Petroleum hydrocarbon degrading bacterium immobilized composite material with pH self-buffering and long-acting slow-release functions and application of petroleum hydrocarbon degrading bacterium immobilized composite material in contaminated soil remediation and plant synergy

Abstract

The invention belongs to the crossing field of environmental engineering, soil remediation and ecological restoration, and relates to a petroleum hydrocarbon degrading bacterium immobilized composite material with pH self-buffering and long-acting slow-release functions and application of the petroleum hydrocarbon degrading bacterium immobilized composite material in contaminated soil remediation and plant synergy. According to the composite material disclosed by the invention, iron alginate gel is used for embedding charcoal to form a composite carrier, and a petroleum hydrocarbon degrading flora is specifically fixed. The composite material disclosed by the invention can be used for efficiently degrading petroleum hydrocarbon; organic acid impact in petroleum hydrocarbon degradation can be effectively resisted, and the microenvironment is stabilized; the long-acting slow release of the petroleum hydrocarbon degrading bacteria is realized, and the bacteria become dominant bacterial communities in the later period of remediation; meanwhile, as a slow-release carbon-based fertilizer and an iron source, the carbon-based fertilizer and the iron source cooperate with functional bacteria metabolites, so that the activity of key enzymes such as soil dehydrogenase and FDA hydrolase is remarkably improved, plant growth is promoted, and systematic remediation integrating single degradation to degradation-improvement-growth promotion is achieved. The material is simple and convenient to prepare, and a new technology is provided for green and sustainable remediation of petroleum hydrocarbon pollution.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of environmental engineering, soil remediation and ecological restoration, and specifically relates to a multifunctional composite material for efficient remediation and simultaneous improvement of petroleum hydrocarbon contaminated soil, its preparation method and application. Background Technology

[0002] Total Petroleum Hydrocarbons (TPH) are a highly complex mixture composed of alkanes, cycloalkanes, polycyclic aromatic hydrocarbons, and heteroatom hydrocarbons. Due to their strong hydrophobicity, reluctance to degrade, and high toxicity, they have become one of the most serious soil pollution problems. Therefore, the development of efficient, green, and sustainable TPH-contaminated soil remediation technologies is urgently needed.

[0003] Bioremediation technology is considered one of the most promising methods for remediating TPH-contaminated soil due to its environmental friendliness and relatively low cost. The core of this technology lies in using the metabolic action of microorganisms to degrade or transform pollutants into harmless substances. However, directly adding free functional microorganisms (bacterial agents) to contaminated soil often faces the following severe challenges: (1) Difficulty in colonization: Exogenous microorganisms are subject to strong competition from native microorganisms and stress from complex environmental factors, making it difficult for them to effectively colonize and become the dominant microbial community, resulting in unstable and short-lived remediation effects; (2) Environmental stress: High concentrations of TPH are toxic to microorganisms, and during aerobic degradation, they produce a large number of intermediate metabolites (such as low-molecular-weight organic acids), leading to a sharp acidification of the soil microenvironment (significant decrease in pH), which further inhibits the activity of the degrading microorganisms themselves, forming a vicious cycle of "degradation-acidification-inhibition of degradation", which is a long-term bottleneck restricting the improvement of bioremediation efficiency.

[0004] To protect functional microorganisms and enhance their regenerative efficacy, immobilized microbial technology has emerged. This technology provides physical protection and a suitable microenvironment by confining or positioning microorganisms within a specific carrier space. Among numerous carrier materials, natural polysaccharides (such as sodium alginate) and multivalent metal ions (such as Ca) are commonly used. 2+ Fe 3+ Hydrogels formed by cross-linking have attracted much attention due to their good biocompatibility and mild operating conditions. In recent years, in order to further improve the performance of the carrier, researchers have tried to introduce biochar, which has a porous structure, adsorption properties and nutrient retention function, into the gel system to form a composite carrier.

[0005] In the prior art, various alginate-biochar composite gel materials have been disclosed for environmental remediation. Patent CN115747196A discloses a "biochar-loaded, bacteria-laden iron alginate gel," which uses Shewanella bacteria and biochar co-encapsulated within an iron alginate gel for the anaerobic remediation of hexavalent chromium-contaminated soil. This technology utilizes the dissimilatory metal reduction capabilities of Shewanella bacteria and the immobilization effect of the iron alginate gel, achieving good results in the specific scenario of heavy metal reduction.

[0006] However, in-depth research and practice have revealed that directly applying the above-mentioned or similar immobilization technologies to the aerobic bioremediation of complex organic pollutants such as petroleum hydrocarbons has fundamental limitations and unresolved technical challenges: First, there is a mismatch in functional orientation: Existing technologies such as CN115747196A mainly target the reduction and fixation of heavy metals. Their microbial action mode (dissimilar reduction), environmental requirements (anaerobic or facultative anaerobic), and target pollutants (inorganic metal ions) are fundamentally different from the aerobic oxidative degradation process of petroleum hydrocarbons.

[0007] Second, there is a lack of mechanisms to address the characteristic byproduct (acidification): existing designs for iron alginate / biochar composite carriers primarily focus on physical encapsulation and protection, and providing adsorption sites. They do not functionalize the carriers to address the organic acid shocks that inevitably occur during the biodegradation of petroleum hydrocarbons, which lead to a continuous decrease in soil pH. Ordinary carriers lack active and long-lasting pH buffering capacity, making it impossible to break the aforementioned acidification inhibition cycle, resulting in remediation efficiency that is difficult to maintain or even declines in the later stages.

