Bacterial self-assembly to form core-shell micro-nano confined ca-mofs-microbial aggregates and applications thereof

Abstract

This invention belongs to the interdisciplinary field of environmental microbial engineering and biomineralization materials, specifically relating to a bacterial self-assembly forming a core-shell micro / nano confined Ca-MOFs-microbial aggregate and its applications. The structure is induced by the metabolic activity of *Cryptospirobacter spp.* A4N01 in a calcium-containing system. Organic components produced by the strain's metabolism coordinate with calcium ions, self-assembling on the surface of the microbial aggregate to form a Ca-MOFs shell, thus constructing a core-shell micro / nano confined structure with the microbial aggregate as the core and Ca-MOFs as the shell. This structure forms a confined reaction space at the nanoscale to micrometer scale, promoting substrate enrichment, regulating substance diffusion, and enhancing microbial metabolic processes, thereby improving microbial community stability and biotransformation efficiency. It has significant practical application value in extreme environmental pollution control fields such as oil and gas field produced water treatment and saline-alkali soil remediation.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of environmental microbial engineering and biomineralization materials, specifically relating to a bacterial self-assembly forming core-shell micro / nano confined Ca-MOFs-microbial aggregates and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Marine environments, produced water from oil and gas fields, and saline-alkali soils often exhibit characteristics such as high salinity, high hardness, and the coexistence of complex organic matter. These environments are typically extreme environments. Under such conditions, microbial communities are susceptible to osmotic pressure stress and restricted substrate diffusion, leading to decreased metabolic activity, unstable community structure, and reduced biotransformation efficiency of pollutants. Microorganisms often enhance their environmental adaptability by forming microbial aggregates, creating relatively stable spatial microenvironments at the microscale. This allows substrates and metabolic intermediates to accumulate in localized areas, thereby promoting metabolic synergy among microorganisms and improving community stability.

[0004] Metal-organic frameworks (MOFs) are a class of porous materials formed by the coordination of metal ions and organic ligands, possessing high specific surface area, tunable pore size, and good structural stability. However, the inventors have found that most MOFs still rely on chemical synthesis methods, and their sustainability and biocompatibility in complex aquatic environments remain unsatisfactory. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for bacterial self-assembly to form core-shell micro / nano-confined Ca-MOFs-microbial aggregates and their applications. This invention utilizes the metabolic activities of specific microorganisms to induce coordination and self-assembly of calcium ions with organic components produced by microbial metabolism at the biological interface, without the need for urea or the addition of exogenous precipitates, thereby constructing a stable core-shell micro / nano-confined Ca-MOFs-microbial aggregate structure. This structure forms a nanoscale to micrometer-scale reaction space with confinement effects, promoting substrate enrichment, regulating substance diffusion pathways, and enhancing microbial metabolic processes, thereby improving the stability and biotransformation efficiency of microbial communities. Based on the above research results, this invention is thus completed.

[0006] To achieve the above-mentioned technical objectives, the present invention relates to the following technical solutions:

[0007] One aspect of the present invention provides *Marinaphthous* ( Psychrobacter aquimarisApplication of A4N01 in the preparation of core-shell micro / nano confined Ca-MOFs-microbial aggregates.

[0008] The seawater psychrophilic bacteria ( Psychrobacter aquimaris A4N01 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2021120 and has been published in Chinese patent CN113403234A.

[0009] Specifically, the present invention has found through research that in a culture or treatment system containing calcium ions, the strain coordinates with calcium ions through organic components produced by its metabolic activities, and induces the formation of Ca-MOFs structures on the cell surface or in the extracellular polymer matrix, thereby forming a core-shell type micro-nano confined structure by self-assembly outside the bacterial aggregate.

[0010] Therefore, in the aforementioned application, calcium ions must be present during the preparation of core-shell micro / nano confined Ca-MOFs-microbial aggregates. The concentration of calcium ions can be no less than 10 mg / L, further ranging from 10-1000 mg / L, and even further from 10-500 mg / L. No specific limitation is made here. Of course, to maintain the basic growth and metabolism of the strain, the preparation of core-shell micro / nano confined Ca-MOFs-microbial aggregates also includes carbon-containing organic matter (organic carbon source). Any organic compound that can provide the strain with carbon (to form the cytoskeleton) and energy (obtained through oxidative decomposition) is acceptable, such as sugars, amino acids (peptone), and organic acid salts (e.g., sodium acetate). No specific limitation is made here. However, this preparation process does not rely on urea hydrolysis and does not require the introduction of exogenous nucleating species such as carbonates or phosphates.

