Method for enhancing bioremediation of organic groundwater contamination using microorganisms

Abstract

The application belongs to the field of environmental protection, and particularly relates to a method for strengthening microbial remediation of groundwater organic pollution, which adopts an anaerobic degradation organic pollutant strain capable of adapting to the groundwater environment, and the name of the strain is Klebsiella oxytoca K0106, and the preservation number is CCTCC NO:M 20221835; the strain can perform anaerobic biodegradation on the organic pollutant by taking nitrate as an electron acceptor, and organic pollutants and surfactants are used as common carbon sources to provide energy required for microbial growth; the type and optimal dosage of the surfactants can be determined based on the solubilization, toxicity and carbon source competition of the surfactants on the organic pollutants. In the special groundwater environment, the application degrades the organic pollutants in the groundwater by using the microbial remediation technology which has the advantages of environmental friendliness, no secondary pollution, cost-effectiveness and the like, and the organic pollutants are particularly polycyclic aromatic hydrocarbons.

Description

Technical Field

[0001] This invention relates to the field of microbial remediation of contaminated groundwater, and more particularly to a method for enhancing microbial remediation of organic pollution in groundwater. Background Technology

[0002] Groundwater is a crucial component of water resources and a major source of water for industry, agriculture, and urban use. However, the development and utilization of energy sources such as oil and coal have led to severe petroleum hydrocarbon pollution in groundwater. Polycyclic aromatic hydrocarbons (PAHs), persistent petroleum hydrocarbon pollutants that are difficult to degrade, are widely distributed in groundwater. Naphthalene, a typical PAH, is widely detected in groundwater in areas with petroleum and related chemical industries. Long-term intake of naphthalene can cause hemolytic anemia and liver and kidney damage. Therefore, developing a long-term effective remediation technology for PAH-contaminated groundwater is essential. Compared to chemical or physical remediation technologies, microbial remediation is more economical, efficient, produces no secondary pollution, and is environmentally friendly, making it the most commonly used and promising remediation technology for PAH-contaminated groundwater. However, due to the strong lipophilic and hydrophobic properties of PAHs, they are easily adsorbed into aquifer media, and groundwater is often in hypoxic or even anaerobic conditions, resulting in weak PAH migration and low biodegradation efficiency. Therefore, improving the mobility and biodegradation efficiency of PAHs is of significant applied research value for developing bioremediation technologies for PAH-contaminated groundwater.

[0003] CN112481149A discloses a bacterial strain that degrades high-cyclic PAHs, belonging to the genus *Microbacterium*. This strain is Gram-positive, with yellow, round, raised colonies that are smooth and moist with regular edges. The application of this high-cyclic PAH-degrading strain in synergy with a surfactant in the remediation of PAH-contaminated soil or groundwater is also disclosed.

[0004] CN101851582B discloses a microbial agent for removing and / or degrading PAHs and its application. This invention provides a microbial agent whose active ingredients are Rhodococcus sp. LU3 CGMCC No. 3610, Microbacterium sp. LU1 CGMCC No. 3581, Ochrobacterium sp. LU2 CGMCC No. 3582, and Bordetellasp. LU4 CGMCC No. 3611. Experiments with this invention show that after 4 weeks, the removal rate of 16 PAHs in soil with this microbial agent added increased by an average of 30% compared to soil without the agent. Specifically, the removal rate of 6 low-ring (two / three-ring) PAHs increased by an average of 26%, and the removal rate of 10 medium-high ring (four-ring and above) PAHs increased by an average of 32%.

[0005] Microbial remediation technology has become a research hotspot in the field of organic polluted groundwater remediation in recent years due to its ability to meet the requirements of in-situ or ex-situ remediation, and its advantages such as being more economical, efficient, free of secondary pollution, and environmentally friendly. However, the special environmental conditions of groundwater limit the application and promotion of microbial remediation technology: First, the background value of dissolved oxygen in groundwater is low, which cannot meet the oxygen demand of microorganisms, resulting in a low microbial proliferation rate and thus affecting the degradation efficiency; Second, the strong hydrophobicity of PAHs severely restricts their migration in groundwater, thereby reducing the bioavailability of PAHs and resulting in poor degradation effect.

[0006] Furthermore, due to differences in understanding among those skilled in the art, and because the applicant researched a large number of documents and patents when developing this invention, but space limitations prevented a detailed listing of all details and contents, this does not mean that the present invention lacks the features of these prior art techniques. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background section. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for enhancing microbial remediation of organic pollutants in groundwater, wherein the organic pollutants can be PAHs, especially naphthalene. This invention first utilizes nitrates, which are commonly found in groundwater, as electron acceptors for microbial degradation of organic pollutants to improve the degradation efficiency of microorganisms in an anaerobic environment; secondly, it introduces surfactants to enhance the solubility of organic pollutants in groundwater, thereby improving the migration capacity of the organic pollutants and enabling them to be better utilized by microorganisms.