[0008] Third, the remediation goals are singular, neglecting ecological restoration: Existing technologies mostly use "pollutant removal rate" as a single endpoint indicator, and the functional design of their carrier systems also revolves around this single goal. However, ideal soil remediation requires not only the removal of pollutants, but also the simultaneous restoration of soil ecological functions (such as enzyme activity) and productivity (such as promoting plant growth). Existing carrier materials (such as the carrier in CN115747196A) do not integrate soil improvement and plant growth promotion functions, and cannot achieve the leap from "purification" to "restoration".

[0009] In summary, there is currently a lack of immobilized microbial composite materials specifically designed to address the remediation characteristics of petroleum hydrocarbon-contaminated soils. Summary of the Invention

[0010] In view of the shortcomings of existing technologies that use sodium alginate iron-biochar carriers for immobilizing microorganisms, such as limited functionality, inability to cope with the special characteristics of petroleum hydrocarbon remediation processes, and lack of ecological restoration capabilities, this invention provides a composite material for the remediation and improvement of petroleum hydrocarbon contaminated soil. Through specific component design and structural construction, this material produces an unexpected synergistic remediation effect that goes beyond the simple summation of the components.

[0011] The technical solution of this invention is a petroleum hydrocarbon-degrading bacteria immobilized composite material with pH self-buffering and long-term slow-release functions. The composite material contains ferric alginate gel and biochar loaded with petroleum hydrocarbon-degrading bacteria. The ferric alginate gel encapsulates the biochar loaded with petroleum hydrocarbon-degrading bacteria, forming a three-dimensional network porous structure. This petroleum hydrocarbon-degrading bacteria immobilized composite material with pH self-buffering and long-term slow-release functions is used to remediate petroleum hydrocarbon-contaminated soil and possesses pH self-buffering capabilities.

[0012] Fe in the ferric alginate gel network 3+ The biochar surface, which is loaded with petroleum hydrocarbon-degrading bacteria, forms a unique buffer system with hydroxyl and other functional groups. This buffer system can effectively neutralize the organic acids produced by petroleum hydrocarbon degradation and stabilize the pH of the soil microenvironment within a range suitable for microbial metabolism. This is the first time that this invention has revealed and applied this key feature to this specific scenario.

[0013] The petroleum hydrocarbon-degrading bacteria immobilized composite material of this invention, possessing pH self-buffering and long-lasting sustained-release functions, has a three-dimensional network porous structure. This structure not only provides physical protection for microorganisms but also achieves long-lasting buffering through the synergistic effect of physical confinement and chemical bonding of the petroleum hydrocarbon-degrading bacteria. This ensures that the functional bacteria can still effectively colonize and become the dominant bacterial population in the mid-to-late stages of remediation, solving the problem of remediation efficacy decaying over time.

[0014] The petroleum hydrocarbon-degrading bacteria immobilized composite material of this invention, with pH self-buffering and long-lasting slow-release functions, can effectively enhance the activity of various key enzymes in soil, such as dehydrogenases, FDA hydrolases, and polyphenol oxidases. This is attributed to the stable microenvironment and continuous microbial stimulation, which synergistically activate the basic metabolic functions of the soil ecosystem.

[0015] This invention utilizes a petroleum hydrocarbon-degrading bacteria immobilized composite material with pH self-buffering and long-term slow-release functions. Biochar serves as a carbon-based fertilizer, ferric alginate as a slow-release iron source, and the petroleum hydrocarbon-degrading bacteria as a biostimulant. The synergistic effect of these three components significantly promotes the biomass of plants growing in the remediated soil. This achieves a revolutionary "remediation while production" model.

[0016] The biochar used in the biochar loaded with petroleum hydrocarbon-degrading bacteria is made from raw materials such as straw, sawdust, sludge, or algae.

[0017] The petroleum hydrocarbon degrading bacteria are selected from any one or any combination of bacteria, fungi, and yeast, preferably bacteria, more preferably aerobic petroleum hydrocarbon degrading bacteria, and further selected from Pseudomonas, Bacillus, Serratia, Acinetobacter, Bacillus belye, Rhodococcus, Achromobacter, Burkholderia, Alcaligenes, Gordon's, Alkane-eating bacteria, Marineella, Sphingosomal, Trichomonas, Flavobacterium, and Mycobacterium. The microbiota includes any one or any combination of the genera *R. roxburghii*, ...

[0018] The preparation of the petroleum hydrocarbon degrading bacteria immobilized composite material with pH self-buffering and long-lasting sustained-release functions described above in this invention includes: (1) The biochar loaded with petroleum hydrocarbon degrading bacteria and sodium alginate were thoroughly mixed in water to form a mixed slurry; (2) The mixed slurry was added dropwise to the iron salt solution to cross-link the reaction and obtain a petroleum hydrocarbon degrading bacteria immobilized composite material with pH self-buffering and long-term slow-release function.

[0019] In step (1), sodium alginate is first added to water to form a sodium alginate solution, and then biochar loaded with petroleum hydrocarbon degrading bacteria is added to the sodium alginate solution and mixed thoroughly to form a mixed slurry.