[0011] A second aspect of the present invention provides a core-shell micro / nano confined Ca-MOFs-microbial aggregate, wherein the core-shell micro / nano confined Ca-MOFs-microbial aggregate is composed of *Bacillus psychrophilus* (a type of marine psychrophilic bacterium). Psychrobacter aquimaris A4NO1 is induced to form in calcium-containing systems through metabolic activity; Furthermore, the core-shell micro-nano confined Ca-MOFs-microbial aggregate structure has a microbial aggregate as its core, and a Ca-MOFs structural shell formed on its surface by the partitioning of calcium ions and organic components produced by microbial metabolism, thereby constituting a core-shell micro-nano confined structure; the organic components produced by microbial metabolism include extracellular polymers, organic acids, or organic components containing carboxyl, hydroxyl, or amino groups produced by strain metabolism.

[0012] The core-shell micro / nano confined Ca-MOFs-microbial aggregates are amorphous or have low crystallinity.

[0013] A third aspect of the present invention provides a method for preparing the above-mentioned core-shell micro / nano confined Ca-MOFs-microbial aggregates, the method comprising: Inoculating the bioreactor system with seawater psychrophilic bacteria Psychrobacter aquimaris The A4N01 is cultured; wherein the bioreaction system contains at least calcium ions.

[0014] Furthermore, the bioreaction system can be a calcium-ion-containing seawater environment, oilfield produced water environment, saline-alkali soil environment, or other extreme saline environments, without specific limitations. The calcium ion concentration can be no less than 10 mg / L, further 10-1000 mg / L, and even further 10-500 mg / L. Of course, in order to maintain the basic growth and metabolism of the strain, the preparation of core-shell micro / nano confined Ca-MOFs-microbial aggregates also contains carbon-containing organic matter (organic carbon source). The above-mentioned bioreaction system only needs to provide the strain with organic compounds that can provide carbon (to form the cytoskeleton) and energy (obtained through oxidative decomposition), such as sugars, amino acids (peptone), and organic acid salts (such as sodium acetate), without specific limitations.

[0015] In this invention, calcium ions are induced to accumulate on the cell surface or in the extracellular polymer matrix and partition with organic components through the metabolic activity of the strain under culture conditions. As the coordination and self-assembly process proceeds, a Ca-MOF structural layer is formed on the surface of the microbial aggregate, thereby forming a core-shell micro-nano confined structure.

[0016] A fourth aspect of the present invention provides the application of the above-described core-shell micro / nano confined Ca-MOFs-microbial aggregates in any one or more of the following: (a) Constructing a functional microbiome; (b) Treatment of produced water from oil and gas fields; (c) Remediation of saline-alkali soil; (d) Promote the bioconversion or removal of organic matter, total petroleum hydrocarbons or ammonium nitrogen.

[0017] The beneficial technical effects of one or more of the above technical solutions are as follows: The aforementioned technical solution is the first to utilize the metabolic characteristics of *Bacillus psychrophilus* A4N01 to achieve the bio-self-assembly of a core-shell micro / nano confined Ca-MOFs-microbial aggregate structure. This function of the strain is a first-time discovery. The formation of the core-shell micro / nano confined structure does not require urea hydrolysis or the addition of exogenous nucleating species such as carbonates and phosphates; it can be achieved solely using the system's own calcium ions and the strain's metabolic organic components. This approach is green, environmentally friendly, and low-cost, making it suitable for in-situ applications in complex and extreme environments. The core-shell micro / nano confined structure forms a confined reaction space at the nanoscale to micrometer scale, enabling substrate enrichment, regulating substance diffusion pathways, significantly increasing the contact probability between reactants and microbial metabolic active sites, enhancing microbial metabolic processes, and effectively solving the problem of low microbial metabolic activity in extreme environments.

