Compounding method and application of cadmium-tolerant petroleum hydrocarbon degrading flora

Abstract

The invention provides a compounding method and application of a group of cadmium-resistant petroleum hydrocarbon degrading flora, belongs to the technical field of microorganisms, can realize effective degradation of petroleum hydrocarbon, can adapt to a cadmium-containing degrading environment, and further can realize effective degradation of the petroleum hydrocarbon in the cadmium-containing environment. The method comprises the following steps: preparing a pseudomonas oleovorans solution and a pseudomonas monteilii solution, and performing cadmium-tolerant domestication through a petroleum hydrocarbon degradation culture medium to obtain a domesticated pseudomonas oleovorans solution and a domesticated pseudomonas monteilii solution; and finally compounding the two bacterial solutions to obtain the cadmium-resistant petroleum hydrocarbon degrading flora.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a compounding method and application of a group of cadmium-resistant petroleum hydrocarbon-degrading bacteria. Background Art

[0002] Petroleum hydrocarbons are hydrocarbon compounds in petroleum, which are the main components of petroleum. Aromatic hydrocarbons in petroleum are extremely toxic to humans and animals. Cadmium (Cd) is a highly carcinogenic metal that can cause toxic reactions even at low concentrations. When petroleum hydrocarbons are detected in soil, heavy metal Cd pollution often occurs. Both enter the human body through various means, causing serious health problems for people.

[0003] For the above problems, the current more economical and efficient method is to use microbial agents, but this is prone to the problem of reduced degradation efficiency of strains under heavy metal Cd pollution stress, which in turn leads to the death of the strains and makes it impossible to achieve effective degradation of petroleum hydrocarbons in a cadmium-containing environment. Summary of the invention

[0004] The present invention provides a compounding method and application of a group of cadmium-resistant petroleum hydrocarbon-degrading bacterial flora, which can achieve effective degradation of petroleum hydrocarbons in a cadmium-containing environment.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a compounding method for a cadmium-resistant petroleum hydrocarbon degradation bacterial community, comprising: activating Pseudomonas oleovorans and Pseudomonas monsoni, and preparing Pseudomonas oleovorans liquid and Pseudomonas monsoni liquid, wherein the 16S rRNA sequence of Pseudomonas oleovorans is shown in SEQ ID NO: 1, and the 16S rRNA sequence of Pseudomonas monsoni is shown in SEQ ID NO: 2. The Pseudomonas oleovorans liquid and the Pseudomonas monsoni liquid are inoculated into a petroleum hydrocarbon degradation medium, and the Pseudomonas oleovorans and Pseudomonas monsoni are cultured in the petroleum hydrocarbon degradation medium to obtain cultured Pseudomonas oleovorans and cultured Pseudomonas monsoni. The cultured Pseudomonas oleovorans and the cultured Pseudomonas monsoni are respectively subjected to cadmium resistance acclimation to obtain acclimated Pseudomonas oleovorans liquid and acclimated Pseudomonas monsoni liquid. The cadmium-resistant petroleum hydrocarbon degradation bacterial community is obtained by compounding the acclimated Pseudomonas oleovorans liquid and the acclimated Pseudomonas monsoni liquid.

[0006] In one implementation of the first aspect, activating Pseudomonas oleovorans and Pseudomonas monsoni, and preparing Pseudomonas oleovorans liquid and Pseudomonas monsoni liquid, comprising: inoculating Pseudomonas oleovorans and Pseudomonas monsoni into LB liquid culture medium respectively, and culturing to the logarithmic phase of growth. After performing multiple centrifugation on Pseudomonas oleovorans grown to the logarithmic phase and Pseudomonas monsoni grown to the logarithmic phase, Pseudomonas oleovorans cells and Pseudomonas monsoni cells are collected. The OD values ​​of Pseudomonas oleovorans cells and Pseudomonas monsoni cells are adjusted by MSM liquid culture medium respectively. 600 to 1.0, and obtain Pseudomonas oleovorans liquid and Pseudomonas monterinautica liquid.

