuous values, requiring the candidate water bodies to simultaneously meet the following parameter limits: water temperature between 20°C and 22°C; salinity between 18 ppt and 33 ppt; pH between 8.0 and 8.4; dissolved oxygen concentration between 6.8 mg / L and 7.1 mg / L; and chlorophyll a concentration measured by fluorescence spectroscopy between 1.69 μg / L and 2.75 μg / L. When the output parameters of a candidate water body fell within all of these limits, the area was designated as the target water body.

[0060] Boundaries were demarcated within the target water area to separate the oyster farming area and the control area, with a spacing set between them. The spacing was set based on a distance greater than the maximum tidal displacement distance of the target water area within one tidal cycle, in order to prevent excrement and shed biomass from the oyster farming area from spreading into the control area with the water flow and interfering with the control data.

[0061] Multiple sets of floating rafts were deployed within the oyster farming area. Each raft consisted of a floating frame, polyethylene buoys providing buoyancy, and a farming net cage suspended below the floating frame. Hong Kong oysters were placed inside the farming net cages. The rafts were limited to a length and a width of 6 meters. The control area maintained its original water condition, without any floating raft components or individual oysters.

[0062] To ensure that the filter-feeding and excretion activities of Hong Kong oysters have a sufficiently strong impact on the local water area, resulting in a change in the concentration of DNA fragments in the water sample that reaches the detection limit of the sequencing instrument, the total coverage area of ​​the floating rafts deployed within the oyster farming area must be greater than 30% of the total surface area of ​​the oyster farming area. This area ratio is calculated according to the following formula: ; In the formula, Characterizes the percentage of the total surface area of ​​the oyster farming area covered by floating rafts; The total number of floating rafts deployed within the oyster farming area; The length of a single raft is represented by a value of 6, and the unit is meters. The width of a single raft is a measured value of 6, in meters. The total area of ​​the designated oyster farming area on a horizontal plane is measured in square meters.

[0063] By increasing or decreasing the number of floating rafts deployed. Adjust the output Numerical value, making >30%. After the floating raft deployment is completed, the aquaculture cycle will begin.

[0064] Reference Figures 1-7 After the breeding cycle, water samples were collected from the oyster farming area and the control area, respectively. The water samples were filtered to obtain filtrate and precipitate, which included the biological intervention process of the aquatic micro-ecological environment and the mechanical separation operation.

[0065] During the established culture cycle, Hong Kong oysters perform shell-opening movements, driving the movement of their internal cilia to generate water flow into the mantle cavity. This water flow draws in phytoplankton and organic particles from the surrounding water. After being intercepted by the gill structure and biochemically degraded through the digestive tract, the carbon contained within the inhaled material flows along three different pathways: the first part of the carbon is assimilated and incorporated into the soft tissues and calcium carbonate shell of the Hong Kong oyster; the second part of the carbon, undigested, is expelled through the excretory pore, forming biofeces and pseudofeces. The density of the expelled biofeces and pseudofeces is greater than that of seawater, and under the influence of gravity, they settle towards the bottom of the water and adhere to the sediment, performing biological sedimentation. The aforementioned continuous filter-feeding and excretion processes release water-soluble metabolic products into the oyster culture area and alter the distribution of particulate matter, forming organic substrate input into the local water body.

[0066] After completing the set culture cycle, operators used a calibration tool equipped with a positioning module to set up three independent sampling points in the oyster culture area. The coordinates of the three sampling points formed an equilateral triangle on the horizontal plane. Using the same setting logic, three independent control sampling points were set up in the control area. At each independently set sampling point in the culture area and the control area, a polytetrafluoroethylene (PTFE) water collection container made of a metal ion-free material was deployed. The water collection container was lowered to a depth of 0.5 to 1.0 meters below the water surface, and the valve was opened to collect surface seawater. The collected surface seawater was transferred to a brown polyester light-proof bottle for sealed storage. The brown polyester light-proof bottle blocks photons in the visible light frequency band from entering, inhibiting the carbon consumption reaction based on photosynthesis of the autotrophic microorganisms contained in the sealed water. Subsequently, the brown polyester light-proof bottle containing the surface seawater was placed in a low-temperature storage box maintained at 0°C to 4°C, and the surface seawater was transported as a water sample to the filtration treatment station.