[0018] Meanwhile, the core-shell micro / nano confined structure exhibits good stability and can serve as a spatial framework for enriching and constructing functional microbiomes. It maintains good structural integrity and functional activity even in high-salt, high-calcium produced water from oil and gas fields and saline-alkali soils, demonstrating high pollutant removal efficiency and strong microbial community stability with no significant performance degradation. The preparation method is simple and mild, enabling in-situ self-assembly of the core-shell micro / nano confined structure without complex equipment or processes, facilitating large-scale application. It holds significant industrial value in the bioremediation of extreme environments such as marine environments, produced water from oil and gas fields, and saline-alkali soils. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0020] Figure 1A The macroscopic morphology of strain A4N01 in sodium acetate hydrochloride medium; Figure 1B Macroscopic morphology of strain A4N01 in LB sodium chloride medium without calcium ions; Figure 2 The sedimentation properties of strain A4N01 and Ca-MOFs-microbial aggregate structure; Figure 3A SEM images of strain A4N01 and the structure of Ca-MOFs-microbial aggregates; Figure 3B SEM images of strain A4N01 and Ca-MOFs-microbial aggregates in other fields of view; Figure 3C FTIR spectra of strain A4N01 in different culture systems; Figure 3D XRD patterns of strain A4N01 in different culture systems; Figure 4A The effect of the formation or non-formation of Ca-MOFs-microbial aggregates by strain A4N01 on ammonium nitrogen removal; Figure 4B The effect of strain A4N01 forming or not forming Ca-MOFs-microbial aggregates on total organic carbon removal; Figure 5A The effect of preformed Ca-MOFs-microbial aggregates of strain A4N01 on ammonium nitrogen removal; Figure 5B The effect of preformed Ca-MOFs-microbial aggregates of strain A4N01 on total organic carbon removal; Figure 6 SEM images of the functional microbiome for oilfield produced water treatment developed by strain A4N01; Figure 7A To develop the microbiome of strain A4N01 for removing ammonium nitrogen from oilfield produced water; Figure 7B To study the effectiveness of strain A4N01 in removing total petroleum hydrocarbons from produced water in oilfields; Figure 8 SEM image of microbial aggregates of strain A4N01 applied to saline-alkali soil; Figure 9 The remediation efficacy of strain A4N01 on total petroleum hydrocarbons in saline-alkali soil was investigated. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0023] 1. Bioreaction System In this invention, the term "bioreaction system" refers to a reaction environment containing strains capable of inducing the formation of core-shell micro / nano-confined Ca-MOFs-microbial aggregates and their metabolic activities. The bioreaction system can be a liquid system, a semi-solid system, or a solid-liquid coexistence system, preferably an aqueous system. The bioreaction system may contain live bacterial cells, or in some embodiments, may not contain intact bacterial cells; its core characteristic is that the system contains at least the biochemical processes generated by the metabolic activities of the strains, thereby providing reaction conditions for the construction of core-shell micro / nano-confined Ca-MOFs-microbial aggregates.

[0024] In some embodiments, the bioreaction system contains organic matter and calcium ions, enabling the system to meet the conditions for self-assembly of Ca-MOFs-microbial aggregates.

[0025] 2. Ca-MOF structural precursor components In this invention, the term "Ca-MOF structure precursor component" refers to a functional component capable of participating in or inducing the formation of Ca-MOF structures. The precursor component includes at least extracellular metabolites produced by the metabolism of the strain, preferably including extracellular polymers, organic acids, or other organic groups. In the presence of calcium ions, the precursor component can promote the local enrichment of calcium ions through complexation, adsorption, charge neutralization, or bridging, and lower the energy barrier required for their nucleation, thereby inducing the formation of Ca-MOF structures at the biological interface.

[0026] In some embodiments, the Ca-MOFs structure can be formed on the cell surface or the surface of microbial aggregates and serve as the basis for the subsequent formation of core-shell structures.

[0027] 3. Non-cellular active components In this invention, the term "non-cellular active component" refers to a functional component that does not depend on the presence of intact viable bacterial cells but still has the ability to participate in the formation of Ca-MOF structures. The non-cellular active component can be derived from the liquid phase obtained after solid-liquid separation of a bioreactor system, such as supernatant, filtrate, or other systems that do not contain intact bacterial cells. The non-cellular active component may include extracellular metabolites or extracellular polymers produced by the strain, which can still participate in the formation of Ca-MOF structures and promote the formation of microbial aggregate structures in the presence of calcium ions.

[0028] In some embodiments, the non-cellular active component may be used independently to form Ca-MOFs structures, or in synergy with a live bacterial system to form Ca-MOFs-microbial aggregate structures.

