Pseudomonas stutzeri and application thereof in repairing heavy metal and petroleum combined pollution

Abstract

The invention relates to the technical field of wastewater biological treatment, in particular to pseudomonas stutzeri and application thereof in remediation of heavy metal and petroleum combined pollution, the pseudomonas stutzeri is preserved in China Center for Type Culture Collection, the address is Wuhan University, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M 20232233. The pseudomonas stutzeri is inoculated into underground water subjected to combined pollution of heavy metal and petroleum, the heavy metal is hexavalent chromium, and in the underground water subjected to combined pollution of the heavy metal and the petroleum, the initial concentration range of the hexavalent chromium is 0-100 mg / L, and the initial volume content range of crude oil is 0.05%-2%. When the pseudomonas stutzeri is used for repairing heavy metal and petroleum combined pollution, the strain selection range can be increased, and the applicable pollutant concentration range can be expanded.

Description

Technical Field

[0001] The present application relates to the technical field of wastewater biological treatment, and in particular to Pseudomonas stutzeri and its application in repairing heavy metal and petroleum composite pollution. Background Art

[0002] Petroleum is a complex mixture of hydrocarbons and other organic compounds, including some organic metal components. The heavy metal components in crude oil have multiple sources. First, heavy metal elements themselves are one of the components of petroleum, and they often exist in the form of complexes with petroleum hydrocarbons; second, industrial products such as drilling fluid additives are used in the process of oil extraction, and the extraction process also carries heavy metal mineral debris from the formation, thereby introducing heavy metal pollution into petroleum hydrocarbons. Some heavy metal-contaminated soils are contaminated by petroleum hydrocarbons due to machining and other operations, resulting in petroleum hydrocarbon-heavy metal composite pollution.

[0003] Physical, chemical and biological methods are the three main methods for treating petroleum pollutants. Among them, physical methods can only dilute, aggregate or migrate pollutants to other environments but cannot completely eliminate pollution; chemical methods are difficult to completely degrade oil and will cause secondary pollution. The biological method has a mild effect and can completely degrade petroleum pollutants, treat contaminated soil in situ, and has low costs. It overcomes the shortcomings of physical and chemical methods and shows its unique advantages. Bioremediation mainly uses microorganisms to degrade harmful organic components into harmless substances, such as promoting the cracking of petroleum hydrocarbon pollutants, micelle formation, increasing their fluidity, etc., to achieve the purpose of biodegradation of petroleum hydrocarbons, and reducing hexavalent chromium to trivalent chromium. Bioremediation has the advantages of high efficiency, low investment, wide range of pH and temperature conditions, and effective recovery of some precious metals.

[0004] At present, relevant technical research mainly focuses on the research and development of microbial agents and microbial soil conditioners for repairing petroleum pollution or heavy metals. For example, Liu Yulong et al. provided an in-situ remediation method for heavy metal and petroleum hydrocarbon composite pollution in groundwater (CN106915869A), which uses oxygen-releasing materials and immobilized bacterial agents to remove heavy metal and petroleum pollution respectively. However, there are few studies on the synergistic remediation of petroleum and heavy metal composite pollution in groundwater by microorganisms. It is urgent to propose an improved solution based on microbial functional bacteria to synergistically repair heavy metal and petroleum composite pollution in groundwater. Summary of the invention

[0005] The purpose of the present invention is to provide a Pseudomonas stutzeri and its application in repairing heavy metal and petroleum composite pollution, aiming to solve the technical problem of petroleum hydrocarbon and heavy metal composite pollution in water bodies in the prior art.

[0006] In a first aspect, the present application discloses a Pseudomonas stutzeri, wherein the Pseudomonas stutzeri is the Pseudomonas stutzeri with a deposit number of CCTCCNO: M 20232233.

[0007] In a second aspect, the present application discloses a method for repairing heavy metal and petroleum combined pollution, which is applied to the repair of groundwater contaminated by heavy metals and petroleum, comprising: inoculating the Pseudomonas stutzeri described in the first aspect into groundwater contaminated by heavy metals and petroleum.

[0008] In an optional embodiment, the initial volume content of crude oil in the groundwater contaminated by heavy metals and petroleum is in the range of 0.05%-2%.