[0020] In step (1), the mass ratio of the biochar loaded with petroleum hydrocarbon degrading bacteria to sodium alginate is 1:1-5, preferably 1:1-3, and more preferably 1:2; the concentration of sodium alginate is 0.01-0.1 g / mL, preferably 0.01-0.05 g / mL, more preferably 0.01-0.03 g / mL, and even more preferably 0.02 g / mL.

[0021] In step (1), the preparation of biochar loaded with petroleum hydrocarbon degrading bacteria includes: mixing and adsorbing biochar and petroleum hydrocarbon degrading bacteria in a liquid culture medium to obtain biochar loaded with petroleum hydrocarbon degrading bacteria.

[0022] The ratio of biochar to liquid culture medium is 1g:10-50mL, preferably 1g:15-35mL, more preferably 1g:15-25mL, and even more preferably 1g:20mL. The liquid culture medium is selected from, but is not limited to, LB liquid medium, beef extract peptone liquid medium, bean sprout juice liquid medium, SCDLP liquid medium, YEPD liquid medium, potato dextrose liquid medium, etc., and is scientifically selected according to the strain being cultured. This is a conventional technique well-known to those skilled in the art.

[0023] The raw materials for biochar production are derived from straw, sawdust, sludge, or algae, etc. The preparation includes: carbonizing the raw materials at 500±50℃ under an inert or nitrogen atmosphere to obtain biochar. The heating rate is 4-6℃ / min, preferably 5℃ / min. The carbonization time is 2-3 hours, preferably 2 hours. As one embodiment, carbonization is carried out at 5℃ / min to 500℃ for 2 hours. The inert atmosphere is selected from any one or any combination of argon, helium, neon, krypton, and xenon.

[0024] The total bacterial count of the petroleum hydrocarbon degrading bacteria is ≥10. 7 CFU / g, wherein the petroleum hydrocarbon degrading bacteria are selected from any one or any combination of bacteria, fungi, and yeast, preferably bacteria, more preferably aerobic petroleum hydrocarbon degrading bacteria, and further selected from, but not limited to, *Pseudomonas*, *Bacillus*, *Serratia*, *Acinetobacter*, *Bacillus belyssae*, *Rhodococcus*, *Achromobacter*, *Burkholderia*, *Alcaligenes*, *Gordonella*, *Alkanephaga*, *Hydrogenobacter*, *Sphingomonas*, *Trichophyton*, *Flavobacterium*, and *Bacillus*. The bacterial culture comprises any one or any combination of the genera *Bacillus*, *Rolstonia*, *Trichomonas*, *Gluttony*, *Carnosus*, *Aureobacillus*, *Trichomonas*, *Acidophilus*, *Aureobacillus*, *Micrococcus*, *Aeromonas*, *Oligotrophosporium*, *Corynebacterium*, *Alcaligenes*, *Goldenella*, *Fibrobacter*, *Lycobacterium*, and *Hydrocyclobacter*, further selected from *Pseudomonas* and / or *Bacillus*, more preferably a mixed flora of *Pseudomonas* and *Bacillus*, to utilize their metabolic complementarity. In the mixed flora of *Pseudomonas* and *Bacillus*, the mixing ratio of *Pseudomonas* to *Bacillus* is 1:0.9-1.2, preferably 1:1; the *Pseudomonas* is selected from *Pseudomonas aeruginosa*, and the *Bacillus* is selected from *Bacillus licheniformis*.

[0025] Furthermore, the mixture is adsorbed at 30°C for 16-48 hours. Mixing is achieved by shaking or stirring at a speed of 150-200 r / min, preferably 150 r / min.

[0026] In step (2), the molar ratio of sodium alginate in the mixed slurry to iron salt in the iron salt solution is 1-5:1, preferably 2-3:1, more preferably 2.5-3:1, and even more preferably 2.5:1; the concentration of the iron salt solution is 0.005-0.05 g / mL, preferably 0.008-0.03 g / mL, more preferably 0.008-0.02 g / mL, more preferably 0.008-0.012 g / mL, and even more preferably 0.01 g / mL. The iron salt in the iron salt solution is a ferric salt, which is selected from any one or any combination of ferric chloride, ferric sulfate, or ferric nitrate, preferably ferric chloride, such as FeCl3·6H2O.

[0027] The preparation method of the petroleum hydrocarbon degrading bacteria immobilized composite material with pH self-buffering and long-term sustained-release function described above in this invention first adsorbs and immobilizes petroleum hydrocarbon degrading bacteria on biochar to form a bacterial-carbon composite; then mixes the bacterial-carbon composite with sodium alginate solution and adds it dropwise into an iron salt solution for cross-linking and molding; this two-step process of "adsorption followed by encapsulation" constructs a dual stable structure of "biochar-microorganism" and "ferric alginate gel-biochar".

[0028] The petroleum hydrocarbon-degrading bacteria immobilized composite material of the present invention, possessing pH self-buffering and long-lasting slow-release functions, simultaneously remediates petroleum hydrocarbon pollution and promotes plant growth. It can be used to remediate petroleum hydrocarbon pollution and / or promote plant growth, or to prepare products for petroleum hydrocarbon pollution remediation and / or plant growth promotion. These products include, but are not limited to, additives, fertilizers, cultivation substrates, and soil conditioners. The present invention provides a product for petroleum hydrocarbon pollution remediation and / or plant growth promotion, wherein the product contains the aforementioned petroleum hydrocarbon-degrading bacteria immobilized composite material with pH self-buffering and long-lasting slow-release functions. These products include, but are not limited to, additives, fertilizers, cultivation substrates, and soil conditioners.