[0029] 4. Core-shell micro / nano confined structures In this invention, the term "core-shell micro / nano confined structure" refers to a composite structure formed by the coordination self-assembly of organic components produced by the metabolism of the strain with calcium ions under calcium ion-induced conditions.

[0030] In some implementations, microbial aggregates serve as the structural core, while Ca-MOF structures form a coating layer on their surface, thus constituting a core-shell type micro / nano confined structure. This structure can macroscopically appear as granular or flocculent aggregates, and microscopically form confined reaction spaces ranging from nanoscale to micrometer scale with a certain degree of enclosure or semi-enclosure.

[0031] In some implementations, the Ca-MOFs-bacterial aggregates can exist as independent structural units and be used to construct functional microbiomes applicable to water or soil.

[0032] strain information The strain described in this invention is *Cryptospirobacter marinum* (Sinocyclohexane). Psychrobacter aquimaris The strain A4N01 was deposited at the China Center for Type Culture Collection (CCTCC) on January 20, 2021, with accession number CCTCC No: M 2021120. It should be noted that this invention is not directed at the strain itself, but rather relates to the novel functional performance and applications of this strain in self-assembling under calcium ion induction to form core-shell micro / nano confined Ca-MOFs-microbial aggregates. For ease of description, the term "seawater psychrophilic bacillus" is used in this specification. Psychrobacter aquimaris A4N01 can also be simply referred to as strain A4N01 or A4N01.

[0033] Fabrication of core-shell micro / nano confined structures The core-shell micro / nano confined Ca-MOFs-microbial aggregate structure of this invention is prepared through a calcium ion-triggered biomineralization and self-assembly process. This preparation process does not rely on urea hydrolysis and does not require the introduction of exogenous nucleating species such as carbonates or phosphates. The formation of the structure mainly includes the following steps: 1. Constructing a bioreaction system. Inoculate the strain A4N01, which is capable of inducing the formation of Ca-MOFs-microbial aggregates, into the culture system, or introduce the structural precursors produced by this strain into the reaction system. The system must contain both organic matter and calcium ions to provide the reaction conditions for subsequent Ca-MOF structure formation. For example, LB medium with hydrochloric acid as the salinity regulator, or other suitable culture media containing calcium ions for A4N01 growth.

[0034] 2. Under the influence of the bacterial strain's metabolic activities and its extracellular metabolites, calcium ions gradually accumulate on the cell surface, in the extracellular polymer matrix, or in the microenvironment of bacterial aggregates, and coordinate with organic components, thereby inducing the formation of a Ca-MOFs precursor layer at the biological interface.

[0035] 3. As the coordination and mineralization processes continue, the Ca-MOFs structure gradually self-assembles on the surface of the microbial aggregates and forms a coating layer, thereby constructing a core-shell micro / nano confined structure with the microbial aggregates as the core and the Ca-MOFs structure as the outer shell. Macroscopically, this structure can manifest as granular or flocculent aggregates, while microscopically it forms confined reaction spaces at the nanoscale to micrometer scale with a certain degree of enclosure or semi-enclosure.

[0036] In some embodiments, the formation process of the structure can be carried out under continuous or intermittent culture conditions; the formed Ca-MOFs-microbial aggregate structure can be separated and collected by sedimentation, centrifugation, filtration, or used directly in the in-situ system.

[0037] In a further embodiment, the core-shell micro-nano confined Ca-MOFs-microbial aggregate structure can remain stable under complex environmental conditions and exist as a structural unit with spatial confinement effect, thereby providing a stable microenvironment for the microbial community and promoting its continuous growth and metabolic activities.

[0038] Core-shell micro / nano confinement structures promote the conversion of organic matter and ammonium nitrogen. The core-shell micro / nano confined Ca-MOFs-microbial aggregate structure formed in this invention not only serves as a stable biomineralization structure but also creates a confined microenvironment within the system, thereby promoting the biotransformation of organic matter and ammonium nitrogen. The core-shell structure forms nanoscale to microscale spatial regions with a certain degree of enclosure or semi-enclosure at the microscale. These regions can, to some extent, restrict the diffusion of reactants, allowing organic matter, ammonium nitrogen, and their related metabolic intermediates to accumulate locally. Due to the shortened diffusion path and increased local concentration, the probability of contact between reactants and microbial metabolic active sites increases, thus promoting microbial metabolic processes. The core-shell micro / nano confined Ca-MOFs-microbial aggregate can form a local microenvironment distinct from the overall system conditions.