[0009] In an optional embodiment, the heavy metal is hexavalent chromium, and in the groundwater contaminated by heavy metals and petroleum, the initial concentration of the hexavalent chromium is in the range of 0-100 mg / L.

[0010] In an alternative embodiment, the Pseudomonas stutzeri is expressed in OD 600 The inoculum amount with a value of 0.5-2 is inoculated into the groundwater contaminated by heavy metals and petroleum.

[0011] In an optional embodiment, the method further comprises adding a carbon source to the groundwater contaminated by heavy metals and petroleum.

[0012] In an optional embodiment, the carbon source is one of ethanol, glucose, sodium acetate, lactose or any combination thereof.

[0013] In an optional embodiment, the COD value of the added carbon source is 100-500 mg / L.

[0014] In an optional embodiment, the method further comprises adding a surfactant to the groundwater contaminated by heavy metals and petroleum, wherein the volume of the surfactant added is 1.5-2 times the initial volume content of the crude oil.

[0015] In an optional embodiment, the Pseudomonas stutzeri is inoculated into groundwater contaminated by heavy metals and petroleum, and then cultured in a constant temperature shaking incubator at 28° C.-32° C. and 120-150 r / min.

[0016] Based on the above technical solution, the beneficial effects of this application compared with the prior art are as follows:

[0017] 1. The Pseudomonas stutzeri in the present application has the ability to synergistically repair the combined pollution of Cr(VI) and petroleum in groundwater. The removal rate of Cr(VI) can reach 100%, and the degradation rate of petroleum hydrocarbons can reach more than 90%. It shows good application prospects in treating water bodies with combined pollution of petroleum and Cr(VI), thus enriching the range of bacterial species selection for the repair of combined pollution of heavy metals and petroleum in groundwater.

[0018] 2. The Pseudomonas stutzeri in the present application shows good tolerance to different concentrations of Cr(VI), and the applicable concentration range reaches 0-100 mg / L. When the concentration is 100 mg / L, the removal rate of Cr(VI) can still reach about 70%. Therefore, the Pseudomonas stutzeri in the present application is suitable for contaminated sites with high Cr(VI) concentration and severe pollution. Higher concentrations of Cr(VI) will not cause the growth and metabolic activity of Pseudomonas stutzeri to be lost.

[0019] 3. The Pseudomonas stutzeri in the present application adopts a carbon source co-metabolism mechanism. The carbon source has a significant promoting effect on the synergistic remediation of petroleum hydrocarbons and heavy metal complex pollution in water bodies by Pseudomonas stutzeri, and can accelerate the pollution remediation process.

[0020] 4. The Pseudomonas stutzeri in the present application can repair the combined pollution of Cr(VI) and petroleum in groundwater under both aerobic and anaerobic conditions and has strong adaptability to the environment. DETAILED DESCRIPTION

[0021] The inventors referred to the prior art and used modified strains of Pseudomonas aeruginosa to treat refinery wastewater. When hexavalent chromium (hereinafter referred to as Cr(VI)) was added, the modified strain achieved a 100% reduction rate of Cr(VI) while achieving petroleum hydrocarbon degradation. However, the inventors found that when this strain synergistically degraded petroleum hydrocarbons and Cr(VI), the initial concentration of Cr(VI) applicable to it was very low, only 1×10 -3 mg / L, which is not suitable for solving polluted sites with serious chromium pollution.

[0022] In order to increase the breadth of bacterial strain selection and expand the applicable pollutant concentration range, the inventors further developed and made the present invention.

[0023] In the first aspect, the present application provides a Pseudomonas stutzeri, which is Pseudomonas stutzeri KLHJ-01 with a preservation number of CCTCC NO: M20232233, and is preserved in the China Center for Type Culture Collection, located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, and the preservation time is November 15, 2023.

[0024] The Pseudomonas stutzeri provided in the present application is obtained by inoculating a pure bacterial strain of Pseudomonas stutzeri into groundwater containing crude oil and Cr(VI) for domestication and cultivation.