[0029] The present invention provides a method for remediating petroleum hydrocarbon pollution and / or promoting plant growth, comprising the steps of adding the petroleum hydrocarbon degrading bacteria immobilized composite material with pH self-buffering and long-term slow-release function described above, or a petroleum hydrocarbon pollution remediation and / or plant growth promotion product described above, to the target material.

[0030] Furthermore, the amount of the petroleum hydrocarbon degrading bacteria immobilized composite material with pH self-buffering and long-lasting slow-release function or the petroleum hydrocarbon pollution remediation and / or plant growth promotion product of the present invention can be flexibly and scientifically adjusted according to the needs of petroleum hydrocarbon pollution or plant growth.

[0031] The plant in question is rapeseed.

[0032] The petroleum hydrocarbon-degrading bacteria immobilized composite material of this invention, possessing pH self-buffering and long-term sustained-release functions, not only efficiently immobilizes and protects functional bacteria but also intelligently responds to environmental acidification during the remediation process, maintaining microenvironmental stability. It not only achieves long-term sustained release and dominant colonization of functional bacteria, ensuring the durability of the remediation effect but also synergistically improves soil properties, activates soil enzyme systems, and promotes plant growth while degrading pollutants, ultimately realizing integrated ecological restoration of contaminated soil through "remediation-improvement-production." Developing such multifunctional integrated composite materials is of great significance for promoting the practical application and upgrading of petroleum hydrocarbon-contaminated soil remediation technology. Specifically, this invention solves the following technical problems: 1. How to make the immobilized carrier not only protect microorganisms, but also intelligently respond to and buffer the soil acidification problem that inevitably occurs during the biodegradation of petroleum hydrocarbons, so as to provide a continuous and stable metabolic environment for functional bacteria; 2. How to achieve long-term, controlled release and dominant colonization of functional microorganisms, overcome the problem of exogenous bacterial agents being easily eliminated, and ensure the durability of the repair effect; 3. How to go beyond the single pollutant removal target and design a composite material that can simultaneously activate soil ecological functions, improve soil properties and promote plant growth, so as to achieve a leap from "purification" to "ecological restoration".

[0033] The beneficial effects of this invention are significant and unexpected: Functional Integration and Scenario Targeting: This invention is not a simple replacement of existing bacterial strains with existing carriers, but rather a functional redesign of the carrier system specifically for the complex scenario of "aerobic bioremediation of petroleum hydrocarbons." It is the first to integrate pH self-buffering as one of its core functions into an iron alginate-biochar carrier, solving a key bottleneck problem in this scenario—a feature not taught or inspired by existing technologies.

[0034] Synergistic effects and multiple benefits: This invention produces significant synergistic effects: (a) Synergistic effect between carrier stability and microbial activity: Stable pH environment and physical protection enable higher and more persistent microbial activity; (b) Synergistic effect between degradation and improvement: While efficiently degrading petroleum hydrocarbons (e.g., 65.99% removal rate in 30 days in the example), it significantly improves the activity of key soil enzymes (e.g., dehydrogenase activity increased several times); (c) Synergistic effect between remediation and production: The material itself acts as a nutrient source (carbon, iron) and a biostimulant, which can significantly promote plant growth (e.g., rapeseed), and the biomass can even exceed that of the uncontaminated soil control group, achieving a unity of ecological and production benefits.

[0035] Long-lasting and ecologically guiding: Through a long-lasting slow-release mechanism, exogenous functional bacteria are guided to successfully colonize into dominant bacterial groups in the soil in the middle and late stages (such as a significant increase in the relative abundance of Pseudomonas spp.), which changes the remediation process from "externally driven" to "system self-sustaining", ensuring the long-term stability of the remediation effect.

[0036] In summary, this invention provides an integrated solution to a systemic problem. Its inventiveness lies not in altering a single isolated component, but in unexpectedly stimulating and utilizing the unique and powerful synergistic effect among iron alginate, biochar, and petroleum hydrocarbon-degrading bacteria in the novel application scenario of petroleum hydrocarbon-contaminated soil remediation through specific component selection and structural design. This simultaneously overcomes several long-standing technical challenges, such as environmental acidification inhibition, difficulty in bacterial colonization, and limited remediation function, achieving unexpected and superior results, demonstrating outstanding substantive characteristics and significant progress. Attached Figure Description

[0037] Figure 1 This is a SEM image of sesame straw biochar.

[0038] Figure 2 SEM image of petroleum hydrocarbon-degrading bacteria immobilized on biochar.

[0039] Figure 3 This is a SEM image of the iron alginate hydrogel.

[0040] Figure 4 SEM image of petroleum hydrocarbon degrading bacteria embedded in iron alginate.

[0041] Figure 5 SEM image of petroleum hydrocarbon degrading bacteria immobilized on biochar embedded in iron alginate.

[0042] Figure 6 SEM image of petroleum hydrocarbon degrading bacteria immobilized on biochar embedded in iron alginate.

[0043] Figure 7 The effects of different treatment groups on the remediation of petroleum hydrocarbons in soil.

[0044] Figure 8 The changes in soil pH in different treatment groups.

[0045] Figure 9 To investigate the changes in dehydrogenase activity in soil from different treatment groups 30 days later.