[0039] In some embodiments, the organic matter can be a simple carbon source such as sodium acetate or glucose, or it can be complex organic matter in oilfield produced water or organic matter in saline-alkali soil.

[0040] In some embodiments, the self-assembly process of the core-shell micro / nano confined Ca-MOFs-microbial aggregate structure can be carried out in the same system as the conversion process of organic matter and ammonium nitrogen; in other embodiments, the structure can also be formed first and play its confining role in subsequent reactions.

[0041] In some embodiments, the structure can remain stable under continuous or intermittent operating conditions and participate in microbial metabolic processes, thereby promoting the conversion of organic matter and ammonium nitrogen.

[0042] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0043] The following embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation of this disclosure. Those skilled in the art can make various modifications or equivalent substitutions without departing from the spirit and scope of the present invention.

[0044] The formulations of the culture media used in the following examples are as follows: LB hydrochloric acid activation medium: 10 g / L tryptone, 5 g / L yeast extract, 35 g / L hydrochloric acid.

[0045] LB sodium chloride activated medium: 10 g / L tryptone, 5 g / L yeast extract, 35 g / L sodium chloride.

[0046] Sodium acetate hydrochloride culture medium: 1600 mg / L anhydrous sodium acetate, 200 mg / L ammonium chloride, 20 mg / L potassium dihydrogen phosphate, 35 g / L hydrochloride.

[0047] Sodium acetate and sodium chloride culture medium: 1600 mg / L anhydrous sodium acetate, 200 mg / L ammonium chloride, 20 mg / L potassium dihydrogen phosphate, 0~1330 mg / L anhydrous calcium chloride, 35 g / L sodium chloride.

[0048] The above-mentioned culture medium corresponds to a salinity of 3.5%, and the salinity and calcium ion concentration used are based on seawater as a reference. As used herein, "salinity %" in the examples refers to the salt content expressed as a percentage by weight (w / v) of seawater.

[0049] In some embodiments of the present invention, strain A4N01 is first inoculated into LB hydrochloric acid activation medium or LB sodium chloride activation medium and cultured until the cells reach the logarithmic growth phase. Subsequently, the cultured cells are collected by centrifugation and washed with a 3.5% sodium chloride solution, repeated three times to remove residual culture medium components and achieve uniform salinity conditions. The washed cells are then transferred to sodium acetate culture medium, with an inoculation amount of 10% of the culture medium volume unless otherwise specified. Depending on the salinity source, sodium acetate hydrochloric acid culture medium or sodium acetate-sodium chloride culture medium may be used.

[0050] Unless otherwise stated, in order to evaluate the pollutant degradation efficiency of the self-assembled core-shell micro-nano confined Ca-MOFs-microbial aggregates formed by strain A4N01, the culture system used in the following examples involving the determination of "gravity sedimentation performance" and / or "total organic carbon removal rate" and / or "ammonium nitrogen removal rate" is sodium acetate culture medium; and different salinity sources and / or soluble calcium salts are added to the culture medium.

[0051] Example 1: Self-assembled particle structure of strain A4N01 After strain A4N01 was cultured to the logarithmic growth phase in LB hydrochloride activation medium, it was transferred to sodium acetate hydrochloride culture medium for further culture for 48 hours. At the same time, strain A4N01 was cultured to the logarithmic growth phase in LB sodium chloride activation medium and then inoculated into sodium acetate-sodium chloride culture medium without calcium ions for control culture.

[0052] In a culture medium using hydrochloric acid as the salinity source, strain A4N01 gradually formed dispersed, visible fine particles over time. These particles exhibited good stability in the culture medium and showed a clear sedimentation trend, displaying typical self-assembled particle structure characteristics, such as... Figure 1A As shown. In contrast, in the culture medium using sodium chloride as the salinity source, the culture system maintained a uniform dispersion throughout, and no obvious particles, flocs, or sedimentation structures were observed, such as... Figure 1B As shown.

[0053] Example 2: Sedimentation properties of self-assembled particles of strain A4N01 The bacterial culture of strain A4N01, activated with LB hydrochloric acid activation medium in Example 1, was washed with 3.5% sodium chloride and then inoculated into sodium acetate hydrochloric acid culture medium. Samples were taken at 0, 4, 8, 24, and 48 hours of incubation, with 15 mL of culture medium taken each time as test samples. The samples were placed under static conditions, and the turbidity change of the supernatant was measured at preset time points to characterize the sedimentation performance of the bacteria and their granular structures under gravity. The results are as follows: Figure 2 As shown.