[0025] Specifically, a sterilized 250 mL glass conical flask was used as a reactor, pure Pseudomonas stutzeri was cultured, and the lower layer microorganisms obtained by centrifugation were inoculated into groundwater containing crude oil and Cr(VI) for acclimation culture, wherein the volume percentage of crude oil was 1%, the concentration of Cr(VI) was 50 mg / L, and the concentration of Cr(VI) was K 2 Cr 2 O 7 , and the pH of groundwater was adjusted to about 7. After inoculation of microorganisms, the conical flask was sealed with a sterile filtered breathable sealing film, wrapped with tin foil and stored in a constant temperature shaker at 28-32°C for 10 days at 150r / min, and then 5mL of the first acclimation culture solution was transferred to a fresh 200mL acclimation medium, and the culture was continued under the same conditions, and repeated 3 times. The strain after acclimation culture is a strain resistant to the combined pollution environment of petroleum hydrocarbon-Cr(VI). 1mL of the culture solution was mixed with 1mL of glycerol aqueous solution (V / V=1:1) and frozen in a -80°C refrigerator as the strain used in subsequent experiments.

[0026] In the second aspect, the present application also discloses a method for repairing heavy metal and petroleum composite pollution, which is applied to the repair of groundwater compositely polluted by heavy metals and petroleum, comprising: inoculating the Pseudomonas stutzeri described in the first aspect into groundwater compositely polluted by heavy metals and petroleum. Wherein, the heavy metal is Cr(VI), and the ability of Pseudomonas stutzeri to repair groundwater composite pollution of heavy metals and petroleum is determined by the change in the concentration of Cr(VI) and TPH (total petroleum hydrocarbons). The Pseudomonas stutzeri in the present application can reduce Cr(VI) to trivalent chromium and degrade TPH in groundwater into non-polluting or less polluting compounds, such as carbon dioxide and water.

[0027] Specifically, Pseudomonas stutzeri was inoculated into artificially simulated groundwater containing crude oil and Cr(VI), and cultured under aerobic and anaerobic environments, respectively. The aerobic environment used a 250 ml sterile conical flask sealed with a sterile sealing film as a reactor, and the anaerobic environment used a 250 ml sterile serum bottle as a reactor. The simulated groundwater was 200 ml, and the culture was carried out in a constant temperature shaking incubator at 28-32°C and 120-150 r / min.

[0028] Specifically, when the Pseudomonas stutzeri of the present application is used to repair the combined pollution of heavy metals and petroleum in groundwater, the initial volume content of crude oil in the groundwater contaminated by heavy metals and petroleum is in the range of 0.05%-2% (V / V), specifically 0.05%, 1%, 1.5% or 2%; the initial concentration of Cr(VI) is in the range of 0-100 mg / L, specifically 10 mg / L, 25 mg / L, 50 mg / L, 75 mg / L or 100 mg / L; the Pseudomonas stutzeri is measured by OD 600 An inoculum amount of 0.5-2 is inoculated into groundwater contaminated by heavy metals and petroleum, and specifically can be 0.5, 1, 1.5 or 2.

[0029] In an alternative embodiment, Pseudomonas stutzeri is grown in a groundwater medium with an OD of 0.1. 600 =1 was inoculated in groundwater containing 1% (V / V) crude oil and 10 mg / L Cr(VI). Within 29 days, the final degradation rates of Cr(VI) under aerobic conditions were 28.80%, and the final degradation rates of TPH were 83.08%. The final degradation rates of Cr(VI) under anaerobic conditions were 55.52%, and the final degradation rates of TPH were 84.12%, indicating that Pseudomonas stutzeri has the ability to synergistically remove petroleum hydrocarbon-Cr(VI) composite pollution, and Pseudomonas stutzeri shows good tolerance to different concentrations of Cr(VI). Higher concentrations of Cr(VI) will not completely lose the growth and metabolic activity of Pseudomonas stutzeri, so it is suitable for sites with high Cr(VI) concentrations and serious pollution.

[0030] Further, when the Pseudomonas stutzeri of the present application is applied to repair the heavy metal and petroleum composite pollution of groundwater, it also includes adding a carbon source to the groundwater contaminated by heavy metals and petroleum, and the carbon source is one of ethanol, glucose, sodium acetate, lactose or any combination thereof. That is, Pseudomonas stutzeri can also use one or more of the soluble carbon sources of glucose, lactose, sodium acetate, ethanol, etc. as a carbon source for growth metabolism, while degrading TPH and reducing Cr (VI) in groundwater. Wherein, the COD value of the added carbon source is 100-500mg / L, specifically 100mg / L, 200mg / L, 300mg / L or 500mg / L. In the case of adding 4 soluble carbon sources of glucose, lactose, sodium acetate, and ethanol carbon sources, the Cr (VI) removal rate can reach about 100%, and the TPH removal rate can reach about 85%. Therefore, the carbon source has a significant promoting effect on the reduction of Cr (VI) by Pseudomonas stutzeri.