[0067] A negative pressure filtration circuit, including a vacuum pump, a collection bottle, and a porous funnel, is constructed within the filtration station. A glass fiber filter membrane with a pore size of 0.45 μm is extracted, laid flat, and sealed onto the cross-section of the porous funnel. The vacuum pump is activated to establish a negative pressure in the structural region below the glass fiber filter membrane. A measured volume of water sample is injected into the porous funnel. Under the pressure difference, the water sample passes through the glass fiber filter membrane and undergoes separation: liquid water molecules and dissolved substances smaller than 0.45 μm penetrate the pores and enter the collection bottle below, where they are collected separately and defined as the filtrate; suspended particles larger than or equal to 0.45 μm, macromolecular organic polymers, and microbial cell entities are intercepted and adhere to the upper surface of the glass fiber filter membrane, forming a solid retention layer, which, along with the glass fiber filter membrane bearing the retention layer, is collected and defined as the retained material.

[0068] To verify whether the total amount of substances obtained from a fixed volume of water sample at a single sampling point meets the lower limit requirement of the deoxyribonucleic acid extraction process, a high-precision microbalance was used to measure the mass change of the filter membrane, and the mass concentration of total suspended particulate matter in the water at a single sampling point was calculated using the following mathematical formula: ; In the formula, This represents the total suspended particulate matter concentration (TPS) calculated for a water sample collected at a single designated sampling point, expressed in mg / L. This represents the measured total mass of the glass fiber filter membrane, along with the solid retention layer on its surface, after the aforementioned vacuum filtration operation and the removal of liquid water by evaporation using a constant-temperature heating device. The unit is mg. This represents the initial measured mass of a blank glass fiber filter membrane in a dry, unloaded state before the filtration operation is performed, expressed in mg. This indicates the volume of water sample injected into the porous funnel for separation in a single operation, expressed in liters (L).

[0069] Reference Figures 1-7 The operation process is divided into chemical instrument detection operations for the filtrate and biomolecular sequence reading operations for the retained material, which output macroscopic carbon concentration data and microscopic ecological sequence data respectively.

[0070] The filtrate is extracted and introduced into the inlet of the total organic carbon analyzer. An acidic reagent is injected into the filtrate to induce a chemical reaction, converting carbonate components into carbon dioxide gas. A non-dispersive infrared detector is used to measure the volume of the emitted gas and output the dissolved inorganic carbon content. The liquid after the inorganic carbon is discharged is then introduced into a high-temperature combustion tube for oxidation, converting dissolved organic matter into carbon dioxide. The non-dispersive infrared detector is used again to measure the gas volume and output the dissolved organic carbon content.

[0071] Since the aforementioned steps established three independent sampling points in both the oyster farming area and the control area, three independent carbon measurement values ​​were obtained. To eliminate the measurement bias of a single point, the average carbon content measurement value for each area was calculated using the following mathematical formula: ; In the formula, This represents the average measured carbon content output for either the oyster farming area or the control area, expressed in mg / L. This indicates the output measured using a total organic carbon analyzer. The independent carbon content values ​​at each sampling point are expressed in mg / L. This indicates the total number of sampling points set, with a value of 3; This represents the sorting index number of each independent sampling point, and its value is a positive integer.

[0072] Based on the actual measurement and formula calculation output of the total organic carbon analyzer, the dissolved organic carbon content in the oyster farming area is limited to the range of 3.56 mg / L to 3.82 mg / L; the dissolved inorganic carbon content in the oyster farming area is limited to the range of 0.84 mg / L to 0.96 mg / L.

[0073] In the biomolecular sequence reading section, a glass fiber filter membrane containing the retentate is immersed in elution buffer and subjected to ultrasonic oscillation to separate the microbiome attached to the surface of the retentate. Chemical lysis buffer and glass microbeads are added to the separated microbiome, and mechanical oscillation is performed to disrupt the cell membrane and cell wall, thereby lysing the microbiome and releasing deoxyribonucleic acid (DNA).