[0008] PAHs pollution in groundwater is prevalent worldwide. Compared with traditional in-situ groundwater remediation technologies, this invention employs a microbial remediation technology that offers advantages such as greater cost-effectiveness, no secondary pollution, and environmental friendliness in degrading PAHs in groundwater. Among these technologies, naphthalene, a common PAH in groundwater pollution, can be targeted as the pollutant.

[0009] Due to the unique anoxic and anaerobic environment of groundwater and the strongly hydrophobic nature of pollutants, the microbial degradation of organic pollutants is limited. Therefore, to improve the efficiency of microbial degradation, this invention utilizes nitrates, which are commonly found in groundwater, to provide electron acceptors and nitrogen sources for the anaerobic degradation of organic pollutants by microorganisms, thereby maximizing the degradation efficiency of organic pollutants under anaerobic conditions. In addition, to address the problem of pollutants in groundwater being difficult to migrate and easily adsorbed by media, surfactants are introduced to solubilize organic pollutants and enhance the microbial remediation effect.

[0010] This invention discloses a bacterial strain capable of anaerobic degradation of organic pollutants that can adapt to groundwater environments. Its name is Klebsiella oxytoca K0106, and its accession number is CCTCC NO:M20221835. It was deposited at the China Center for Type Culture Collection on November 29, 2022.

[0011] According to a preferred embodiment, the nucleotide sequence of the 16S rDNA of the strain is shown in SEQ ID NO.1.

[0012] Furthermore, the amplification primer sequences are as follows:

[0013] 27F: 5'-GAGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 2)

[0014] 1492R: 5'-TACGGCTACCTTGTTACGAC-3' (SEQ ID NO. 3).

[0015] Furthermore, the sequencing results are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively. The sequencing results were assembled using ContigExpress, and inaccurate parts at both ends were removed to obtain the assembly result shown in SEQ ID NO.1.

[0016] According to a preferred embodiment, the biological characteristics of the strain are as follows: the isolated and purified strain is milky white, with a smooth and rounded surface, Gram-negative, light red, short rod-shaped, with a length of 1.01 μm to 1.86 μm and a width of 0.5 nm.

[0017] The present invention also discloses a method for enhancing microbial remediation of organic pollution in groundwater. The method uses the aforementioned bacterial strains to remediate organic pollutants in groundwater, wherein the bacterial strains are capable of anaerobic degradation of organic pollutants, including at least naphthalene.

[0018] According to a preferred embodiment, the strain is capable of anaerobic biodegradation of organic pollutants using nitrate as an electron acceptor, wherein the optimal concentration of nitrate is at least 300 mg / L.

[0019] According to a preferred embodiment, the strain can improve the bioavailability and biodegradation efficiency of highly hydrophobic organic pollutants with the help of surfactants. Tea saponin is selected as a surfactant suitable for the strain based on the solubilizing effect, toxicity and carbon source competition of the surfactant. The optimal dosage of tea saponin is twice the critical micelle concentration (CMC).

[0020] Surfactants, when dispersed in solution as monomers at low concentrations, exhibit amphiphilic properties, allowing them to arrange themselves in an orderly manner at the gas-aqueous or organic-aqueous interface, becoming intermediates at the contact interface and reducing the surface tension and interfacial tension of the solution. As the surfactant concentration increases, the monomers in the solution attract each other and aggregate to form micelles. The critical concentration value for micelle formation is called the critical micelle concentration (CMC).

[0021] According to a preferred embodiment, the strain is domesticated, cultured, and enriched using the following steps:

[0022] An organic pollutant solution was added to the acclimation medium containing activated sludge. After removing the solvent and dissolved oxygen, the medium was shaken and cultured under anaerobic conditions to complete the microbial proliferation.

[0023] Then, the turbid liquid was added back into a new culture medium, and the same culture conditions were maintained. The initial concentration of organic pollutants was continuously increased and controlled, so that the microorganisms were domesticated for 4 generations under the extreme conditions of continuously increasing organic pollutant concentration.

[0024] Take a sample from the fourth generation culture medium and spread it onto a solid culture medium containing organic contaminants.

[0025] According to a preferred embodiment, the method includes at least the following steps:

[0026] (1) Adjusting the pH value of organically polluted groundwater

[0027] Adjust the pH of organically polluted groundwater to the range of 6.8–7.0;

[0028] (2) Controlling the temperature of organically polluted groundwater

[0029] Remediation work should be carried out when the temperature of organically contaminated groundwater is higher, at least above 15°C.

[0030] (3) Adjusting the inoculum concentration

[0031] The inoculum concentration (volume percentage) of the strain is adjusted to at least 5%;

[0032] (4) Adjusting nitrate concentration

[0033] Adjust the nitrate dosage to ensure that the nitrate concentration in the groundwater is at least 300 mg / L.

[0034] According to a preferred embodiment, the type and dosage of surfactant are determined based on a comprehensive analysis of the strain type, nitrate dosage, type of organic pollutant, and initial concentration. The determined surfactant is at least capable of enhancing the solubilization of organic pollutants without producing toxicity, serving as a common carbon source with the organic pollutants to provide energy for microbial growth.