[0031] Furthermore, when the Pseudomonas stutzeri of the present application is applied to repair the heavy metal and petroleum composite pollution of groundwater, a surfactant is added to the groundwater compositely polluted by heavy metals and petroleum, and the volume of the surfactant added is specifically 1.5-2 times the initial volume content of the crude oil. In an optional embodiment, the surfactant can be rhamnolipid, and the surfactant can accelerate the dissolution rate of petroleum hydrocarbons and groundwater.

[0032] When the Pseudomonas stutzeri of the present application is used to repair the heavy metal and petroleum composite pollution of groundwater, a constant temperature shaking incubator is carried out at 28°C-32°C and 150r / min. The culture temperature can be specifically 28°C, 30°C or 32°C. The groundwater temperature is controlled within a certain range to provide a suitable external environment for the growth and metabolism of Pseudomonas stutzeri, so as to improve the growth and metabolic activity of the strain as much as possible, and ensure the strain's ability to efficiently degrade petroleum hydrocarbons and reduce Cr (VI). Specifically, the temperature of the groundwater can be adjusted by setting the temperature of a constant temperature shaking incubator or by using a water bath for heating. The heating method is not specifically limited here.

[0033] Furthermore, when the Pseudomonas stutzeri of the present application is used to treat groundwater containing petroleum and heavy metals, the pH value of the groundwater is 6-10, specifically 6, 7, 8, 9 or 10, etc., preferably 7, to maintain normal growth and metabolism of Pseudomonas stutzeri.

[0034] The present invention is further described in detail below in conjunction with specific embodiments:

[0035] Example 1

[0036] 1. Screening of functional strains

[0037] (1) Cultivation of strains

[0038] Bacillus subtilis and Pseudomonas stutzeri strains were cultured in LB medium until the logarithmic growth phase.

[0039] (2) Extraction of strains

[0040] Using sterile LB medium as blank reference, the absorbance of the culture solution was measured with a cuvette at a wavelength of 600nm, i.e. OD 600 The calculated volume of bacterial solution was placed in a centrifuge tube and centrifuged at 6000 r / min for 20 min. The supernatant was discarded and the lower sediment was rinsed three times with sterile deionized water to finally obtain the microorganisms in the lower layer of the centrifuge tube.

[0041] (3) Inoculation strain

[0042] Four reactors were prepared, two of which were used for aerobic environment experiments and the other two for anaerobic environment experiments. The aerobic experiment used a 250 mL sterile conical flask sealed with a sterile sealing film as the reactor, and the anaerobic experiment used a 250 ml sterile serum bottle as the reactor. The total amount of simulated groundwater in the reactor was 200 mL. 0.2% (V / V) crude oil, 0.4% (V / V) rhamnolipid and two bacteria were added to the artificially simulated groundwater. The bacterial culture medium was OD 2. 600 =1 inoculation amount, and cultured in a constant temperature shaker at 30°C and 150r / min. The concentration changes of TPH in the aerobic and anaerobic system with the addition of Bacillus subtilis and Pseudomonas stutzeri are shown in the following table:

[0043] Table 1 Changes in TPH concentration in aerobic and anaerobic systems with the addition of Pseudomonas stutzeri and Bacillus subtilis

[0044]

[0045]

[0046] As shown in Table 1, on the sixth day, the aerobic experimental group of Pseudomonas stutzeri first produced a significant degradation effect on petroleum hydrocarbons. During the entire cycle, under the same experimental conditions, the degradation effect of Pseudomonas stutzeri on petroleum hydrocarbons was shorter and more efficient. Pseudomonas stutzeri was selected for the subsequent groundwater remediation process.