[0074] The released deoxyribonucleic acid (DNA) was placed in the detection chamber of a UV spectrophotometer, and the absorbance ratio at wavelengths of 260 nm and 280 nm was measured to test the purity of the released DNA. DNA with an absorbance ratio between 1.8 and 2.0 was considered to have passed the purity test. Using an ultrasonic generator, the purified DNA was broken into short sequence fragments ranging from 300 bp to 500 bp in length. Adapter molecules with sequencing tags were ligated to both ends of these fragments to construct a sequencing library. The sequencing library was then input into the sequencing channel of a high-throughput sequencing instrument, and high-throughput metagenomic sequencing was performed to output the metagenomic sequencing sequences.

[0075] After quality control filtering of metagenomic sequencing sequences to remove low-quality sequences, a total sequence set containing DNA base arrangement information was obtained, and the total number of sequences was recorded. Sequence alignment algorithms were used to compare the total sequence set with microbial classification databases, functional metabolic pathway databases, and functional gene databases line by line to find matches, and the absolute number of sequences matching the target entries was counted. Based on the alignment results, the following mathematical formulas were used to obtain the abundance values ​​of microbial species, relative abundance values ​​of metabolic functions, and abundance values ​​of carbon cycling functional genes in the oyster farming area and the control area, respectively: ; In the formula, This represents the percentage of the calculated abundance value. This indicates the total number of sequences contained within the total sequence set; This represents the absolute number of sequences that matched the target sequence in a sequence comparison. When calculating microbial species abundance values, Replace these with the absolute sequence counts of methane consumers, organic matter degraders, and nitrogen-carbon cyclers, respectively; when calculating the relative abundance values ​​of metabolic functions... Replace these with the absolute sequence counts of amino acid metabolism, energy metabolism, and carbohydrate metabolism, respectively; when calculating the abundance values ​​of carbon cycle functional genes... Replace them with the absolute sequence number of the acyl-CoA dehydrogenase gene and the absolute sequence number of the mannosidase gene, respectively.

[0076] Reference Figures 1-7Before performing the numerical comparison and judgment operation, in order to eliminate the random fluctuation bias introduced by the environmental sampling process, nonparametric statistical tests and spatial matrix dimensionality reduction operations are performed on the multiple sets of numerical data obtained from the oyster farming area and the control area to establish a data statistical judgment flow to verify whether each value has reached the set judgment threshold.

[0077] Data sets containing dissolved organic carbon content, dissolved inorganic carbon content, microbial species abundance, relative abundance of metabolic functions, and abundance of carbon cycling genes were extracted as input variables. The data from the corresponding oyster farming area and control area were combined, sorted in ascending order, and assigned rank labels. For observations with the same value, an average rank label was assigned. Based on the rank assignment results, a Kruskal-Wallis test was performed, and the test statistic was calculated using the following mathematical formula: ; In the formula, This represents the numerical value of the Kruskal-Wallis nonparametric test statistic in the accounting output; This indicates the total number of merged sample observations that participated in the test. This indicates the total number of independent test groups; Indicates the first The number of sample observations contained within each test group; Indicates the first The sum of the rank indices corresponding to all independent sample observations within each test group; This represents the sorting index number of each independent sampling point, and its value is a positive integer.

[0078] Finish After numerical calculation, the P-value is output by comparing it with the chi-square distribution critical value table. The statistical decision threshold is set as follows. When the average values ​​of all measurements in the oyster farming area are greater than those in the control area, and the corresponding group's test output is also satisfied, the result is... When this condition is met, the characteristic variable is confirmed to meet the criteria for being greater than.

[0079] In addition to performing numerical comparisons for individual indicator variables, the system performs spatial distance matrix calculations to measure the overall structural shift in response to high-dimensional changes in micro-ecological communities and gene sequence distribution. The system extracts the statistical matrix of all absolute sequence counts corresponding to each sampling point and uses the following mathematical formula to calculate the Bray-Curtis dissimilarity index between each pair of sampling points: ; In the formula, Indicates the first The location of the sampling point is related to the first sampling point. The Bray-Curtis dissimilarity index values ​​for the distribution of microecological sequences at each sampling point were calculated, with the value range limited to between 0 and 1. This represents the total number of dimensions in the input matrix, indicating the classification of microbial species or carbon cycle functional genes. Indicates the first In the set of deoxyribonucleic acid fragment sequences at each sampling point location, the sequence belonging to the first... The absolute sequence count of each category; Indicates the first In the set of deoxyribonucleic acid fragment sequences at each sampling point location, the sequence belonging to the first... The absolute sequence count of each category; The sort index number represents the category, indicating the number to which it belongs. Classification of microbial species or carbon cycle functional genes.