[0007] In an implementation of the first aspect, a liquid of Pseudomonas oleovorans and a liquid of Pseudomonas montonii are inoculated into a petroleum hydrocarbon degradation medium, and the petroleum hydrocarbon degradation medium is used to culture the Pseudomonas oleovorans and the Pseudomonas montonii, comprising: adding the liquid of Pseudomonas oleovorans and the liquid of Pseudomonas montonii to the petroleum hydrocarbon degradation medium at a volume ratio of 1:25 to 1:10, and extracting the cultured Pseudomonas oleovorans and the cultured Pseudomonas montonii on the 3rd to 6th day.

[0008] In an implementation of the first aspect, the preparation method of the petroleum hydrocarbon degradation medium is: adding MSM liquid culture medium to petroleum hydrocarbon at a ratio of 50ul:9.95ml to obtain the petroleum hydrocarbon degradation medium.

[0009] In one implementation of the first aspect, the cultured Pseudomonas oleovorans and the cultured Pseudomonas monteri are subjected to cadmium resistance acclimation, including: adjusting the OD of the cultured Pseudomonas oleovorans and the cultured Pseudomonas monteri respectively by using MSM liquid culture medium. 600 to 1.0, and the cultured Pseudomonas oleifera and Pseudomonas monteri were obtained. 0 ml, 0.4 ml, 0.8 ml, 1.2 ml, 1.6 ml and 2 ml of 1 g / L Cd were added to 10 ml of LB liquid medium respectively. 2+ solution, and obtain Cd 2+ The LB liquid medium containing cadmium was prepared with a gradient concentration of 0 mg / L, 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L and 200 mg / L. The cultured Pseudomonas oleovorans and Pseudomonas monteri were inoculated into the Cd solution at a volume ratio of 1:25 to 1:10. 2+ The gradient concentrations of cadmium were 0 mg / L, 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L and 200 mg / L in LB liquid medium. The growth of Pseudomonas oleovorans and Pseudomonas monteri in LB liquid medium containing cadmium was observed, and the cadmium-resistant Pseudomonas oleovorans and Pseudomonas monteri with the best growth were extracted respectively.

[0010] In one implementation of the first aspect, a cadmium-resistant petroleum hydrocarbon-degrading bacterial community is obtained by compounding a tamed Pseudomonas oleifera solution and a tamed Pseudomonas montenosii solution, comprising: adjusting the OD values ​​of the cadmium-resistant Pseudomonas oleifera and the cadmium-resistant Pseudomonas montenosii respectively by using MSM liquid culture medium. 600 The cadmium-resistant Pseudomonas edible oil liquid and the cadmium-resistant Pseudomonas monsoni liquid were obtained. The cadmium-resistant Pseudomonas edible oil liquid and the cadmium-resistant Pseudomonas monsoni liquid were mixed in a volume ratio of 1:0.8 to 1:1.2 to obtain a cadmium-resistant petroleum hydrocarbon-degrading bacterial community.

[0011] In a second aspect, the present invention provides an application of a group of cadmium-resistant petroleum hydrocarbon-degrading bacteria, which is a group of cadmium-resistant petroleum hydrocarbon-degrading bacteria obtained by compounding based on the method provided in the first aspect and is used to degrade petroleum hydrocarbons.

[0012] In an implementation of the second aspect, the cadmium-resistant petroleum hydrocarbon-degrading bacterial community is used to reduce petroleum hydrocarbons in cadmium-contaminated soil and cadmium-contaminated water.