[0047] 2. Feasibility verification experiment of Pseudomonas stutzeri removing TPH and Cr(VI)

[0048] Specifically, the Pseudomonas stutzeri strain was cultured after acclimation and culture, and the OD of the groundwater medium in the reactor was 600 =1 was inoculated into artificially simulated groundwater containing 1% (V / V) crude oil and 10 mg / L Cr(VI), and cultured under aerobic and anaerobic environments respectively. The aerobic experiment used a 250 ml sterile conical bottle sealed with a sterile sealing film as the reactor, and the anaerobic experiment used a 250 ml sterile serum bottle as the reactor. The simulated groundwater in each reactor was 200 ml, and cultured in a constant temperature oscillator at 30°C and 150 r / min. The concentration changes of TPH and Cr(VI) in the anaerobic and aerobic system with Pseudomonas added are shown in the following table:

[0049] Table 2 Changes in TPH and Cr(VI) concentrations in the anaerobic and aerobic system with the addition of Pseudomonas

[0050]

[0051] As shown in Table 2, within 29 days, the final degradation rate of Cr(VI) under aerobic conditions was 28.80%, and the final degradation rate of TPH was 83.08%; the final degradation rate of Cr(VI) under anaerobic conditions was 55.5%, and the final degradation rate of TPH was 84.12%. The surface Pseudomonas stutzeri has the ability to synergistically remove petroleum hydrocarbon-Cr(VI) complex pollution.

[0052] 3. Experiment on the removal characteristics of TPH and Cr(VI) by Pseudomonas stutzeri under different carbon sources

[0053] Specifically, four soluble carbon sources, glucose, lactose, sodium acetate, and ethanol, were set as co-metabolic matrices. The added amount of soluble carbon source COD (chemical oxygen demand) was 150 mg / L. The initial Cr(VI) concentration was 10 mg / L, the initial crude oil concentration of groundwater was 0.2% (V / V), and 0.4% rhamnolipid (V / V) was added as a biosurfactant. The four carbon sources were set up in anaerobic and aerobic experimental groups, for a total of 8 experimental groups. A 250 mL sterile conical flask was used as an aerobic reactor, and a 250 mL sterile serum bottle was used as an anaerobic reactor. The simulated groundwater in each reactor was 200 mL. The culture solution of the domesticated Pseudomonas stutzeri strain was taken to measure the OD value of the groundwater medium in the reactor. 600 =1 was inoculated into the above 8 experimental groups and cultured in a constant temperature shaker at 30°C and 150r / min. Specifically, the changes in Cr(VI) concentrations in aerobic and anaerobic systems with four soluble carbon sources as co-metabolism substrates are shown in the following table:

[0054] Table 3 Changes in Cr(VI) concentrations in aerobic and anaerobic systems using four soluble carbon sources as co-metabolism substrates

[0055]

[0056] Furthermore, the changes in TPH concentration in the aerobic and anaerobic systems using four soluble carbon sources as co-metabolism substrates are shown in the following table:

[0057] Table 4 Changes in TPH concentration in aerobic and anaerobic systems using four soluble carbon sources as co-metabolism substrates

[0058]

[0059] As shown in Tables 3 and 4, the experimental groups with four soluble carbon sources, glucose, lactose, sodium acetate, and ethanol, had good reduction effects on Cr(VI), and the removal rates were all about 100%, which was relatively complete. The effects of different carbon sources on TPH degradation were not very different, and the TPH removal rates were all above 85%. Among them, the experimental group with ethanol as the external carbon source under aerobic conditions had the best TPH removal effect, reaching 91.67%.

[0060] In summary, under aerobic conditions, the order of different carbon sources for promoting TPH removal is: ethanol>glucose>sodium acetate>lactose; under anaerobic conditions, the order of different carbon sources for promoting TPH removal is: lactose>ethanol>glucose>sodium acetate. Based on the results of carbon source screening under aerobic and anaerobic conditions, ethanol has the best synergistic removal effect on crude oil-heavy metal composite pollution of Pseudomonas stutzeri, so it was decided to use ethanol as the optimal carbon source for subsequent experiments.