[0046] Figure 10 To investigate the changes in FDA hydrolase activity in soil from different treatment groups 30 days after remediation.

[0047] Figure 11 To investigate the changes in polyphenol oxidase activity in soil from different treatment groups 30 days after treatment.

[0048] Figure 12 Hierarchical cluster analysis of different soil samples at the bacterial genus level. Detailed Implementation

[0049] The following detailed description, in conjunction with the accompanying drawings and specific embodiments, illustrates the present invention's immobilized composite material for petroleum hydrocarbon-degrading bacteria, possessing pH self-buffering and long-lasting slow-release functions, and its application in contaminated soil remediation and phytoremediation. The advantages and features of the invention will become clearer from the following description and claims.

[0050] Example 1: The method for preparing petroleum hydrocarbon-degrading bacteria immobilized by alginate-embedded biochar in this embodiment includes the following steps: (1) Preparation of sesame straw biochar (BC) Weigh a certain amount of pre-treated sesame straw biomass powder into an alumina crucible, cover it and place it in a tube furnace, purge with nitrogen, set the heating rate to 5℃ / min, heat from room temperature to 500℃ and hold for 2 hours, and allow it to cool naturally after the reaction is complete. The resulting black solid powder is biochar.

[0051] (2) Activation of petroleum hydrocarbon degradation compound bacteria (B) Inoculate 2 mL of petroleum hydrocarbon-degrading mixed bacteria (a 1:1 mixture of Pseudomonas aeruginosa and Bacillus licheniformis) into 100 mL of autoclaved LB broth and incubate at 30 °C and 150 rpm for 24 h.

[0052] (3) Preparation of biochar-immobilized petroleum hydrocarbon degrading bacteria (BC-B) Weigh 1g of biochar and add it to LB liquid medium at a ratio of 1:20 (w / v) and sterilize for 30 min. Centrifuge the activated petroleum hydrocarbon degrading mixed bacteria from (2) at 3500 r / min for 10 min, and add the precipitate to the LB solution of biochar. The total bacterial count should be ≥10. 7 The biochar was then incubated at CFU / g and adsorbed in a constant temperature shaker at 30℃ and 150 r / min for 48 h. Finally, the biochar-immobilized microorganisms were centrifuged, freeze-dried, and stored at low temperature for later use.

[0053] (4) Preparation of petroleum hydrocarbon degrading bacteria encapsulated with iron alginate (Fe-SA-B) Weigh 2g of sodium alginate powder into 100mL of deionized water, stir continuously to remove air bubbles, and mix evenly. Centrifuge the activated petroleum hydrocarbon degrading mixed bacteria from (2) at 3500r / min for 10min, and add the precipitate to 2% sodium alginate gel to disperse evenly. Then, weigh 1g of FeCl3•6H2O and dissolve it in 100mL of deionized water to prepare an ionic crosslinking agent. Finally, add the sodium alginate gel mixture containing petroleum hydrocarbon degrading bacteria dropwise to the iron ion crosslinking agent solution and crosslink for 30min. The resulting gel spheres are the iron alginate-encapsulated petroleum hydrocarbon degrading bacteria composite material. Freeze-dry them and store them at low temperature for later use.

[0054] (5) Preparation of petroleum hydrocarbon degrading bacteria immobilized by iron alginate-encapsulated biochar (Fe-SA-BC-B) Weigh 2g of sodium alginate powder into 100mL of deionized water, stir continuously to remove air bubbles, and mix evenly. Add the biochar-immobilized microbial composite material obtained after centrifugation in (3) to 2% sodium alginate gel and disperse it evenly. Then, weigh 1g of FeCl3•6H2O and dissolve it in 100mL of deionized water to prepare an ionic crosslinking agent. Finally, add the sodium alginate gel mixture containing biochar-immobilized petroleum hydrocarbon degrading bacteria dropwise to the iron ion crosslinking agent solution and crosslink for 30min. The resulting gel spheres are the iron alginate-embedded biochar-immobilized petroleum hydrocarbon degrading bacteria composite material. Freeze-dry it and store it at low temperature for later use.

[0055] Example 2: The samples prepared in Example 1 were imaged and analyzed using a scanning electron microscope (SEM) to observe the appearance morphology of different materials and the adhesion of microorganisms.

[0056] The biochar, biochar-immobilized petroleum hydrocarbon-degrading bacteria, ferric alginate hydrogel, ferric alginate-embedded petroleum hydrocarbon-degrading bacteria, and ferric alginate-embedded biochar-immobilized petroleum hydrocarbon-degrading bacteria from Example 1 were analyzed by field emission scanning electron microscopy (SEM). Figure 1 Sesame straw biochar Figure 2 For biochar immobilization of petroleum hydrocarbon degrading bacteria, Figure 3 It is an iron alginate hydrogel. Figure 4 Ferrous alginate-encapsulated petroleum hydrocarbon-degrading bacteria, Figure 5 and Figure 6 Ferrous alginate-encapsulated biochar-immobilized petroleum hydrocarbon-degrading bacteria. Figure 1 It is evident that sesame straw biochar possesses a loose and porous carbonaceous framework structure, providing favorable conditions for microbial attachment and proliferation. As expected, the petroleum hydrocarbon-degrading strains adhered well to the biochar, thus successfully achieving the preparation of biochar-immobilized petroleum hydrocarbon-degrading bacteria. Figure 2Most cells are dispersed on the surface of the biochar. However, due to their small size, some cells may also have entered the porous structure of the biochar. Interestingly, extracellular polymers are clearly observed around the cells. These extracellular polymers effectively mitigate the toxic effects of toxins on cells and enhance the adhesion between cells and biochar. Figure 3 This is a cross-sectional morphology image of iron alginate hydrogel microspheres, which mainly exhibit a sheet-like porous structure. Figure 4 It is evident that the petroleum hydrocarbon degrading bacteria were successfully encapsulated within the alginate-iron hydrogel network structure. This not only protects the encapsulated petroleum hydrocarbon degrading bacteria from adverse microenvironments but also prolongs the release time of the petroleum hydrocarbon degrading bacteria at the target site, effectively enhancing the long-term effectiveness of the bioremediation material. Figure 5 and Figure 6 The magnified image shows a portion of the petroleum hydrocarbon-degrading bacteria immobilized on biochar embedded in iron alginate. This indicates that the biochar and petroleum hydrocarbon-degrading bacteria can break through the three-dimensional network structure of the hydrogel beads and be released during the expansion of the iron alginate microspheres, thus achieving a slow-release effect.