[0035] According to a preferred embodiment, when the surfactant is tea saponin, the concentration of tea saponin is controlled to be higher than the CMC to enhance the solubilizing effect on naphthalene, so that naphthalene can have a carbon source competitive advantage by increasing its apparent solubility. When the concentration of tea saponin is increased to 2 CMC, the system simultaneously exhibits the solubilizing effect of tea saponin on naphthalene and the carbon source competitive effect, so that tea saponin can promote the biodegradation of naphthalene without negatively impacting the growth and reproduction of microorganisms.

[0036] Biological Preservation Instructions

[0037] The Klebsiella oxytoca K0106 provided by this invention was deposited at the China Center for Type Culture Collection on November 29, 2022, with accession number CCTCCNO:M 20221835, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China. Attached Figure Description

[0038] Figure 1 (a) dilution and plating of naphthalene-degrading strains; (b) Gram staining;

[0039] Figure 2 This is an electron micrograph of naphthalene-degrading bacteria;

[0040] Figure 3 This is the growth curve of naphthalene-degrading bacteria;

[0041] Figure 4 This is a 16S rDNA electrophoresis gel image;

[0042] Figure 5It is a microbial phylogenetic tree;

[0043] Figure 6 (a) Degradation curves of naphthalene under different pH conditions; (b) Effect of pH on degradation rate and average degradation rate; (c) Changes in pH before and after the reaction;

[0044] Figure 7 (a) Degradation curves of naphthalene under different temperature conditions; (b) Effect of temperature on degradation rate and average degradation rate;

[0045] Figure 8 (a) Curves of microbial degradation of naphthalene at different inoculum concentrations; (b) Effect of inoculum concentration on degradation rate and average degradation rate;

[0046] Figure 9 (a) Naphthalene degradation curves at different initial naphthalene concentrations; (b) the effect of initial naphthalene concentration on degradation rate and average degradation rate;

[0047] Figure 10 (a) Naphthalene degradation curves under different nitrate addition amounts; (b) The effect of nitrate addition amount on degradation rate and average degradation rate;

[0048] Figure 11 These are (a) commonly used logarithmic change curves of tea saponin concentration in solution; (b) commonly used logarithmic change curves of Tween 80 concentration in solution; and (c) commonly used logarithmic change curves of TX-100 concentration in solution.

[0049] Figure 12 The solubilizing effect of three surfactants, tea saponin, Tween 80, and TX-100, on naphthalene is as follows:

[0050] Figure 13 The growth curves of microorganisms when three surfactants, tea saponin (a), Tween 80 (b), and TX-100 (c), are used as a single carbon source are shown.

[0051] Figure 14 The growth curves of microorganisms are shown when three surfactants, tea saponin (a), Tween 80 (b), and TX-100 (c), and naphthalene are used as a common carbon source.

[0052] Figure 15 The effects of different concentrations of three surfactants on the microbial degradation of naphthalene are shown in the figures: (a) tea saponin; (b) Tween 80; and (c) TX-100. Detailed Implementation

[0053] The following is a detailed explanation with reference to the accompanying drawings.

[0054] This invention discloses a method for enhancing microbial remediation of organic pollutants in groundwater. The organic pollutants specifically refer to PAHs, a class of toxic organic pollutants mainly produced by the incomplete combustion of coal, tar, petroleum and natural gas, vehicle exhaust, tobacco, or smoked foods. These pollutants are widely distributed in the air, surface water, soil-groundwater system, and sediments, and have toxic, carcinogenic, and mutagenic effects on humans. Naphthalene, composed of two benzene rings fused together with two adjacent carbon atoms, is the simplest and most common type of PAH. Preferably, this embodiment uses the microbial remediation of naphthalene-contaminated groundwater as an example.

[0055] Microbial remediation and degradation of PAHs in soil or groundwater typically utilizes naturally occurring microorganisms in the environment or employs highly efficient and targeted degrading bacteria obtained through artificial domestication and screening of contaminated soil, groundwater, and activated sludge. These bacteria are then introduced into the soil or groundwater, where they degrade the pollutants into harmless substances through microbial action. The degradation effect of different bacterial species varies depending on the species.

[0056] According to a preferred embodiment, the present invention provides a microorganism capable of anaerobic degradation of organic pollutants that can adapt to the groundwater environment. It can be obtained by using activated sludge from the secondary sedimentation tank of a sewage treatment plant as the source of bacteria, and then enriching, separating and purifying it.