[0054] With prolonged incubation time, the sedimentation performance of strain A4N01 in the seawater hydrophobic system significantly improved. The system reached a relatively stable sedimentation state after about 8 hours of incubation, and the sedimentation process was basically completed within about 5 minutes.

[0055] Example 3: Identification of self-assembled particles The particles formed in the culture system of Example 1 were collected by centrifugation, washed with deionized water, and freeze-dried for identification and testing of self-assembled particles. Figure 3ASEM results showed that the obtained particles exhibited a micron-sized aggregate structure with a porous surface, containing bacterial entry and exit channels, and calcium-containing organic-inorganic composite structures formed at different stages could be observed; simultaneously Figure 3B The SEM image shows an organic core-shell micro / nano confined structure in the early stage of self-assembly.

[0056] Figure 3C FTIR analysis showed the presence of characteristic absorption peaks such as carboxyl, hydroxyl, and amino groups in the sample. Furthermore, these characteristic peaks significantly decreased after cultivation in a calcium-containing culture medium, indicating that the organic components produced by microbial metabolism are related to calcium. 2+ Coordination occurs, forming a Ca-MOF structure. Figure 3D The XRD test results showed that the sample did not show obvious sharp crystal diffraction peaks, but instead exhibited broad and diffuse peak characteristics, indicating that the core-shell micro-nano confined structure is an amorphous Ca-MOF structure.

[0057] Example 4: Core-shell micro / nano confined structures accelerate the degradation of organic matter and ammonium nitrogen To verify the enhancing effect of the self-assembled core-shell micro / nano confined structure on the degradation process of organic matter and ammonium nitrogen, this example compares the degradation performance of strain A4N01 on organic carbon and ammonium nitrogen under different salinity conditions.

[0058] The bacterial suspension of strain A4N01, activated in Example 1 using LB hydrochloric acid activation medium and LB sodium chloride activation medium, was washed and then inoculated into sodium acetate hydrochloric acid culture medium and sodium acetate-sodium chloride culture medium (without calcium ions) respectively, to maintain consistency in salinity source. After continuous culture under the above conditions for 48 hours, the removal of ammonium nitrogen and total organic carbon in the system was detected, and the results are as follows: Figure 4A and Figure 4B As shown.

[0059] The A4N01 strain, activated and cultured using hydrochloric acid as a salinity source, achieved a removal rate of 81.51% for ammonium nitrogen and 94.82% for total organic carbon. However, the A4N01 strain, activated and cultured using sodium chloride as a salinity source without calcium ions, showed a decrease in ammonium nitrogen removal rate to 49.37% and total organic carbon removal rate to 39.20%. This indicates that in a hydrochloric acid system containing calcium ions, strain A4N01 can enhance the biodegradation of organic matter and ammonium nitrogen through self-assembly to form a core-shell micro / nano confined structure.

[0060] Example 5: The effect of core-shell micro / nano confined structure preforming on the degradation of ammonium nitrogen and organic matter. To verify the effect of pre-formation of the core-shell micro / nano confined structure during the activation stage on the degradation of organic matter and ammonium nitrogen, this example investigated the biocatalytic degradation performance of strain A4N01, which was activated in LB hydrochloric acid activation medium and LB sodium chloride activation medium, respectively, during the cultivation stage.

[0061] The bacterial suspensions of strain A4N01, activated to the logarithmic growth phase in LB hydrochloride activation medium and LB sodium chloride activation medium respectively in Example 1, were washed and then inoculated into sodium acetate and sodium chloride culture medium. Calcium ions were further introduced into the culture medium, with calcium ion concentration gradients of 0, 10, 20, 50, 100, 160, 320, and 480 mg / L. The strain A4N01 activated in LB hydrochloride activation medium formed self-mineralized particles and corresponding micro / nano confined structures during the activation stage; however, the strain activated in LB sodium chloride activation medium did not form such confined structures. After continuous cultivation in sodium chloride culture medium for 48 hours, the removal of ammonium nitrogen and total organic carbon in each system was detected, and the results are as follows: Figure 5A and Figure 5B As shown.