[0061] 4. Experiment on the effect of ethanol on different pollutants

[0062] With ethanol (0.0914 mL / L) with COD = 150 mg / L as the external carbon source, the culture solution of the domesticated Pseudomonas stutzeri strain was mixed with the groundwater medium OD 600 =1 was inoculated into 8 experimental groups, using a 250mL sterile conical flask as an aerobic reactor and a 250mL sterile serum bottle as an anaerobic reactor, with 200mL of simulated groundwater in each reactor. The experimental group settings are shown in Table 5:

[0063] Table 5 Ethanol effect on different pollutants Experimental supplementary experimental groups

[0064]

[0065] Each experimental group was cultured at a constant temperature of 30°C and 150 r / min. The concentration changes of TPH and Cr(VI) in different experimental groups under aerobic and anaerobic systems are shown in Tables 6 and 7:

[0066] Table 6 Changes in TPH concentrations in different experimental groups under aerobic and anaerobic systems

[0067]

[0068]

[0069] Table 7 Changes in Cr(VI) concentrations in different experimental groups under aerobic and anaerobic systems

[0070]

[0071] As shown in Tables 6 and 7, the TPH degradation rates of experimental groups 1, 2, 3, and 4 with ethanol added were significantly higher than those of the four experimental groups without carbon source added. Oxygen conditions had little effect on the degradation of Cr(VI) by Pseudomonas stutzeri, and the TPH degradation rate under aerobic conditions was higher than that under anaerobic conditions. In summary, the co-metabolism substrate ethanol can improve the degradation efficiency of crude oil-Cr(VI) composite pollution by Pseudomonas stutzeri, and has a significant promoting effect on the degradation of petroleum hydrocarbons, which is consistent with the results of the carbon source screening experiment.

[0072] 5. Experiment to determine the optimal degradation conditions

[0073] The experiment on the removal characteristics of TPH and Cr(VI) by Pseudomonas stutzeri under different carbon sources showed that ethanol was the optimal carbon source, so ethanol was selected as the external carbon source.

[0074] Specifically, the culture solution of the domesticated Pseudomonas stutzeri strain was taken and the OD of the groundwater medium in the reactor was 600 =0.5 inoculation volume was inoculated in the reactor, and 100mL artificial groundwater was added. The initial volume content of crude oil in the groundwater was 0.05% (V / V), the initial Cr(VI) concentration was 25mg / L, ethanol with COD=100mg / L was used as the added carbon source, and the amount of surfactant rhamnolipid added was twice the initial volume content of crude oil (volume), and cultured in a constant temperature shaker at 30°C and 150r / min under aerobic conditions. It should be noted that the aerobic condition uses a sterile conical flask sealed with a sterile sealing film as the reactor.

[0075] The difference between Example 2 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 =1 was inoculated into the reactor, the initial Cr(VI) concentration in the groundwater was 50 mg / L, and ethanol with COD=500 mg / L was used as the added carbon source.

[0076] The difference between Example 3 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 =1.5 inoculation volume was inoculated in the reactor, the initial Cr(VI) concentration in the groundwater was 75 mg / L, ethanol with COD=200 mg / L was used as the external carbon source, and culture was carried out under anaerobic conditions, 30°C, 150 r / min constant temperature shaking incubator. It should be noted that the anaerobic conditions used sterile serum bottles as reactors.

[0077] The difference between Example 4 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 =2 was inoculated into the reactor, the initial Cr(VI) concentration in the groundwater was 100 mg / L, and ethanol with COD=300 mg / L was used as the external carbon source. The culture was carried out under anaerobic conditions at 30°C and 150 r / min constant temperature shaking incubator.

[0078] The difference between Example 5 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 = 1 was inoculated into the reactor, the initial crude oil concentration in the groundwater was 0.05% (V / V), and ethanol with COD = 300 mg / L was used as the external carbon source. The culture was carried out under anaerobic conditions at 30°C and 150 r / min constant temperature shaking incubator.

[0079] The difference between Example 6 and Example 1 is that the initial crude oil concentration in the groundwater is 0.2% (V / V), the initial Cr(VI) concentration is 50 mg / L, ethanol with COD=200 mg / L is used as the added carbon source, and the culture is carried out under anaerobic conditions, 30°C, and 150 r / min constant temperature shaking incubator.

[0080] The difference between Example 7 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 =2 was inoculated into the reactor, the initial crude oil concentration in the groundwater was 0.2% (V / V), the initial Cr(VI) concentration was 75 mg / L, and ethanol with COD=500 mg / L was used as the added carbon source.

[0081] The difference between Example 8 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 =1.5 was inoculated into the reactor, the initial crude oil concentration in the groundwater was 0.2% (V / V), and the initial Cr(VI) concentration was 100 mg / L.