[0057] For example, the strain can be obtained in the laboratory using the following methods:

[0058] S1. Preparation of culture medium:

[0059] Acclimation medium (MSM): NaNO3 (2.0 g / L), Na2HPO4 (0.4228 g / L), KH2PO4 (0.41 g / L), MgCl2·6H2O (0.1 g / L), CaCl2 (0.083 g / L), NaCl (0.3 g / L), with 1 mL of trace elements added. Trace elements: FeCl₂·4H₂O (4.5 mg / L), H₃BO₃ (0.15 mg / L), ZnCl₂ (0.15 mg / L), CuCl₂·2H₂O (0.038 mg / L), MnCl₂·4H₂O (0.1138 mg / L), CoCl₂·6H₂O (0.05 mg / L), Na₂SeO₃ (0.026 mg / L), NiCl₂·6H₂O (0.092 mg / L), NaWO₄·2H₂O (0.033 mg / L), Biotin (0.004 mg / L), Nicotinic acid (0.04 mg / L). After preparation, adjust the medium to neutral using 1 mol / L HCl and 1 mol / L NaOH.

[0060] Isolation medium: Use the same nutrient substrate as the acclimatization medium and add 1.5% agar to make a solid medium.

[0061] Expanded culture medium: glucose (8.5 g / L), urea (3 g / L), K2HPO4·3H2O (3.28 g / L), MgSO4·7H2O (2.05 g / L), NaCl (4 g / L), trace elements (3 ml / L).

[0062] Furthermore, all culture media used in the experiment must be sterilized before use. The autoclave temperature was set to 121°C and the time was 30 minutes. The culture media removed from the autoclave must be cooled under natural conditions before use.

[0063] S2. Domestication, culture, and enrichment of microbial strains:

[0064] Add 2.5g of sludge to a conical flask containing 200mL of acclimatization culture medium. Add a mixed solution of 50mg / L naphthalene and acetone, shake well, and agitate for 30min to remove the acetone. Purge the flask with argon gas for 10min to remove dissolved oxygen. Place the flask on a constant temperature water bath shaker at 30℃ and 160r / min for shaking culture. After 5 days of culture, allow the sludge to settle. When the flask is visibly turbid and the liquid feels suspended, it indicates microbial proliferation in the culture medium. Then, use a sterile dropper to take 10mL of the turbid liquid and add it back to a new culture medium, maintaining the same culture conditions. Continuously increase the initial concentration of naphthalene, controlling it within the range of 50mg / L to 200mg / L, allowing the microorganisms to be acclimatized for 4 generations under the extreme conditions of continuously increasing naphthalene concentration. Take 1mL of the sample from the 4th generation culture medium and spread it onto a solid culture medium containing 100mg / L naphthalene to observe whether colonies grow.

[0065] S3. Isolation and purification of bacterial strains:

[0066] In an anaerobic incubator, take 7 sterilized centrifuge tubes, numbered 1 to 7, and add 9 mL of sterile water to each. Add 1 mL of the fourth-generation acclimated bacterial culture to centrifuge tube 1 and mix well. Then, take 1 mL of the liquid from centrifuge tube 1 and add it to centrifuge tube 2, and so on, diluting stepwise until a final solution of 10⁻⁶ is obtained. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7Seven bacterial suspensions at different concentration gradients were prepared. 50 μL of each of these seven concentration gradients was transferred to selective medium and inoculated using a sterile triangular inoculating loop. This inoculation was repeated three times. After inoculation, the selective medium was incubated in an anaerobic incubator at 30°C in the dark for 3–4 days. After the incubation period, single colonies were selected using a sterile inoculating loop and placed into selective medium for streak incubation for 3–4 days. Finally, a small number of strains were transferred to clean glass slides for morphological analysis under a microscope. If morphological characteristics were inconsistent, single colonies were continued to be streak-cultured until strains with consistent morphological characteristics were obtained.

[0067] Pick a single strain obtained from the above streaking with an inoculation loop and place it in an expansion medium. Incubate in an anaerobic constant temperature incubator at 30℃ in the dark for 3 days. Then, take out the medium and centrifuge at 7000 rpm for 10 min. Discard the supernatant and keep the bottom bacterial cells. Wash the cells three times with 0.85% NaCl solution and store them with 70% glycerol at -20℃.

[0068] Depend on Figure 1 (a) It can be seen that the isolated and purified strain is milky white and has a smooth, round surface; Figure 1 (b) It can be seen that bacteria that turn pale red after staining in the Gram staining test are Gram-negative bacteria. Figure 2 The image shows the results of further observation of the bacterial morphology of the purified colonies using scanning electron microscopy. The isolated and purified strains appear as short rod-shaped bacteria with a length of 1.01 μm to 1.86 μm and a width of approximately 0.5 nm.

[0069] Furthermore, the obtained bacterial strain can be subjected to property testing and biochemical identification to obtain its growth curve and determine its microbial genus. For example, the property testing and biochemical identification of the bacterial strain can be performed in the laboratory using the following methods:

[0070] S4. Plotting the growth curve of the microbial strain:

[0071] Prepare 14 sterilized test tubes, numbered 0 to 13. Add 9 ml of MSM medium with 50 mg / L naphthalene as the sole carbon and energy source to each tube, then add OD. 600 1 mL of bacterial suspension (0.1 g / mL) was used to seal test tubes 1-13 and incubate them on a biological shaker at 160 rpm and 30°C. Test tube 0 was stored at 4°C as a blank control. Every 12 hours, test tubes 1-13 were sequentially removed and their growth was measured using a UV-Vis spectrophotometer at λ=600 nm to determine the microbial growth. The results are shown below. Figure 3 As shown.