[0062] Compared to strains activated in LB sodium chloride activation medium, strain A4N01, which had its core-shell micro / nano confined structure pre-formed in LB hydrochloric acid activation medium, exhibited higher ammonium nitrogen and organic matter removal efficiencies in subsequent cultivation stages. The ammonium nitrogen removal rate increased from 41.15%–60.23% to 68.88%–93.60% compared to strains without pre-formed confined structures; the total organic carbon removal rate increased from 5.92%–37.17% to 84.52%–96.07%.

[0063] Example 6: A method for developing a functional microbiome for oilfield produced water treatment using strain A4N01 Produced water from the oilfield was pretreated to remove large suspended solids and free oil phase, ensuring the water met the basic conditions for biological treatment. A4N01 bacterial cultures activated to the logarithmic growth phase using LB seawater activation medium (as described in Example 1) were washed and inoculated into a sequencing batch reactor (SBR), with the concentration of activated bacteria controlled at approximately 10 g / L. Associated microorganisms derived from the produced water were introduced, allowing them to gradually accumulate and synergistically grow within the core-shell micro-nano confined structure self-assembled by A4N01. The reactor had an effective volume of 3.14 L and operated continuously, with an air diffuser at the bottom for aeration and mixing. Each cycle consisted of four steps: influent, aeration, settling, and effluent, including 15 minutes of influent, 23 hours of aeration, 30 minutes of settling, and 15 minutes of effluent. Each cycle lasted 24 hours, with a volume exchange rate of 50.0% and a hydraulic retention time of 48 hours. No sludge was discharged during operation.

[0064] The sequencing batch bioreactor uses produced water from the oilfield for cultivation throughout the process. The produced water contains a high concentration of calcium ions, which is beneficial for the self-assembly of A4N01 to form a core-shell micro-nano confined structure. During operation, appropriate amounts of sodium acetate and ammonium chloride are added to maintain the growth and reproduction of microorganisms. The water quality parameters of the produced water influent are shown in Table 1.

[0065] Table 1. Produced water quality parameters Inlet water composition concentration salinity 5.0% calcium ions 6254 mg / L magnesium ions 995 mg / L Total petroleum hydrocarbons 65.74 mg / L ammonium chloride 200 mg / L Anhydrous sodium acetate 1600 mg / L Potassium dihydrogen phosphate 20 mg / L To gradually regulate and develop the microbial community centered on strain A4N01, a gradient contact strategy using produced water was employed to construct the microbial community. This gradient contact strategy involved gradually increasing the proportion of produced water in the influent system during the reaction operation, with an operation period of 90 days. The operating parameters of the sequencing batch reactor are shown in Table 2.

[0066] Table 2 Operating parameters of sequencing batch reactor Operating days Produced water load salinity 0-20 50% 2.5% 21-60 75% 3.6% 60-90 100% 5.0% The resulting microbial community was collected by centrifugation, washed with deionized water, and freeze-dried to obtain SEM images, such as... Figure 6 As shown, the microbial aggregates are generally composed of nearly spherical or irregularly shaped particles, with a size of approximately tens of micrometers. These aggregates are formed by the tight aggregation of numerous short rod-shaped or ellipsoidal microbial cells, with fine granular deposits adhering to the surrounding cells, indicating that microorganisms and mineralized structures form self-assembled complex aggregates.

[0067] Example 7: The effect of microbial community developed from strain A4N01 on the treatment efficiency of oilfield produced water In Example 6, the concentrations of total petroleum hydrocarbons and ammonium nitrogen were continuously monitored to evaluate the treatment efficiency of the microbiome on produced water. The results are as follows: Figure 7A and Figure 7B As shown in the figure, throughout the entire operation phase, the removal capacity of the microbial community for total petroleum hydrocarbons and ammonium nitrogen remained relatively stable, without significant fluctuations or performance degradation. The average removal rate of total petroleum hydrocarbons was 86.75%, and the average removal rate of ammonium nitrogen was 90.18%.

[0068] Example 8: Application of strain A4N01 in saline-alkali soil Take the bacterial suspension of strain A4N01 activated with LB hydrochloric acid activation medium in Example 1, wash it, let it settle naturally and pour off the excess liquid, retain the core-shell micro-nano confined structure and bacterial suspension, and inoculate it into saline-alkali soil samples (salinity about 0.6-0.8%) at 0.25 L / kg (bacterial suspension / soil). After adding the bacterial suspension, turn the soil every 5 days and water it every 3 days to control the soil moisture content at 20%-30%.