[0013] The beneficial effects of the present invention are: The present invention provides a compounding method for cadmium-resistant petroleum hydrocarbon-degrading bacterial flora. After activating Pseudomonas oleovorans and Pseudomonas monsoni, the two bacteria are respectively subjected to petroleum hydrocarbon degradation experiments and cadmium-resistant acclimation, and the cadmium-resistant Pseudomonas oleovorans and Pseudomonas monsoni with the best growth conditions are screened out, thereby compounding to obtain a cadmium-resistant petroleum hydrocarbon-degrading bacterial flora. Compared with a single bacterium, the bacterial flora can not only realize the effective degradation of petroleum hydrocarbons, but also adapt to the degradation environment containing cadmium, and thus can realize the effective degradation of petroleum hydrocarbons in the cadmium-containing environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a flow chart of a compounding method for a group of cadmium-resistant petroleum hydrocarbon-degrading bacteria provided in an embodiment of the present application; Figure 2 It is a growth curve diagram of Pseudomonas oleovorans and Pseudomonas monterinum provided in the examples of the present application; Figure 3 This is a schematic diagram of the degradation of petroleum hydrocarbons by Pseudomonas oleovorans provided in the examples of the present application; Figure 4 This is a schematic diagram of the degradation of petroleum hydrocarbons by Pseudomonas monteridium provided in the examples of the present application; Figure 5 The cadmium-resistant oil-eating Pseudomonas and cadmium-resistant Pseudomonas monteri provided in the embodiments of the present application are 2+ OD of the cells grown in LB liquid medium containing cadmium for 24 h 600 Schematic diagram of value changes; Figure 6This is a kinetic degradation diagram of petroleum hydrocarbons by a cadmium-resistant petroleum hydrocarbon-degrading bacterial community within seven days provided in the examples of the present application; Figure 7 It is a schematic diagram of the degradation ability of two single bacteria and cadmium-resistant petroleum hydrocarbon-degrading bacteria provided in the examples of the present application on petroleum hydrocarbons in a cadmium-containing environment; Figure 8 This is a kinetic degradation diagram of petroleum hydrocarbons by a cadmium-resistant petroleum hydrocarbon-degrading bacterial community provided in an embodiment of the present application in a cadmium-containing environment within seven days; Fig. 9 Schematic diagram of the effect of the total concentration of petroleum hydrocarbons on the cadmium-resistant petroleum hydrocarbon-degrading bacterial community provided in the examples of the present application; Fig.10 Schematic diagram of the effect of pH value on cadmium-resistant petroleum hydrocarbon-degrading bacteria provided in the examples of the present application; Fig.11 Schematic diagram of the effect of temperature on cadmium-resistant petroleum hydrocarbon-degrading bacteria provided in the examples of the present application; Fig.12 This is a schematic diagram of the effect of salinity on cadmium-resistant petroleum hydrocarbon-degrading bacteria provided in the examples of the present application. DETAILED DESCRIPTION

[0015] In the embodiments of the present application, words such as "exemplary" or "for example" are used to indicate examples, illustrations or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present application should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0016] Since the existing single-bacteria agents cannot effectively degrade petroleum hydrocarbons in an environment contaminated by heavy metal Cd, the embodiments of the present application provide a compounding method and application of a group of cadmium-resistant petroleum hydrocarbon-degrading bacterial communities. After activating Pseudomonas oleovorans and Pseudomonas mondensii in the embodiments of the present application, petroleum hydrocarbon degradation experiments and cadmium-resistant acclimation are carried out on the above two bacteria, respectively, and the cadmium-resistant Pseudomonas oleovorans and cadmium-resistant Pseudomonas mondensii with the best growth conditions are screened out, thereby compounding to obtain a cadmium-resistant petroleum hydrocarbon-degrading bacterial community. Compared with single bacteria, this bacterial community can not only achieve effective degradation of petroleum hydrocarbons, but also adapt to cadmium-containing degradation environments, and thus can achieve effective degradation of petroleum hydrocarbons in cadmium-containing environments.