[0057] Example 3: Bioremediation experiment on petroleum hydrocarbon-contaminated soil.

[0058] Preparation of test contaminated soil: The crude oil contaminated soil from Liaohe Oilfield was thoroughly mixed with clean soil to prepare test contaminated soil with a total petroleum hydrocarbon concentration of 19.88 g / kg. The soil was sealed and stored for later use.

[0059] Remediation steps: To determine the remediation effects of different materials on petroleum hydrocarbons in soil, five treatment groups were set up, with each treatment group repeated three times. The remediation experiment was carried out for 30 days in a constant temperature incubator at 30℃.

[0060] (1) Control group (CK): Soil without any added petroleum hydrocarbons; (2) Free bacteria (B): Add free bacteria to petroleum hydrocarbon contaminated soil at a rate of 10% v / w of the soil.

[0061] (3) Biochar-immobilized petroleum hydrocarbon degrading bacteria (BC-B): Petroleum hydrocarbon contaminated soil with added biochar-immobilized petroleum hydrocarbon degrading bacteria at a concentration of 5% w / w of the soil.

[0062] (4) Iron alginate-encapsulated petroleum hydrocarbon degrading bacteria (Fe-SA-B): The amount of petroleum hydrocarbon contaminated soil with iron alginate-encapsulated petroleum hydrocarbon degrading bacteria added is 5% w / w of the soil.

[0063] (5) Iron alginate-encapsulated biochar immobilized petroleum hydrocarbon degrading bacteria (Fe-SA-BC-B): The amount of petroleum hydrocarbon-contaminated soil with iron alginate-encapsulated biochar immobilized petroleum hydrocarbon degrading bacteria added is 5% w / w of the soil.

[0064] During the remediation experiment, the relative humidity of the five treatment groups was maintained at around 60%. Soil samples were taken at 6, 12, 18, 24, and 30 days to determine the residual petroleum hydrocarbon content and to measure the soil pH.

[0065] Figure 7 As shown in the figure, after 30 days of remediation with exogenous petroleum hydrocarbon-degrading bacteria, the removal rate of petroleum hydrocarbons was 29.57%. The removal rate of petroleum hydrocarbons in the biochar-immobilized petroleum hydrocarbon-degrading bacteria treatment group was further increased, mainly due to the synergistic adsorption and biodegradation of biochar and microorganisms. Simultaneously, due to the interaction between the biochar carrier and the microorganisms, the metabolites and related enzymes secreted by the petroleum hydrocarbon-degrading bacteria can be retained in the carrier structure, further participating in the degradation process of petroleum hydrocarbons. Both iron alginate-encapsulated microbial pellets and iron alginate-encapsulated biochar-immobilized microbial pellets showed high petroleum hydrocarbon removal effects, at 52.16% and 65.99%, respectively, indicating that encapsulation treatment can effectively alleviate the stress of petroleum hydrocarbons on microorganisms. In the initial stage of soil remediation (0-12 days), the removal rate of petroleum hydrocarbons was low, but it significantly increased during the 15-30 day remediation process. This is because during the expansion of the alginate iron pellets, biochar and microorganisms can break through the hydrogel mesh and be released. This slow-release effect fully ensures that petroleum hydrocarbons continuously reach the active target sites of biochar and microorganisms.

[0066] Changes in soil pH in different treatment groups, such as Figure 8 As shown. During the 30-day remediation period, the soil pH of the group treated with iron alginate-embedded biochar immobilized microbial pellets remained within a narrow range of 6.70-6.88, with the smallest variation (<0.2 units). In contrast, the pH of the free bacteria group continuously decreased to 5.98 due to acid production, and the pH of the biochar immobilized microbial pellets also showed a significant decrease in the later stage. This result directly confirms that the soil pH of the group treated with iron alginate (Fe...) remained within a narrow range of 6.70-6.88, with the smallest variation (<0.2 units). 3+ The unique buffering system formed by the biochar surface hydroxyl groups has a significant and unexpected buffering effect on the acidification inhibition during the degradation of petroleum hydrocarbons.