[0080] Using all of the aforementioned outputs A high-dimensional distance matrix structure containing the spatial relationships of all sampling points is numerically constructed. This high-dimensional distance matrix structure is then input into a non-metric multidimensional scaling algorithm module to iteratively find the minimum loss value, mapping the high-dimensional spatial data to a two-dimensional plane coordinate system. To quantify the degree of deformation and distortion caused by the two-dimensional plane mapping to the original high-dimensional distance data, the pressure coefficient is calculated using the following mathematical formula: ; In the formula, This represents the pressure coefficient measurement result output by the non-metric multidimensional scaling analysis operation. Represents the first element in the original high-dimensional matrix structure. The sampling point and the first The original measurement distance between each sampling point was calculated using the aforementioned method. Numerical value; This indicates the position of the first element in the two-dimensional plane after the non-metric multidimensional scaling analysis mapping operation is completed. The coordinates of the sampling point and the first The Euclidean distance between the coordinates of each sampling point.

[0081] The system determines when the output is When the test value is less than 0.2, it is determined that the dimensionality reduction distance structure retains the community difference characteristics of the original data. In a two-dimensional plane coordinate system, it is determined that the coordinates of the three sampling points in the oyster farming area aggregate into a cluster, and are spatially separated from the coordinate cluster of the three sampling points in the control area, which constitutes objective evidence to confirm the spatial heterogeneity of the micro-ecological structure altered by aquaculture intervention.

[0082] Reference Figures 1-7The system receives and statistically validates a set of ten numerical values. These ten values ​​are mapped to dimensions of physicochemical concentration, microbial community, metabolic pathway, and functional genes, respectively. The comparison module subtracts the ten corresponding measurement values ​​belonging to the control area from the ten input values ​​belonging to the oyster farming area, generating ten independent difference calculation results.

[0083] To perform unified state determination and avoid logical deviations caused by single-index determination, a comprehensive state determination mathematical relationship based on product operations is set within the control module. The following mathematical formula is used to perform Boolean logic determination on ten independent difference calculation results: ; In the formula, This represents the final state control command signal output by the system, and its value is limited to either integer 0 or integer 1. When the output value is 1, the control process to maintain the breeding state is triggered; when the output value is 0, the current process is terminated. This indicates the total number of independent test groups; Indicates the first The result is calculated by subtracting the measured values ​​from the control area's measured values ​​from the measured values ​​in the oyster farming area. This represents the set step decision function. When the input variable... When greater than 0, The function outputs the integer 1; when the input variable... When less than or equal to 0, The function outputs the integer 0.

[0084] The ten criteria strictly correspond to the dissolved organic carbon content, dissolved inorganic carbon content, methane consumer abundance, organic matter degrader abundance, nitrogen-carbon cycler abundance, relative abundance of amino acid metabolism, relative abundance of energy metabolism, relative abundance of carbohydrate metabolism, acyl-CoA dehydrogenase gene abundance, and mannosidase gene abundance obtained from the aforementioned calculations. The comprehensive state determination mathematical formula outputs the following values ​​only if all ten calculated differences are greater than 0: The result is 1.

[0085] Systematic measurement data showed that in the control area, due to the lack of input from Hong Kong oyster excrement, the water maintained its original biochemical characteristics. The results confirmed that the absolute number of DNA fragments involved in carbon retention mechanisms in the control area was greater than the number of corresponding fragment sequences obtained from the oyster farming area. These carbon retention mechanism-related gene fragments were combinations of the carB, gmd, rpoS, algU, and pil genes. Through the above reverse numerical measurement comparison, it was confirmed that a shift from macromolecular carbon retention mechanisms to rapid degradation metabolic mechanisms occurred within the oyster farming area that met the comprehensive judgment threshold.