[0017] like Figure 1 As shown, a compounding method of a group of cadmium-resistant petroleum hydrocarbon-degrading bacteria provided in the embodiments of the present application includes S101-S104; S101, activating Pseudomonas oleovorans and Pseudomonas mongrowingii, and preparing Pseudomonas oleovorans liquid and Pseudomonas mongrowingii liquid, wherein the 16S rRNA sequence of Pseudomonas oleovorans is shown as SEQ ID NO: 1, and the 16S rRNA sequence of Pseudomonas mongrowingii is shown as SEQ ID NO: 2; In the examples of the present application, the 16S rRNA sequence of Pseudomonas oleovorans (i.e., SEQ ID NO: 1) is as follows: The 16S rRNA sequence of Pseudomonas monteri (i.e., SEQ ID NO: 2) is as follows: In one implementation, S101 includes S1011-S1013; S1011, inoculating Pseudomonas oleovorans and Pseudomonas monterinautii into LB liquid culture medium respectively, and culturing to the logarithmic growth phase; S1012, centrifuging the Pseudomonas oleovorans grown to the logarithmic phase and the Pseudomonas mongrowingii grown to the logarithmic phase for multiple times, and collecting the Pseudomonas oleovorans cells and the Pseudomonas mongrowingii cells; S1013, respectively, adjusting the OD of Pseudomonas oleovorans and Pseudomonas monterinautica cells by MSM liquid culture medium 600 to 1.0, to obtain Pseudomonas oleovorans liquid and Pseudomonas monsecchii liquid; Specifically, Pseudomonas oleovorans and Pseudomonas monteri stored in cryopreservation tubes were streaked onto LB solid medium using an inoculation loop, and cultured in a 30°C constant temperature incubator until a single colony grew; Then pick a single colony on the LB solid medium and activate it in the LB liquid medium. Set the culture conditions as follows: temperature 30°C, rotation speed 150rpm, initial pH value 7.0, and shade throughout the process; take samples regularly to measure the OD of the bacterial solution. 600 value until the logarithmic growth phase turns into the decline phase (such as Figure 2 As shown), determine the time when the colony grows to the logarithmic growth phase; After obtaining the time when the colonies of Pseudomonas oleovorans and Pseudomonas monsoni grow to the logarithmic growth phase, centrifuge the Pseudomonas oleovorans and Pseudomonas monsoni that have grown to the logarithmic growth phase at 7500-8000 r / min for 5-10 min, remove the supernatant, add an equal amount of MSM culture medium, resuspend, and centrifuge again at 7500-8000 r / min for 5-10 min; repeat the above steps 2-3 times, collect the last bacterial cells and adjust the OD with MSM liquid culture medium 600 =1.0 to obtain Pseudomonas oleovorans liquid and Pseudomonas monteridium liquid; The formula of LB liquid medium is as follows: 5.0 g yeast extract; 10.0 g tryptone; 10.0 g sodium chloride (NaCl); add ultrapure water to 1 L, adjust pH to 7.0, and sterilize at 121°C for 20 minutes; LB solid medium is based on LB liquid medium and 1.5% (W / V) agar powder is added; The formula of MSM liquid culture medium (also known as inorganic salt culture medium) is: (NH 4 ) 2 SO 4 :1.5 g / L; KH 2 PO 4:0.5 g / L; K 2 HPO 4 ·3H 2 O: 1.91 g / L; NaCl: 0.5 g / L; MgSO 4 7H 2 O: 0.2 g / L; adjust the final pH of the inorganic salt culture medium to 7.0, and add 1.5% (W / V) agar powder to the solid inorganic salt culture medium; S102, inoculating the oleovorable Pseudomonas liquid and the Pseudomonas montonii liquid into a petroleum hydrocarbon degradation medium, and culturing the oleovorable Pseudomonas and the Pseudomonas montonii using the petroleum hydrocarbon degradation medium to obtain cultured Pseudomonas oleovorable and cultured Pseudomonas montonii; It can be understood that in the embodiments of the present application, the petroleum hydrocarbon degradation medium refers to an inorganic salt medium containing petroleum hydrocarbons; Specifically, the oleovorable Pseudomonas solution and the Pseudomonas monteri solution are added to the petroleum hydrocarbon degradation medium at a volume ratio of 1:25 to 1:10, and the cultured Pseudomonas oleovorable and the cultured Pseudomonas monteri are extracted on the 3rd to 6th day; The above extraction method is as follows: streak the bacterial liquid cultured in petroleum hydrocarbon medium for 3-6 days on a solid inorganic salt medium with