[0067] Example 4: Enzyme activities in soil from different treatment groups 30 days after remediation were determined: The activities of dehydrogenase, FDA hydrolase, and polyphenol oxidase in the soil were measured using a UV-Vis spectrophotometer. Dehydrogenase activity was detected using the triphenyltetrazolium chloride (TTC) colorimetric method at a wavelength of 485 nm. FDA hydrolase activity was measured using spectrophotometry at 490 nm using fluorescein diacetate (FDA) as a substrate. Simultaneously, polyphenol oxidase activity was measured using the pyrogallol colorimetric method at a wavelength of 430 nm. (In the figure, 1 represents the control group, 2 represents free bacteria, 3 represents biochar-immobilized petroleum hydrocarbon-degrading bacteria, 4 represents petroleum hydrocarbon-degrading bacteria encapsulated in iron alginate, and 5 represents petroleum hydrocarbon-degrading bacteria encapsulated in iron alginate and immobilized in biochar).

[0068] Changes in dehydrogenase, FDA hydrolase, and polyphenol oxidase in soil from different treatment groups are as follows: Figure 9 , Figure 10 and Figure 11 As shown. After 30 days of remediation, the activities of dehydrogenase, FDA hydrolase, and polyphenol oxidase in the soil of the group treated with iron alginate-embedded biochar immobilized microbial pellets were significantly higher than those in all other treatment groups, at 0.082 mg / g / d, 1220.39 μmol / g / d, and 0.216 mg / g / d, respectively. Particularly noteworthy is that its dehydrogenase activity was not only 2.6 times that of the control group, but also 1.3 times and 1.1 times that of the biochar immobilized petroleum hydrocarbon-degrading bacteria and the iron alginate-embedded petroleum hydrocarbon-degrading bacteria treatment groups, respectively. Figure 9 This data strongly supports and proves that iron alginate, biochar, and immobilized microorganisms are all indispensable. Their synergistic effect creates an excellent soil microecology, thereby achieving an unexpected increase in soil enzyme activity, an effect that cannot be achieved by simply adding up the components.

[0069] Example 5: Soil samples from the control group, free bacteria, biochar-immobilized petroleum hydrocarbon-degrading bacteria, iron-alginate-embedded petroleum hydrocarbon-degrading bacteria, and iron-alginate-embedded biochar-immobilized petroleum hydrocarbon-degrading bacteria treatment groups were named A1, A2, A3, A4, and A5, respectively. A clean soil sample was designated A6. High-throughput sequencing was performed on the different samples to analyze their microbial diversity.

[0070] Table 1 Comparison of microbial Alpha diversity indices from different soil samples Table 1 compares the microbial alpha diversity indices of different soil samples. The Chao1 and Ace indices show no clear pattern, indicating that organic pollutants and bioremediation materials have varying degrees of impact on microbial diversity. A6 had the highest Shannon index and the lowest Simpson index, while A5 had the lowest Shannon index and the highest Simpson index. This means that the clean soil sample A6 had the highest microbial community diversity, while the A5 soil sample treated with petroleum hydrocarbon-degrading bacteria immobilized in ferric alginate-encapsulated biochar had the lowest microbial community diversity. These results suggest that during the bioremediation of petroleum hydrocarbons, the slow-release effect of the ferric alginate hydrogel immobilization carrier on petroleum hydrocarbon-degrading bacteria enhances the metabolic activity of this bacterial community in the later stages of remediation, making it the dominant bacteria in the soil sample. Other free-floating bacterial communities are reduced and disappear under extreme environmental factors and pollutant stress.

[0071] Figure 12 Hierarchical clustering dendrograms of different soil samples at the bacterial genus level were generated. Under unweighted unifrac distance, the bacterial communities of different soil samples were divided into three major groups: A1, A2, and A3 clustered together; A4 and A5 clustered together; and A6 was a separate group. This result is consistent with the bioremediation materials added to the soil. Samples A1, A2, and A3 showed higher relative abundances of *Bacillus* and *Gordonella*, while samples A4 and A5 showed higher relative abundances of *Bacillus*, *Pseudomonas*, and *Bacillus-like* species. These bacterial communities are consistent with the composition of the exogenously introduced immobilized petroleum hydrocarbon-degrading bacteria. Furthermore, the remediation effect of soil sample A5 was higher than that of A4, which was positively correlated with the relative abundance of *Pseudomonas* and *Bacillus-like* species. These results indicate that the iron alginate-embedded biochar immobilized petroleum hydrocarbon-degrading bacteria composite material can slowly release the petroleum hydrocarbon-degrading bacteria and make them the dominant bacterial community in the soil during the later stages of remediation.

[0072] Example 6: Application of Ferrous Alginate-Encased Biochar Immobilized Petroleum Hydrocarbon Degrading Bacteria in Remediating Petroleum Hydrocarbon-Contaminated Soil and Promoting Plant Growth: A rapeseed pot experiment was conducted with three treatment groups: 1) Rapeseed cultivated in clean soil (T1), 2) Rapeseed cultivated in petroleum hydrocarbon-contaminated soil (T2), and 3) Rapeseed cultivated in petroleum hydrocarbon-contaminated soil treated with ferrous alginate-encased biochar immobilized petroleum hydrocarbon-degrading bacteria (T3). The soil petroleum hydrocarbon concentration was 11.44 g / kg, and the amount of ferrous alginate-encased biochar immobilized petroleum hydrocarbon-degrading bacteria composite material added was 5% (w / w) of the soil mass. Ten rapeseed seedlings were transplanted into each pot and managed routinely for 30 days. During the experiment, water management was kept consistent across all treatment groups. After the experiment, five rapeseed plants from each treatment were selected for subsequent physiological and biochemical index measurements, and the removal rate of petroleum hydrocarbons in the soil was determined. The results are shown in Table 2.