[0082] The difference between Example 9 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 =1.5 inoculation volume was inoculated into the reactor, the initial crude oil concentration in the groundwater was 1% (V / V), ethanol with COD = 500 mg / L was used as the external carbon source, and the culture was carried out under anaerobic conditions at 30°C and 150 r / min constant temperature shaking incubator.

[0083] The difference between Example 10 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 =2 was inoculated into the reactor, the initial crude oil concentration in the groundwater was 1% (V / V), the initial Cr(VI) concentration was 50 mg / L, and the culture was carried out in an anaerobic condition at 30°C and 150 r / min constant temperature shaking incubator.

[0084] The difference between Example 11 and Example 1 is that the initial crude oil concentration in the groundwater is 1% (V / V), the initial Cr(VI) concentration is 75 mg / L, and ethanol with COD=300 mg / L is used as the added carbon source.

[0085] The difference between Example 12 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 =1 was inoculated into the reactor, the initial crude oil concentration in the groundwater was 1% (V / V), the initial Cr(VI) concentration was 100 mg / L, and ethanol with COD=200 mg / L was used as the added carbon source.

[0086] The difference between Example 13 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 =2 was inoculated into the reactor, the initial crude oil concentration in the groundwater was 2% (V / V), the initial Cr(VI) concentration was 75 mg / L, and ethanol with COD=200 mg / L was used as the added carbon source.

[0087] The difference between Example 14 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 =2 was inoculated into the reactor, the initial crude oil concentration in the groundwater was 1.5% (V / V), the initial Cr(VI) concentration was 50 mg / L, and ethanol with COD=300 mg / L was used as the added carbon source.

[0088] The difference between Example 15 and Example 1 is that the culture solution of Pseudomonas stutzeri is OD 600 =1 was inoculated into the reactor, the initial crude oil concentration in the groundwater was 2% (V / V), the initial Cr(VI) concentration was 75 mg / L, and the culture was carried out in an anaerobic condition at 30°C and 150 r / min constant temperature shaking incubator.

[0089] The difference between Example 16 and Example 1 is that the initial crude oil concentration in the groundwater is 2% (V / V), the initial Cr(VI) concentration is 100 mg / L, ethanol with COD=500 mg / L is used as the external carbon source, and culture is carried out under anaerobic conditions, 30°C, and 150 r / min constant temperature shaking incubator.

[0090] The intuitive analysis results of the orthogonal experiment of Pseudomonas stutzeri degrading crude oil-Cr(VI) in Examples 1-16 are shown in Table 8, Tables 9 and 10 show the mean response of Cr(VI) removal rate and the mean response of crude oil degradation rate, respectively, Tables 11 and 12 show the inter-subject effect test tables of Cr(VI) removal rate and crude oil degradation rate, respectively, and Tables 13 and 14 show the post hoc analysis tables of variance of Cr(VI) removal rate and crude oil degradation rate based on the SNK algorithm, respectively.

[0091] Table 8 Orthogonal experiment intuitive analysis table

[0092]

[0093]

[0094]

[0095] Table 9 Cr(VI) removal rate mean response table

[0096]

[0097] Table 10 TPH degradation rate mean response table

[0098]

[0099]

[0100] Table 11 Variance analysis of Cr(VI) degradation rate between-subject effect test table

[0101]

[0102] Note: R 2 =0.986

[0103] Table 12 TPH degradation rate variance analysis inter-subject effect test table

[0104]

[0105] Note: R 2 =0.972

[0106] Table 13 Post hoc analysis of variance of Cr(VI) removal rate based on SNK algorithm

[0107]

[0108]

[0109] Table 14 Post hoc analysis of TPH degradation rate variance based on SNK algorithm

[0110]

[0111]

[0112] In summary, it can be seen from Tables 9 and 10 that the correlation between initial crude oil concentration, bacterial biomass, initial Cr(VI) concentration, cometabolism substrate content, oxygen conditions and Cr(VI) removal rate decreases in turn, and the correlation between initial crude oil concentration, cometabolism substrate content, initial Cr(VI) concentration, bacterial biomass, oxygen conditions and TPH degradation rate decreases in turn. Based on the SNK algorithm, the optimal degradation conditions obtained by software simulation are initial crude oil concentration of 0.2%, Cr(VI) concentration of 75 mg / L, cometabolism substrate COD of 200 mg / L, bacterial biomass OD 600 =2. Aerobic conditions.