petroleum hydrocarbon as the sole carbon source, and culture in a constant temperature incubator at 30°C until a single colony grows; The obtained single colony was functionally verified, and the solid inorganic salt culture medium was sent for sequencing. The 16S RNA was compared and found to be consistent with the sequence of the preserved Pseudomonas oleovorans / Pseudomonas mongrowingii, indicating that Pseudomonas oleovorans / Pseudomonas mongrowingii was extracted; In one implementation, the preparation method of the petroleum hydrocarbon degradation medium is: adding MSM liquid culture medium to petroleum hydrocarbon at a ratio of 50ul:9.95ml to obtain a petroleum hydrocarbon degradation medium; Specifically, the petroleum hydrocarbon standard liquid is C 10 -C 40 n-Alkanes mixed standard solution, including 31 pollutants (C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C26 , C 27 , C 28 , C 29 , C 30 , C 31 , C 32 , C 33 , C 34 , C 35 , C 36 , C 37 , C 38 , C 39 and C 40 ); the C 10 -C 40 The normal alkane mixed standard solution was commercially available with a concentration of 1000 mg / L. In the process of preparing the petroleum hydrocarbon degradation medium, C 10 -C 40 The concentration of each pollutant in the normal alkane mixed standard solution is adjusted to 5 mg / L (for example, 5 PPM of C 10 -C 40 The normal alkane mixed standard solution was added to 10 ml of MSM liquid culture medium to obtain a C-1000 medium with a concentration of 5 mg / L for each pollutant and a final concentration of 155 mg / L for total petroleum hydrocarbons. 10 -C 40 Normal alkane mixed standard solution), since the solvent of the normal alkane mixed standard solution is methanol, in order to avoid the influence of methanol on the strain, after the methanol evaporates, MSM liquid culture medium is added to the petroleum hydrocarbon standard solution at a ratio of 50ul:9.95ml to obtain a petroleum hydrocarbon degradation culture medium; Specifically, the edible Pseudomonas oleaginus liquid and the Pseudomonas montenosii liquid obtained by S101 were added to the sterilized and prepared petroleum hydrocarbon degradation medium, and the addition amount of the edible Pseudomonas oleaginus liquid and the Pseudomonas montenosii liquid was 5% of the system. At the same time, a petroleum hydrocarbon degradation medium without adding bacterial suspension was set as a control; three parallels were set for each group, and shaking culture was carried out at a constant temperature of 30°C and 150 rpm under light-proof conditions for 7 days, and samples were taken regularly every day to determine the residual concentration of petroleum hydrocarbons in the culture medium; wherein, the degradation system (i.e., petroleum hydrocarbon degradation medium) was 10 ml, the substrate (i.e., petroleum hydrocarbon) concentration was 155 mg / L, the temperature was 30°C, the pH was 7.0, and no additional salt was added; The extraction method of petroleum hydrocarbons in the petroleum hydrocarbon degradation medium is as follows: add 4 ml of n-hexane extractant to a 10 ml conical flask, shake for 2 min and then stand for 10 min until the two phases separate, collect the upper organic phase and wash the separatory funnel with 5 ml of extractant, combine the washing liquid and the organic phase and add sufficient anhydrous sodium sulfate for dehydration; transfer the dehydrated extract into a 100 ml rotary evaporation bottle, wash the conical flask with 5 ml of extractant and transfer them into a 100 ml rotary evaporation bottle together, and concentrate to near dryness under the conditions of a water bath temperature of 35°C and a vacuum degree of 750 hPa, make the volume to 2 ml with n-hexane, and filter through a 0.22µm organic filter membrane for testing; The degradation process of petroleum hydrocarbons by Pseudomonas oleovorans Figure 3 As shown, the degradation rate of petroleum hydrocarbons by Pseudomonas montenosii was 6.07% on the 3rd day and reached 17% on the 7th day; the degradation rate of low and medium molecular weight petroleum hydrocarbons by the bacterial community was 7.4% on the 3rd day and as high as 23.8% on the 7th day; the degradation rate of high molecular weight petroleum hydrocarbons was only 3.8% on the 3rd day and increased to 6.25% on the 7th day; Pseudomonas