[0069] SEM images of soil samples were obtained after washing, gradient ethanol dehydration, and freeze-drying, as shown below. Figure 8 As shown, a distinct microbial aggregate structure was observed. A composite structure consisting of fine particles and microbial cells formed on the surface of soil particles, in which relatively intact microbial cells were visible attached to the surface of mineralized particles and tightly bound to the surrounding fine-grained sediments. This structure exhibits typical characteristics of a core-shell micro / nano confined Ca-MOFs-microbial aggregate structure.

[0070] Example 9: Remediation efficacy of strain A4N01 on saline-alkali soil Soil samples were collected periodically during the cultivation process, as in Example 8, every 5 days (0, 5, 10, 15, 20, and 25 days), for a total of 5 samplings using a 5-point sampling method. The total petroleum hydrocarbon content in the soil was determined to evaluate the remediation efficacy of the core-shell micro / nano confined structure formed by the self-assembly of A4N01 for petroleum pollutants in soil. Results are as follows: Figure 9 As shown, during the 25-day continuous saline-alkali soil remediation process, the total petroleum hydrocarbon content decreased from 5.16 g / kg to 3.07 g / kg, with a removal rate of 40.54%.

[0071] In summary, this invention transforms calcium ions, which are ubiquitous in the environment, into Ca-MOF structural units, enabling them to self-assemble on the surface of microbial aggregates to form core-shell micro / nano confined structures. This results in the construction of microbial aggregate structures with spatial organization characteristics, achieving the functional transformation from environmental ions to microbial structural frameworks.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Marine psychrophilic bacteria ( Psychrobacter aquimaris Application of A4N01 in the preparation of core-shell micro / nano confined Ca-MOFs-microbial aggregates.

2. The application as described in claim 1, characterized in that, Calcium ions are present in the process of preparing core-shell micro / nano confined Ca-MOFs-microbial aggregates.

3. A core-shell micro / nano confined Ca-MOFs-microbial aggregate, characterized in that, The core-shell micro-nano confined Ca-MOFs-microbial aggregates are composed of marine psychrophilic bacteria ( Psychrobacter aquimaris A4N01 is induced to form in calcium-containing systems through metabolic activity.

4. The core-shell micro / nano confined Ca-MOFs-microbial aggregate as described in claim 3, characterized in that, The core-shell micro-nano confined Ca-MOFs-microbial aggregate structure has a microbial aggregate as its core, and a Ca-MOFs structure shell formed on its surface by the partitioning of calcium ions and organic components produced by microbial metabolism, thus constituting a core-shell micro-nano confined structure.

5. The core-shell micro / nano confined Ca-MOFs-microbial aggregate as described in claim 4, characterized in that, The organic components produced by microbial metabolism include extracellular polymers, organic acids, or organic components containing carboxyl, hydroxyl, or amino groups produced by the metabolism of the strain.

6. The core-shell micro / nano confined Ca-MOFs-microbial aggregates as described in any one of claims 3-5, characterized in that, The core-shell micro / nano confined Ca-MOFs-microbial aggregates are amorphous or have low crystallinity.

7. The method for preparing core-shell micro / nano confined Ca-MOFs-microbial aggregates according to any one of claims 3-6, characterized in that, The preparation method includes: Inoculating the bioreactor system with seawater psychrophilic bacteria Psychrobacter aquimaris A4N01 is cultured; wherein, the bioreaction system contains calcium ions.

8. The preparation method according to claim 7, characterized in that, The bioreactor system consists of a calcium-containing seawater environment, a produced water environment, and a saline-alkali soil environment.

9. The preparation method according to claim 7, characterized in that, Under culture conditions, calcium ions are induced to accumulate on the cell surface or in the extracellular polymer matrix and migrate to organic components through the metabolic activities of the strain. As the coordination and self-assembly process proceeds, a Ca-MOF structural layer is formed on the surface of the microbial aggregate, thereby forming a core-shell micro-nano confined structure.

10. The use of the core-shell micro / nano confined Ca-MOFs-microbial aggregates according to any one of claims 3-6 in any one or more of the following: (a) Constructing a functional microbiome; (b) Treatment of produced water from oil and gas fields; (c) Remediation of saline-alkali soil; (d) Promote the bioconversion or removal of organic matter, total petroleum hydrocarbons or ammonium nitrogen.

Images