[0086] When the received value is 1 Following the signal, continuous environmental maintenance operations will be implemented to preserve the aquaculture status. At the intervention level, the removal of frame components fixed within the oyster farming area is prohibited, and the deployed floating rafts must be retained within the oyster farming area. Boundary dimensions will be continuously monitored, ensuring that the area covered by the deployed floating rafts on the horizontal plane consistently exceeds 30% of the total target water area. At the biological operation level, human harvesting and hauling are prohibited, and cultured Hong Kong oysters must be retained within the oyster farming area.

[0087] By retaining Hong Kong oysters within the water body, the system continuously maintains the cultured Hong Kong oysters through processes such as water intake and discharge, biomass excretion, and biodeposition to facilitate the settling of suspended solids into the bottom sediment. Through these continuous processes of excretion and biodeposition, organic compounds are continuously introduced into the water body of the oyster farming area over time, maintaining the material transfer rate from the aqueous phase to the liquid phase.

[0088] The final output of the above actions and biofeedback process is as follows: maintaining the dissolved organic carbon and dissolved inorganic carbon content in the oyster farming area consistently higher than that in the control area; and simultaneously maintaining the abundance of carbon cycling functional genes such as acyl-CoA dehydrogenase and mannosidase in the oyster farming area consistently higher than that in the control area. Thus, the execution steps for seawater carbon storage regulation, which involve the simultaneous enrichment of dissolved organic and dissolved inorganic carbon and the enrichment of carbon cycling functional genes, have been completed and solidified.

[0089] Specific application examples: This embodiment selects a semi-enclosed inner bay area located in a coastal region as the target water area. Before delineating the area, water quality detection equipment was used to obtain the following parameters: measured water temperature of 21.5℃, measured salinity of 28 ppt, measured pH of 8.2, measured dissolved oxygen concentration of 6.9 mg / L, and measured chlorophyll a concentration of 2.1 μg / L. All five parameters strictly fell within the defined parameter range, confirming that the water area met the initial conditions for conducting Hong Kong oyster farming and microecological testing.

[0090] Oyster farming and control areas were demarcated within the target waters. The straight-line distance between the two areas was set at 3.5 kilometers, greater than the maximum single tidal displacement distance measured in the target waters (2.8 kilometers), to prevent data interference from water exchange. The control area was maintained in its natural water condition. Multiple sets of 6-meter-long and 6-meter-wide floating rafts and Hong Kong oysters were deployed in the oyster farming area. Area calculations showed that the total area of ​​the deployed floating rafts accounted for 35% of the total surface area of ​​the oyster farming area, meeting the 30% coverage threshold requirement. The farming cycle was set at 12 months.

[0091] After the culture cycle ended, three independent sampling points were set up in each of the oyster culture area and the control area in an equilateral triangle structure. Surface seawater was collected using a polytetrafluoroethylene (PTFE) water sampler and stored in a brown polyester light-proof bottle at 4°C. Subsequently, a vacuum pump was used to drive the water sample through a 0.45 μm glass fiber filter membrane, and the liquid filtrate that passed through the filter membrane and the solid residue retained on the filter membrane surface were collected separately.

[0092] The extracted filtrate was input into a total organic carbon (DOC) analyzer. The results showed that the average dissolved organic carbon (DOC) content in the control area was 3.12 mg / L, and the average dissolved inorganic carbon (DIC) content was 0.75 mg / L. Correspondingly, the average DOC content in the oyster farming area reached 3.75 mg / L, and the average DIC content reached 0.88 mg / L. The data verified that due to the continuous filter feeding and excretion process of Hong Kong oysters, the measured concentration of dissolved carbon in the water of the farming area was higher than that in the control area.

[0093] Deoxyribonucleic acid extraction and metagenomic sequencing of the intercepted samples confirmed that microorganisms involved in carbon transfer and degradation pathways were enriched in the oyster farming area. Specifically, the absolute sequence number percentages (species abundance values) of methane consumers, organic matter degraders, and nitrogen-carbon cyclers measured in the oyster farming area were all greater than the abundance values ​​of the same groups in the control area.