montenosii results for the degradation of petroleum hydrocarbons such as Figure 4 As shown, the degradation rate of petroleum hydrocarbons by Pseudomonas oleovorans was 23% on the 3rd day and reached 44% on the 7th day; the degradation rate of low and medium molecular weight petroleum hydrocarbons by the bacteria was 38% on the 3rd day and as high as 69% on the 7th day; the degradation rate of high molecular weight petroleum hydrocarbons was only 2.5% on the 3rd day and increased to 12.6% on the 7th day; S103, respectively acclimating the cultured Pseudomonas oleovorans and the cultured Pseudomonas monteri to cadmium resistance, to obtain an acclimated Pseudomonas oleovorans liquid and an acclimated Pseudomonas monteri liquid; In one implementation, S103 includes S1031-S1034; S1031, respectively, using MSM liquid medium to adjust the OD of cultured Pseudomonas oleovorans and cultured Pseudomonas monteri 600 to 1.0, to obtain a cultured Pseudomonas oleovorans liquid and a cultured Pseudomonas monteridium liquid; S1032, add 0 ml, 0.4 ml, 0.8 ml, 1.2 ml, 1.6 ml and 2 ml of 1 g / L Cd to 10 ml of LB liquid medium respectively. 2+ solution, and obtain Cd 2+ LB liquid medium containing cadmium at gradient concentrations of 0 mg / L, 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L and 200 mg / L; S1033, the cultured Pseudomonas oleifera solution and the cultured Pseudomonas monteridium solution were inoculated into Cd 2+ In LB liquid medium containing cadmium at gradient concentrations of 0 mg / L, 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L and 200 mg / L; S1034, observing the growth of Pseudomonas oleovorans and Pseudomonas monnieri in LB liquid culture medium containing cadmium, and extracting the best growing Pseudomonas oleovorans and Pseudomonas monnieri; Specifically, 0.4 ml, 0.8 ml, 1.2 ml, 1.6 ml, and 2 ml of CdCl were added to 10 ml of LB liquid medium. 2 The concentration of Cd was 1g / L 2+ The solution was prepared so that the Cd ion gradient concentration in the cadmium-containing LB liquid medium was 0 mg / L, 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L, and 200 mg / L; Pseudomonas oleovorans and Pseudomonas monterinia cultured in petroleum hydrocarbon degradation medium were inoculated into LB liquid medium and the OD was obtained in the same way as S1-3. 600 =1.0, added to LB liquid medium containing cadmium at a rate of 5%, with 3 replicates per group; replaced the bacterial suspension with 5% sterile water as a control; cultured in a shaker at 30°C and 150rpm for 1 day; taken out on the second day, destructively sampled, and measured the OD value at 600nm with a UV spectrophotometer; The results are as follows Figure 5 It can be seen that when Cd in LB liquid medium containing cadmium 2+ Concentration 0~160 mg·L -1 Pseudomonas monteri can grow normally, and then with the increase of Cd 2+ With the increase of Cd concentration, the growth of the strain decreased significantly. 2+ The concentration reaches 200 mg·L -1 When the strain was not grown, it showed that the strain was sensitive to Cd 2+ The maximum tolerable concentration is between 120 mg·L -1 ~160 mg·L -1 For Pseudomonas oleovorans, when Cd 2+ Concentration ranges from 0 to 80 mg·L -1 The strain can grow normally, and then with the increase of Cd 2+ With the increase of Cd concentration, the growth of the strain decreased significantly. 2+ The concentration reached 120 mg·L-1 When the strain was not grown, it showed that it was sensitive to Cd 2+ The maximum tolerable concentration is between 40 mg·L -1 ~80 mg·L -1 between; S104, obtaining a cadmium-resistant petroleum hydrocarbon-degrading bacterial community by compounding the domesticated Pseudomonas oleifera solution and the domesticated Pseudomonas monteridium solution; Optionally, S104 includes S1041-S1042; S1041, OD regulation of cadmium-resistant Pseudomonas oleifera and cadmium-resistant Pseudomonas monteri respectively by MSM liquid medium 600 to 1.0, to obtain cadmium-resistant oil-eating Pseudomonas solution and cadmium-resistant Pseudomonas monteridium solution; S1042. Mix the cadmium-resistant oil-eating Pseudomonas solution and the cadmium-resistant Pseudomonas monteridium solution in a volume ratio of 1:0.8 to 1:1.2 to obtain a cadmium-resistant petroleum hydrocarbon-degrading bacterial community.