[0072] Depend on Figure 3It can be seen that the strain isolated and purified by this invention grows slowly in the first 48 hours, then shows logarithmic growth after 48 hours, reaches a stable growth rate after 96 hours, and begins to decline after 108 hours due to carbon source depletion.

[0073] S5. Biochemical identification of the strain:

[0074] (1) PCR amplification: PCR amplification was performed using the following primers:

[0075] 27F: 5'-GAGAGTTTGATCCTGGCTCAG-3'

[0076] 1492R: 5'-TACGGCTACCTTGTTACGAC-3'.

[0077] (2) Electrophoresis detection: The amplified PCR products were subjected to agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue) at 300V for 12 min to obtain the identification gel image, such as... Figure 4 As shown.

[0078] The prepared PCR products were subjected to first-generation sequencing (sequencing primers 27F / 1492R).

[0079] (3) Phylogenetic Tree: Sequencing results were assembled using Contig Express, and inaccurate portions at both ends were removed. The assembled sequences were compared and analyzed in the NCBI database. The 16S rDNA sequences of the bacteria isolated and purified in this invention showed high homology with 14 species of the genus Klebsiella, with the highest homology (100%) with Klebsiella oxytoca. Figure 5 As shown.

[0080] Furthermore, the Klebsiella oxytoca K0106 provided by this invention was deposited on November 29, 2022, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20221835, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China.

[0081] According to a preferred embodiment, the present invention also discloses an application of the above-mentioned microbial strain, namely, applying the obtained microorganisms to remediate organic pollution in groundwater, particularly suitable for remediating PAHs (e.g., naphthalene) in groundwater. In other words, the present invention also discloses a method for microbial remediation of organic pollution in groundwater.

[0082] Furthermore, the degradation efficiency of PAHs by microorganisms is not only affected by the type of strain, but also closely related to factors such as the number of microorganisms, ambient temperature, pH, oxygen content, and pollutant concentration. When the number of microorganisms is low, they cannot fully utilize carbon and energy sources, leading to slow growth. An excessive number of microorganisms will cause internal competition, thus adversely affecting the degradation rate. Temperature is closely related to the enzyme activity of microorganisms, directly affecting the metabolism of PAHs by microorganisms, thereby changing the efficiency of microbial degradation of naphthalene. In extreme environments, excessively low pH affects the binding and catalysis of enzyme molecules and substrate molecules, while excessively high pH affects the ability of microorganisms to absorb nutrients, leading to slower growth and affecting the degradation effect. The concentration of pollutants affects the activity and number of microorganisms. Too low a concentration cannot meet the uptake of microorganisms, while too high a concentration will produce toxic effects and affect the rate of microbial degradation of naphthalene.

[0083] Under normal conditions, microbial degradation of PAHs occurs aerobically through monooxygenase or dioxygenase-mediated oxygenase metabolism. In the aerobic degradation of naphthalene, O2 acts as the terminal electron acceptor in the oxidation reaction, achieving ring-opening of naphthalene via carboxylation. However, with the increasingly widespread distribution of PAHs in the environment, they have been found in some submerged soils, aquatic sediments, and groundwater aquifers. These environments are generally characterized by low or anaerobic conditions. Therefore, developing efficient microbial remediation technologies under anaerobic conditions is one of the urgent challenges to be addressed in the remediation of PAH-contaminated groundwater.

[0084] Furthermore, the degradation of PAHs under anoxic or anaerobic conditions can utilize nitrates, sulfates, iron, manganese reduction, and methanogenesis as terminal electron acceptors. Among these, nitrates, as electron acceptors, have the second-highest redox potential after oxygen, and are readily available and highly soluble. Preferably, nitrate pollution has been detected in industrial wastewater, sewage, and groundwater in recent years; therefore, utilizing environmentally available nitrates as electron acceptors for naphthalene degradation can compensate for the deficiencies of anoxic or anaerobic conditions in groundwater.

[0085] Preferably, different factors have different effects on the efficiency of microbial degradation of PAHs (e.g., naphthalene). These influencing factors may include pH, temperature, inoculum concentration, initial naphthalene concentration, and nitrate dosage. Single-factor experiments can yield results such as… Figures 6 to 10 The results are shown. The method for remediating organic pollution in groundwater according to the present invention can construct a corresponding factor influence system based on the above experimental results, and select appropriate remediation conditions according to the initial concentration of organic pollutants (e.g., initial concentration of naphthalene) and environmental factors at the remediation site.