[0094] Sequence alignment further revealed differences in functional abundance. In the oyster farming area, the relative abundance values ​​of metabolic functions reflecting organic matter processing capacity—amino acid metabolism, energy metabolism, and carbohydrate metabolism—were all higher than in the control area. At the level of carbon cycle functional genes, the abundance values ​​of acyl-CoA dehydrogenase genes and mannosidase genes related to rapid degradation mechanisms were increased in the oyster farming area, exceeding the measured values ​​in the control area.

[0095] Reverse feature validation showed that the abundance of genes related to macromolecular carbon retention mechanisms (specifically including carB, gmd, rpoS, algU, and pil genes) was higher in water samples from the control area, which was not affected by oyster farming, than in the oyster farming area. This control data, from the opposite perspective, confirms that oyster farming behavior essentially causes a shift in the aquatic microecology from macromolecular carbon retention mechanisms to rapid degradation and metabolism mechanisms.

[0096] All measured values ​​from the two groups of regions were input into the Kruskal-Wallis test module. The p-values ​​for all comparison items in the output results were less than 0.05, demonstrating statistical significance and ruling out random sampling error. The Bray-Curtis dissimilarity index was calculated by extracting the absolute sequence quantity statistics matrix and performing non-metric multidimensional scaling (NMDS). The output stress coefficient was 0.11, satisfying the dimensionality reduction retention condition of less than 0.2. In the output two-dimensional plane coordinate map, the coordinates of the three sampling points in the oyster farming area clustered into one cluster (community structure A), while the coordinates of the three sampling points in the control area clustered into another cluster (community structure B). The two sets of coordinate clusters were spatially separated and had no overlapping boundaries. This spatial heterogeneity result objectively confirms that the deployment of the oyster farming system did indeed have a deterministic intervention on the distribution of local aquatic microbial communities.

[0097] The control system receives the above ten key measurement differences (DOC difference, DIC difference, three microbial species abundance differences, three metabolic function relative abundance differences, and two functional gene abundance differences). Since all ten measurement differences are greater than 0, based on the set product operation threshold logic, the overall state judgment result outputs command signal 1.

[0098] According to the instruction signal, the operation to maintain the status was carried out, prohibiting the removal of floating rafts and net cages in the oyster farming area, maintaining a 35% coverage area ratio, and preserving the in-situ farming status of Hong Kong oysters. Thus, by utilizing the continuous biological excretion and sedimentation process, the operational status of transferring carbon components to the liquid phase and enriching functional genes in this sea area was solidified, achieving closed-loop regulation of the seawater carbon storage process.

Claims

1. A method for regulating seawater carbon cycling and carbon storage based on oyster farming, characterized in that, Includes the following steps: Oyster farming areas and control areas were delineated in the target waters. Floating rafts were set up in the oyster farming areas and Hong Kong oysters were farmed there, while no oysters were farmed in the control areas. After the culture cycle, water samples were collected from the oyster culture area and the control area, and the water samples were filtered to obtain filtrate and retentate, respectively. The dissolved organic carbon content and dissolved inorganic carbon content in the filtrate were measured, and the deoxyribonucleic acid of the retentate was extracted for metagenomic sequencing to obtain the abundance of microbial species, relative abundance of metabolic functions, and abundance of carbon cycling genes in the oyster farming area and the control area, respectively. When the dissolved organic carbon content, dissolved inorganic carbon content, abundance of microbial species of methane consumers, organic matter degraders, and nitrogen-carbon cyclers in the oyster farming area, relative abundance of metabolic functions such as amino acid metabolism, energy metabolism, and carbohydrate metabolism, and abundance of carbon cycling functional genes such as acyl-CoA dehydrogenase and mannosidase in the oyster farming area are all greater than those in the control area, the farming state is maintained.

2. The method for regulating seawater carbon cycle and carbon storage based on oyster farming according to claim 1, characterized in that, The target water area specifically includes: The target waters are semi-enclosed inner bays or estuaries; The water temperature of the target water area is between 20°C and 22°C. The salinity of the target water area is limited to between 18 ppt and 33 ppt; The pH of the target water body is limited to between 8.0 and 8.4; The dissolved oxygen concentration in the target water area is limited to between 6.8 mg / L and 7.1 mg / L; The chlorophyll a concentration in the target water area is limited to between 1.69 μg / L and 2.75 μg / L.