[0018] In one implementation, the treated bacterial solution is placed in LB liquid culture medium again for culture, and the OD is obtained by referring to the operation of S1011 above. 600 =1.0, and the two strains were mixed in a volume ratio of 1:1 to obtain a cadmium-resistant petroleum hydrocarbon-degrading bacterial community (also known as a cadmium-resistant petroleum hydrocarbon-degrading bacterial liquid) with the functions of degrading petroleum hydrocarbons and being resistant to Cd.

[0019] The cadmium-resistant petroleum hydrocarbon degradation bacterial liquid was added to the petroleum hydrocarbon degradation medium, wherein the amount of cadmium-resistant petroleum hydrocarbon degradation bacterial liquid added was 5% of the system. At the same time, the petroleum hydrocarbon degradation medium without adding bacterial suspension was set as the control. Three parallels were set for each group, and the shaking culture was cultured at a constant temperature of 30°C and 150 rpm under light-proof conditions for 7 days. Samples were taken regularly every day to measure the residual concentration of petroleum hydrocarbons in the culture medium.

[0020] The results are as follows Figure 6 As shown, the constructed cadmium-resistant petroleum hydrocarbon-degrading bacterial community has a good degradation ability for petroleum hydrocarbons. The degradation rate of petroleum hydrocarbons by the cadmium-resistant petroleum hydrocarbon-degrading bacterial community was 44.69% on the 3rd day, and it could reach 53% on the 7th day. The degradation rate of low and medium molecular weight petroleum hydrocarbons by the cadmium-resistant petroleum hydrocarbon-degrading bacterial community could reach 68.33% on the 3rd day, and 84% on the 7th day; the degradation rate of high molecular weight petroleum hydrocarbons by the cadmium-resistant petroleum hydrocarbon-degrading bacterial community was only 13.80% on the 3rd day, but it increased to 24.89% on the 7th day. It can be seen that the cadmium-resistant petroleum hydrocarbon-degrading bacterial community provided in the embodiment of the present application can achieve a relatively high efficiency of degradation of petroleum hydrocarbons in a cadmium-containing environment within one week.

[0021] Through the above Figure 6 It can be seen that relative to Figure 3 , Figure 4The schematic diagram of the degradation ability of a single bacterium shows that the degradation rate of petroleum hydrocarbons by the cadmium-resistant petroleum hydrocarbon-degrading bacterial community was significantly improved after 7 days.

[0022] Furthermore, in order to verify the effect of cadmium-resistant petroleum hydrocarbon degradation bacteria in a cadmium-containing environment, the present application example conducted a petroleum hydrocarbon degradation experiment on the cadmium-resistant petroleum hydrocarbon degradation bacteria in an environment containing both cadmium and petroleum hydrocarbons, and detected the degradation rate of petroleum hydrocarbons on days 1, 3, 5, and 7. Two single bacteria (i.e., activated oil-eating Pseudomonas and Pseudomonas monsoni) were subjected to a petroleum hydrocarbon degradation experiment in the same environment, and the degradation rate of petroleum hydrocarbons was detected on the 7th day, thereby exploring the difference in the degradation efficiency of petroleum hydrocarbons between the cadmium-resistant petroleum hydrocarbon degradation bacteria and the two single bacteria in an environment containing both cadmium and petroleum hydrocarbons.

[0023] Specifically, for the cadmium-resistant petroleum hydrocarbon degradation bacterial community, the cadmium-resistant petroleum hydrocarbon degradation bacterial liquid obtained by S104 was added to the petroleum hydrocarbon degradation medium in which the cadmium ion concentration in the culture medium was 20 mg / L, wherein the amount of cadmium-resistant petroleum hydrocarbon degradation bacterial liquid added was 5% of the system, and at the same time, the petroleum hydrocarbon degradation medium without adding bacterial suspension was set as a control. Three parallels were set for each group, and the shaking culture was carried out at a constant temperature of 30°C and 150 rpm under light-proof conditions for 7 days. The residual concentration of petroleum hydrocarbons in the culture medium was measured at 1, 3, 5, and 7, and then the degradation rate of petroleum hydrocarbons was calculated.

[0024] For two single bacteria (i.e., activated Pseudomonas oleovorans and Pseudomonas monteri), activated Pseudomonas oleovorans / Pseudomonas monteri were added to the petroleum hydrocarbon degradation medium with a cadmium ion concentration of 20 mg / L in the culture medium, and the addition amount of the two single-resistant bacteria was also 5% of the system. At the same time, the petroleum hydrocarbon degradation medium without adding single bacteria was set as the control. Three parallels were set for each group, and the shaking culture was cultured under constant temperature of 30°C and 150 rpm under light-proof conditions for 7 days. On the 7th day, samples were taken to determine the residual concentration of petroleum hydrocarbons in the culture medium, and then the degradation rate of petroleum hydrocarbons was calculated.