[0086] Furthermore, the method of the present invention can set the following data as initial parameters when selecting suitable repair conditions:

[0087] (1) Adjusting the pH value of organically polluted groundwater

[0088] Adjust the pH of organically polluted groundwater to the range of 6.8–7.0;

[0089] (2) Controlling the temperature of organically polluted groundwater

[0090] Since groundwater temperature is difficult to regulate, remediation work can be carried out when the temperature of organically contaminated groundwater is higher, at least above 15°C.

[0091] (3) Adjusting the inoculum concentration

[0092] The inoculum concentration of the strain is adjusted to at least 5%;

[0093] (4) Adjusting the amount of nitrate added

[0094] Adjust the nitrate dosage to at least 300 mg / L.

[0095] According to a preferred embodiment, some organic pollutants (e.g., naphthalene) are highly hydrophobic, resulting in poor mass transfer rates and limited bioavailability, thus affecting biodegradation efficiency. Surfactants can improve bioavailability during microbial degradation and can also serve as a carbon source for microbial growth along with organic pollutants, ensuring degradation efficiency. However, some surfactants can inhibit the biodegradation of PAHs due to their toxicity to microorganisms. Therefore, when selecting surfactants, it is advisable to choose those with low or even no toxicity to microorganisms and better solubilizing effects compared to other types of surfactants, especially some nonionic surfactants. The selection of surfactants is based at least on the interaction and relationship between PAH biodegradation and the surfactant during the degradation process. In other words, this invention can select the most suitable surfactant and its optimal dosage based on the surfactant's solubilizing effect, toxicity, and carbon source competition.

[0096] Optionally, the surfactant can be selected from nonionic chemical surfactants and nonionic biosurfactants. Nonionic chemical surfactants include Tween 80 and TX-100, while nonionic biosurfactants include tea saponin.

[0097] Preferably, surfactants with different critical micelle concentrations (CMC) exhibit varying degrees of solubilizing activity. When the surfactant concentration exceeds the CMC, the solubility of PAHs is significantly increased. For example... Figure 11The figure shows the relationship between surface tension values ​​and the common logarithm of surfactant concentration in solution, used to determine the CMC value. The CMC value of tea saponin is 100 mg / L, the CMC value of Tween 80 is 16 mg / L, and the CMC value of TX-100 is 145 mg / L.

[0098] Furthermore, the solubilizing ability of surfactants for pollutants is affected by their type and concentration; different surfactants have different solubilizing abilities. The effect of surfactant concentration on solubilization manifests in two different mechanisms: solubilization below the CMC and solubilization above or equal to the CMC. When the surfactant concentration does not exceed the CMC, its hydrophilic and hydrophobic groups are distributed to the interface between the aqueous and organic phases, achieving a solubilizing effect by altering the surface tension of the solution. However, when the surfactant concentration exceeds the CMC, it forms micelles, allowing microorganisms to directly utilize PAHs in the aqueous phase for respiratory metabolism. Figure 12 The figure shows the solubilizing ability of different surfactants at different concentrations for naphthalene. Among them, tea saponin has the best solubilizing effect on naphthalene, which is 6.27 times that of no surfactant added. Tween 80 and TX-100 increased the solubility of naphthalene by 3.32 times and 2.56 times, respectively.

[0099] Preferably, surfactants may have toxic effects on microorganisms, inhibiting their growth and thus affecting the remediation effect of organic pollution in groundwater. The toxicity of surfactants is determined by measuring the effect of different surfactants on biomass when used as the sole carbon source within a set surfactant concentration range (0–5 CMC). Figure 13 The figures show the growth curves of microorganisms when three surfactants are used as a single carbon source, namely (a) tea saponin; (b) Tween 80; and (c) TX-100.

[0100] Furthermore, when tea saponin is the only carbon source in the system, it can serve as a carbon or energy source for microbial growth, and high concentrations of tea saponin have no negative impact on microbial growth and reproduction. When the concentration is below 2 CMC, the biomass reaches its peak after 60 hours. Afterward, the consumption of carbon sources in the system leads to a decline in microbial growth, stabilizing at approximately 0.049 and 0.083 at the end of the experiment, respectively. At a concentration of 5 CMC, the microorganisms have sufficient carbon source and show no decline within the study range; the biomass rises to 0.322 at the end of the experiment.

[0101] When Tween 80 was the sole carbon source in the system, it had no toxic effect on microbial growth, and the biomass showed an increasing trend compared to the control group. The biomass reached its maximum at a concentration of 5 CMC, reaching 0.073.

[0102] When TX-100 is the sole carbon source in the system, it exhibits toxicity to microorganisms, inhibiting their growth, and the higher the concentration, the stronger the toxic effect. At a concentration of 5 CMC, the microbial biomass decreased to below that of the control group, reaching only 0.009.

[0103] Preferably, in the coexistence system of surfactant, naphthalene, and microorganisms formed in groundwater after the addition of a surfactant, both the surfactant and naphthalene can serve as carbon sources required for microbial growth, and they can act as common carbon sources while also exhibiting some carbon source competition. For example... Figure 14 The figures show the growth curves of microorganisms when three surfactants and naphthalene are used as a common carbon source, where (a) tea saponin; (b) Tween 80; and (c) TX-100.