3. The method for regulating seawater carbon cycle and carbon storage based on oyster farming according to claim 1, characterized in that, The raft is 6 meters long and 6 meters wide, and the area covered by the rafts in the oyster farming area accounts for more than 30% of the total area of ​​the target water area.

4. The method for regulating seawater carbon cycle and carbon storage based on oyster farming according to claim 1, characterized in that, The breeding cycle specifically includes: The Hong Kong oysters filter-feed on phytoplankton and organic particles in the water within the oyster farming area; The Hong Kong oyster assimilates carbon into its soft tissue and shell; The Hong Kong oysters enrich their sediments through excretion and biodeposition.

5. The method for regulating seawater carbon cycle and carbon storage based on oyster farming according to claim 1, characterized in that, The collection of water samples from the oyster farming area and the control area specifically includes: Three sampling points were set up within the oyster farming area; Three control area sampling points were set within the control area; Surface seawater was collected from the sampling points in the three aquaculture areas and the three control areas, respectively. The collected surface seawater was stored away from light and used as the water sample.

6. The method for regulating seawater carbon cycle and carbon storage based on oyster farming according to claim 1, characterized in that, The determination of the dissolved organic carbon content and dissolved inorganic carbon content in the filtrate specifically includes: The filtrate was chemically analyzed using a total organic carbon analyzer, and the dissolved organic carbon content and dissolved inorganic carbon content were output. The dissolved organic carbon content in the oyster farming area is limited to the range of 3.56 mg / L to 3.82 mg / L; The dissolved inorganic carbon content in the oyster farming area is limited to the range of 0.84 mg / L to 0.96 mg / L.

7. The method for regulating seawater carbon cycle and carbon storage based on oyster farming according to claim 1, characterized in that, Extracting the deoxyribonucleic acid (DNA) from the retained sample for metagenomic sequencing specifically includes: Separate the microbial community attached to the surface of the retrieval material; The microbiome was subjected to cell disruption and deoxyribonucleic acid (DNA) release. The purity of the released deoxyribonucleic acid was tested. A sequencing library was constructed from deoxyribonucleic acid that passed purity testing. High-throughput metagenomic sequencing is performed on the sequencing library and the metagenomic sequencing sequence is output.

8. The method for regulating seawater carbon cycle and carbon storage based on oyster farming according to claim 1, characterized in that, The abundance values ​​of microbial species, relative abundance values ​​of metabolic functions, and abundance values ​​of genes related to carbon cycling functions in the oyster farming area and the control area specifically include: The abundance values ​​of microbial species are expressed as percentages of the absolute sequence counts of methane consumers, organic matter degraders, and nitrogen-carbon cyclers, respectively, divided by the total sequence count. The relative abundance of metabolic functions is expressed as a percentage of the absolute number of amino acid metabolism sequences, the absolute number of energy metabolism sequences, and the absolute number of carbohydrate metabolism sequences, divided by the total number of sequences. The abundance values ​​of carbon cycle functional genes are expressed as percentages of the absolute sequence number of the acyl-CoA dehydrogenase gene and the absolute sequence number of the mannosidase gene, respectively, divided by the total sequence number.

9. A method for regulating seawater carbon cycle and carbon storage based on oyster farming according to claim 1, characterized in that, Filtering the water sample to obtain filtrate and retentate specifically includes: The water sample was separated using a glass fiber filter membrane with a pore size of 0.45 μm. The liquid passing through the glass fiber filter membrane with a pore size of 0.45 μm is collected as the filtrate; The material adhering to the surface of the glass fiber filter membrane with a pore size of 0.45 μm is collected as the retentate.

10. A method for regulating seawater carbon cycle and carbon storage based on oyster farming according to claim 1, characterized in that, Maintaining the aforementioned aquaculture status specifically includes: The Hong Kong oysters cultivated in the oyster farming area are retained; The cultivated Hong Kong oysters continue to perform excretion and biodeposition processes; Dissolved organic carbon and dissolved inorganic carbon are continuously introduced into the water body of the oyster farming area through the aforementioned excretion and biological sedimentation processes; The dissolved organic carbon content and dissolved inorganic carbon content in the oyster farming area are maintained to be higher than those in the control area.

Images