[0113] In summary, the Pseudomonas stutzeri in the examples of the present application showed good tolerance to Cr(VI) concentrations within the numerical range (25-100 mg / L) selected in the above multi-factor orthogonal experiment, and Pseudomonas stutzeri did not lose growth and metabolic activity at higher concentrations of Cr(VI), and when the Cr(VI) concentration was 100 mg / L, the Cr(VI) removal rate could still reach about 70%. Therefore, the Pseudomonas stutzeri in the present application is suitable for contaminated sites with high Cr(VI) concentrations and severe pollution, and has good application prospects.

[0114] 6. Optimal degradation conditions verification experiment

[0115] The domesticated Pseudomonas stutzeri strain was cultured to the logarithmic growth phase, and 0.4% V / V biosurfactant rhamnolipid, 0.2% V / V crude oil, 75 mg / L Cr(VI), and a co-metabolism substrate with a COD of 200 mg / L were added to the reactor. The bacterial biomass in the reactor was OD 600 =2, a sterilized 250 mL conical flask was used as the reaction container, and the flask was sealed with a sterile filtered breathable sealing film. The cells were cultured in a constant temperature shaking incubator at 30°C and 150 rpm / min under aerobic conditions for 21 days, and the concentration changes of TPH and Cr(VI) were monitored.

[0116] The concentration changes of TPH and Cr(VI) under the optimal degradation conditions are shown in Table 15:

[0117] Table 15 Concentration changes of TPH and Cr(VI) under optimal degradation conditions

[0118]

[0119]

[0120] It can be seen from Table 15 that the final degradation effect of TPH can reach 97.68%, the reduction rate of Cr(VI) is 31.3%, and the composite pollution remediation effect basically meets expectations.

[0121] The above description includes examples of one or more embodiments. Of course, it is impossible to describe all possible combinations of components or methods for the purpose of describing the above embodiments, but it should be recognized by those skilled in the art that the various embodiments may be further combined and arranged. Therefore, the embodiments described herein are intended to cover all such changes, modifications and variations that fall within the scope of protection of the appended claims. In addition, with respect to the term "comprising" used in the specification or claims, the word is covered in a manner similar to the term "including", as explained by the use of "including" as a transitional word in the claims. In addition, any term "or" used in the claims and the specification is intended to mean "non-exclusive or".

Claims

1. A Pseudomonas stutzeri, It is characterized in that The Pseudomonas stutzeri is the Pseudomonas stutzeri with the deposit number CCTCC NO: M20232233.

2. A method for repairing heavy metal and petroleum composite pollution, the method is applied to the repair of groundwater contaminated by heavy metal and petroleum composite pollution, It is characterized in that include: The Pseudomonas stutzeri described in claim 1 is inoculated into groundwater contaminated by heavy metals and petroleum.

3. The method according to claim 2, It is characterized in that In the underground water polluted by heavy metals and petroleum, the initial volume content of crude oil is in the range of 0.05%-2%.

4. The method according to claim 2, It is characterized in that The heavy metal is hexavalent chromium, and in the groundwater contaminated by the heavy metal and petroleum, the initial concentration range of the hexavalent chromium is 0-100 mg / L.

5. The method according to claim 2, It is characterized in that The Pseudomonas stutzeri was expressed as OD 600 The inoculum amount with a value of 0.5-2 is inoculated into the groundwater contaminated by heavy metals and petroleum.

6. The method according to claim 2, It is characterized in that The method further comprises adding a carbon source to the groundwater contaminated by heavy metals and petroleum.

7. The method according to claim 6, It is characterized in that The carbon source is one of ethanol, glucose, sodium acetate, lactose or any combination thereof.

8. The method according to claim 7, It is characterized in that The COD value of the added carbon source is 100-500 mg / L.

9. The method according to claim 3, It is characterized in that The method further comprises adding a surfactant to the groundwater contaminated by heavy metals and petroleum, wherein the volume of the surfactant added is 1.5-2 times the initial volume content of the crude oil.

10. The method according to claim 2, It is characterized in that The Pseudomonas stutzeri is inoculated into groundwater contaminated by heavy metals and petroleum, and then cultured in a constant temperature shaking table at 28-32°C and 150 r / min.

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