[0104] Furthermore, when both naphthalene and tea saponin are present in the system, microorganisms can utilize them according to the following rules based on the different concentration ranges of tea saponin:

[0105] When the concentration of tea saponin is below the CMC, microorganisms can use both tea saponin and naphthalene as a common carbon source. At this time, the surfactant is in a monomeric state and cannot play a good solubilizing role for naphthalene. The microbial biomass increases due to the obvious competition between the two.

[0106] When the concentration of tea saponin is higher than the CMC, the solubilizing effect of tea saponin on naphthalene begins to increase. After the apparent solubility of naphthalene increases, its competitive advantage between the two carbon sources is enhanced, thereby enabling more naphthalene to be utilized and degraded by microorganisms.

[0107] When the concentration of tea saponin is further increased to 5 CMC, the high concentration of surfactant will be preferentially utilized by microorganisms as an energy source, thus reducing the carbon source competition of naphthalene.

[0108] Furthermore, when both Tween 80 and naphthalene are present as carbon sources in the system, microorganisms can exhibit the following utilization rules based on the different concentration ranges of tea saponins:

[0109] When the Tween 80 concentration is below the CMC, microorganisms can simultaneously utilize naphthalene and low concentrations of Tween 80 as energy sources for growth, at which point the competition becomes increasingly prominent.

[0110] When the Tween 80 concentration is equal to or higher than the CMC, the solubilizing effect of Tween 80 on naphthalene outweighs the competition between the two for carbon sources, thereby stabilizing the bacterial concentration at a higher level, allowing microorganisms to utilize naphthalene as a carbon source for growth.

[0111] Furthermore, when both naphthalene and TX-100 are present in the system, microorganisms can utilize them according to the following rules based on the different concentration ranges of tea saponins:

[0112] When the concentration of TX-100 is lower than the CMC, the low concentration of TX-100 has little effect on microbial growth. Some microorganisms can utilize naphthalene in the system as a carbon source, and at this time, the carbon source competition of naphthalene is dominant.

[0113] When the concentration of TX-100 is higher than that of CMC, due to the increased toxicity, the apparent solubility of naphthalene increases, and some microorganisms can continue to reproduce using naphthalene in the early stage. As naphthalene is consumed, the high concentration of TX-100 exerts its toxic effect in the later stage, and the toxic effect becomes stronger as the concentration of TX-100 continues to increase, thus inhibiting the growth and reproduction of microorganisms.

[0114] Preferably, different surfactants, due to their varying solubilizing, toxic, and / or carbon source competing effects, require the selection of appropriate concentrations as the optimal dosage to maximize the microbial remediation effect. For example... Figure 15 The effects of three surfactants at different concentrations on the microbial degradation of naphthalene are shown in the figures: (a) tea saponin; (b) Tween80; and (c) TX-100.

[0115] Furthermore, regarding tea saponin, the biodegradation of naphthalene by different concentrations of tea saponin follows the following rules:

[0116] When the concentration of tea saponin was 0.25 CMC, there was only a carbon source competition mechanism between tea saponin and naphthalene in the system. After a preset time of 96 hours, the biodegradation efficiency of naphthalene was lower than that of the control group, indicating that carbon source competition has a certain inhibitory effect on the degradation of naphthalene.

[0117] When the concentration of tea saponin was 1 CMC and 2 CMC, the system simultaneously exhibited the solubilizing effect of tea saponin on naphthalene and the carbon source competition effect. After a preset time of 96 hours, the biodegradation efficiency of naphthalene was greater than that of the control group, indicating that the solubilizing effect can promote the biodegradation of naphthalene.

[0118] When the concentration of tea saponin was 5 CMC, the system still exhibited solubilizing effect and competition between the two carbon sources. After a preset time, the biodegradation efficiency of naphthalene was higher than that of the control group, but lower than that of naphthalene when the concentration of tea saponin was CMC and 2 CMC. This indicates that both mechanisms exist simultaneously, but the solubilizing effect is slightly stronger than the competition between carbon sources.

[0119] Furthermore, regarding Tween 80, the biodegradation of naphthalene by different concentrations of Tween 80 follows the following rules:

[0120] When the concentration of Tween 80 is less than the CMC, there is a carbon source competition mechanism between Tween 80 and naphthalene. When the surfactant concentration is lower than the CMC, the main carbon source competition in the system will inhibit the degradation efficiency of naphthalene by microorganisms.

[0121] When the concentration of Tween 80 is equal to or higher than CMC, the system exhibits both carbon source competition and solubilization effects. After a preset time of 96 hours, the biodegradation efficiency of naphthalene is significantly improved compared to the control group. At this time, the carbon source competition effect is less than that of the surfactant solubilization effect, indicating that its solubilization effect is beneficial to the biodegradation of naphthalene. Furthermore, Tween 80 at 5 CMC has the strongest solubilizing effect on the microbial degradation of naphthalene.