[0073] Table 2. Petroleum hydrocarbon degradation rate and rapeseed physiological and biochemical indicators in soil under different treatment groups. As shown in Table 2, rapeseed growth was severely inhibited in contaminated soil (T2) without any added materials. Unexpectedly, after adding the material of this invention (T3), not only did the petroleum hydrocarbon degradation rate reach 85.15%, but the rapeseed biomass (0.3603 g) even exceeded that of the control group grown in clean soil (0.2353 g). This result far exceeds the reasonable expectations of those skilled in the art, conclusively demonstrating the "synergistic promotion of plant growth" effect described in claim 5. Analysis suggests that in the material of this invention, biochar acts as a long-acting carbon-based fertilizer, iron alginate slowly releases plant-available iron nutrients, and the biostimulants produced by functional bacteria work together to form a triple synergistic plant growth-promoting model of "repair-nutrient-stimulation," which is unattainable by single-function remediation materials in the prior art.

[0074] In summary, this invention provides a novel solution specifically for the remediation of petroleum hydrocarbon-contaminated soil. Its core innovation lies in the construction of an intelligent remediation system with pH self-buffering, long-term microbial slow-release, and multifunctional synergy through a specific structural composite of petroleum hydrocarbon-degrading bacteria, iron alginate, and biochar. This system not only efficiently degrades pollutants but also actively resists soil acidification during the remediation process, simultaneously activating soil enzyme activity and significantly promoting plant growth. It achieves a leap from simple purification to integrated ecological restoration encompassing degradation, improvement, and plant growth promotion, overcoming the key shortcomings of existing technologies that are limited in function and unable to handle complex bioremediation scenarios. This results in unexpected multiple synergistic effects and outstanding comprehensive ecological benefits.

[0075] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, if these changes fall within the scope of the claims of the present invention and their equivalents, they shall still fall within the protection scope of the present invention.

Claims

1. A petroleum hydrocarbon-degrading bacteria immobilized composite material with pH self-buffering and long-lasting sustained-release functions, characterized in that, The product contains ferric alginate gel and biochar loaded with petroleum hydrocarbon-degrading bacteria, wherein the ferric alginate gel encapsulates the biochar loaded with petroleum hydrocarbon-degrading bacteria, and has a three-dimensional network porous structure.

2. The petroleum hydrocarbon-degrading bacteria immobilized composite material according to claim 1, characterized in that, The following steps are included: (1) mixing biochar loaded with petroleum hydrocarbon degrading bacteria and sodium alginate in water to form a mixed slurry; (2) adding the mixed slurry to an iron salt solution for cross-linking reaction to obtain a petroleum hydrocarbon degrading bacteria immobilized composite material with pH self-buffering and long-term slow-release function.

3. The method for preparing the petroleum hydrocarbon degrading bacteria immobilized composite material according to claim 1, characterized in that, step... include: (1) The biochar loaded with petroleum hydrocarbon degrading bacteria and sodium alginate were thoroughly mixed in water to form a mixed slurry; (2) The mixed slurry was added dropwise to the iron salt solution to cross-link the reaction and obtain a petroleum hydrocarbon degrading bacteria immobilized composite material with pH self-buffering and long-term slow-release function.

4. The preparation method according to claim 3, characterized in that, In step (1), the mass ratio of the biochar loaded with petroleum hydrocarbon degrading bacteria to sodium alginate is 1:1-5, and the concentration of sodium alginate is 0.01-0.1 g / mL; In step (2), the molar ratio of sodium alginate in the mixed slurry to iron salt in the iron salt solution is 1-5:1, and the concentration of the iron salt solution is 0.005-0.05 g / mL.

5. The preparation method according to claim 3, characterized in that, In step (1), the preparation of biochar loaded with petroleum hydrocarbon degrading bacteria includes: mixing and adsorbing biochar and petroleum hydrocarbon degrading bacteria in a liquid culture medium to obtain biochar loaded with petroleum hydrocarbon degrading bacteria.

6. The preparation method according to claim 5, characterized in that, The preparation of the biochar includes: heating the raw material to 500±50℃ under an inert atmosphere or a nitrogen atmosphere to carbonize it, thereby obtaining biochar.

7. The petroleum hydrocarbon degrading bacteria immobilized composite material according to claim 1 or 2 is used for remediating petroleum hydrocarbon pollution and / or promoting plant growth, or for preparing products for remediating petroleum hydrocarbon pollution and / or promoting plant growth.

8. A product for the remediation of petroleum hydrocarbon pollution and / or the promotion of plant growth, characterized in that, The composite material containing petroleum hydrocarbon degrading bacteria as described in claim 1 or 2.

9. A product for remediating petroleum hydrocarbon pollution and / or promoting plant growth according to claim 8, characterized in that, The product is selected from any one of additives, fertilizers, cultivation substrates, and soil conditioners.

10. A method for remediating petroleum hydrocarbon pollution and / or promoting plant growth, characterized in that the steps include... include: Add the petroleum hydrocarbon degrading bacteria immobilized composite material as described in claim 1 or 2, or the petroleum hydrocarbon pollution remediation and / or plant growth promotion product as described in claim 8 or 9, to the target material.

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