[0122] Furthermore, for TX-100, the biodegradation of naphthalene by different concentrations of Tween 80 follows the following rules:

[0123] When the concentration of TX-100 is less than that of CMC, TX-100 has a relatively low toxicity to microorganisms. In the early stage of degradation, naphthalene has a competitive advantage in carbon source competition. In the later stage, the number of microorganisms that can utilize naphthalene continues to decrease, and the degradation efficiency is slightly lower than that of the control group.

[0124] When the concentration of TX-100 is equal to that of CMC, the solubilizing effect of TX-100 on naphthalene is greater than its toxic effect on microorganisms, and the improvement in the biodegradation efficiency of naphthalene after a preset time is greater than that of the control group.

[0125] When the concentration of TX-100 is greater than that of CMC, there are solubilization, carbon source competition and toxicity of TX-100 to bacteria in the system. After the preset time, the degradation rate of naphthalene is lower than that of the control group, indicating that carbon source competition and toxicity of TX-100 have a greater impact on the degradation of naphthalene.

[0126] Preferably, in the remediation of organic pollutants in groundwater using Klebsiella acidogenic bacteria K0106, particularly for the degradation of PAHs (e.g., naphthalene), this invention utilizes nitrate pollution, which is prevalent in groundwater, as an electron acceptor for microbial degradation of organic pollutants to improve the degradation efficiency of microorganisms in an anaerobic environment. The optimal nitrate concentration in the groundwater can be achieved by adding additional nitrate, and other factors can be adjusted to construct a preliminary remediation system. Furthermore, by introducing surfactants into the above remediation system, the solubility of organic pollutants in groundwater is enhanced, thereby improving the migration ability of organic pollutants and enabling them to be better utilized by microorganisms. Based on the solubilizing effect, toxicity, and carbon source competition of surfactants, tea saponin can be selected as the surfactant for the microbial strain. More preferably, the optimal dosage of tea saponin is 2 CMC. Furthermore, an experimental model was constructed according to the above remediation system to simulate the effect of surfactants on the microbial degradation of organic pollutants (such as naphthalene) in the groundwater environment. The addition of 2CMC tea saponin can promote the degradation of naphthalene in the groundwater environment, mainly by promoting the growth of microorganisms and the desorption of naphthalene, so that the method described in this invention can improve the remediation effect on organic pollution in groundwater.

[0127] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and do not constitute a limitation on the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept. Throughout the text, features introduced by "preferredly" are merely optional and should not be construed as mandatory. Therefore, the applicant reserves the right to abandon or delete relevant preferred features at any time.

Claims

1. A method for enhancing microbial remediation of organic pollution in groundwater, characterized in that, The method includes: inoculating organic-contaminated groundwater with Klebsiella oxytoca K0106 (accession number CCTCC NO: M20221835); and adding nitrate to the organic-contaminated groundwater to achieve a nitrate concentration of at least 300 mg / L. mg / L, under anaerobic conditions, Klebsiella acidogenic bacteria K0106 uses nitrate as an electron acceptor to anaerobic biodegrade organic pollutants, including at least naphthalene; tea saponin is added to the organic-polluted groundwater, and the concentration of tea saponin is controlled at twice the critical micelle concentration, so that tea saponin and the organic pollutants serve as a common carbon source. The system simultaneously exhibits the solubilizing effect of tea saponin on the organic pollutants and the carbon source competition between tea saponin and the organic pollutants, and tea saponin does not have a negative impact on the growth and reproduction of Klebsiella acidogenic bacteria K0106, thereby promoting the biodegradation of the organic pollutants by increasing their apparent solubility.

2. The method for enhanced microbial remediation of organic pollution in groundwater according to claim 1, characterized in that, Before inoculating the Klebsiella acidogenic bacteria K0106 into the organically polluted groundwater, the Klebsiella acidogenic bacteria K0106 was acclimatized and enriched. The acclimatization and enrichment included: adding an organic pollutant solution to an acclimatization medium loaded with activated sludge, removing the solvent and dissolved oxygen respectively, and then shaking the medium to complete the microbial proliferation under anaerobic conditions; taking the turbid liquid obtained after proliferation and adding it to a new acclimatization medium, maintaining the same culture conditions, and gradually increasing the initial concentration of organic pollutants generation by generation, so that the microorganisms were acclimatized for 4 generations under the condition of gradually increasing organic pollutant concentration; taking the sample from the 4th generation medium and spreading it onto a solid medium containing organic pollutants.

3. The method for enhanced microbial remediation of organic pollution in groundwater according to claim 1 or 2, characterized in that, The method further includes the following steps: (1) adjusting the pH value of the organic polluted groundwater to 6.8 to 7.0; (2) selecting a time when the temperature of the organic polluted groundwater is at least 15°C for remediation; and (3) adjusting the inoculation concentration of the acid-producing Klebsiella pneumoniae K0106 to at least 5% by volume percentage.

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