[0025] The preparation method of the petroleum hydrocarbon degradation medium in the above process is as follows: add 0.2 ml of CdCl 2 The concentration of Cd was 1g / L 2+ solution so that the cadmium ion concentration in the culture medium is 20 mg / L.

[0026] The results of the above process are as follows Figure 7 and Figure 8 As shown. Figure 8It can be seen that the constructed cadmium-resistant petroleum hydrocarbon-degrading bacterial community has a good degradation ability for petroleum hydrocarbons in the cadmium and petroleum hydrocarbon composite degradation medium. The degradation rate of petroleum hydrocarbons by the cadmium-resistant petroleum hydrocarbon-degrading bacterial community was 14% on the 3rd day, and it could reach 37% on the 7th day. The degradation rate of medium and low molecular weight petroleum hydrocarbons by the cadmium-resistant petroleum hydrocarbon-degrading bacterial community could reach 19.94% on the 3rd day, and 54.7% on the 7th day; the degradation rate of high molecular weight petroleum hydrocarbons by the cadmium-resistant petroleum hydrocarbon-degrading bacterial community was only 14.9% on the 3rd day, and it increased to 6.3% on the 7th day.

[0027] Although Pseudomonas monteridium 2+ The maximum tolerable concentration is between 120 mg·L -1 ~160 mg·L -1 Pseudomonas oleovorans 2+ The maximum tolerable concentration is between 0 and 80 mg·L -1 However, when 20 mg / L of cadmium ions were added, the degradation efficiency of the composite bacterial community on petroleum hydrocarbons was slightly lower than when no cadmium ions were added. The reason may be that when the strain was acclimated to cadmium resistance, it was grown in LB liquid culture medium. LB liquid culture medium has sufficient nutrients and a stable environment, while the above-mentioned petroleum hydrocarbon degradation culture medium only has petroleum hydrocarbons as the only carbon source. The growth conditions of the strain are harsh and the environment is relatively harsh, which leads to a decrease in its degradation efficiency. However, compared with Figure 7 Compared with the single bacteria degradation ability shown in the figure, the degradation rate of petroleum hydrocarbons by the cadmium-resistant petroleum hydrocarbon-degrading bacteria group was still significantly improved after 7 days compared with the single bacteria.

[0028] In summary, in the compounding method of a group of cadmium-resistant petroleum hydrocarbon-degrading bacterial flora provided in the embodiment of the present application, after activating Pseudomonas oleovorans and Pseudomonas monsoni, the above two bacteria are respectively subjected to petroleum hydrocarbon degradation experiments and cadmium-resistant acclimation, so as to obtain a cadmium-resistant petroleum hydrocarbon-degrading bacterial flora by compounding. Compared with a single bacterium, this bacterial flora can not only achieve effective degradation of petroleum hydrocarbons, but also adapt to the degradation environment containing cadmium, and thus can achieve effective degradation of petroleum hydrocarbons in a cadmium-containing environment. In addition, the degradation conditions of S104 to obtain cadmium-resistant petroleum hydrocarbon-degrading bacteria can be optimized. The detailed optimization process is as follows: 1. Optimization of total petroleum hydrocarbon concentration conditions Add petroleum hydrocarbon mixed standard solution to the conical flask, and adjust the OD of the cultured cadmium-resistant petroleum hydrocarbon-degrading bacteria with MSM medium. 600=1.0, at pH=7.0, inoculation size 5%, temperature 30℃, the initial concentration of petroleum hydrocarbons in the culture medium was set to 62mg / L, 86mg / L, 248mg / L, 310mg / L, 465mg / L respectively. After culturing for 7 days, the extracts were taken out and the extraction method was referred to the extraction method of petroleum hydrocarbons in the above petroleum hydrocarbon degradation culture medium. After passing through a 0.22 µm organic filter membrane, the samples were determined by gas chromatography. By comparison, (e.g. Fig. 9 The total degradation rates of petroleum hydrocarbons by the bacterial community under the above concentrations were 46.61%, 49.89%, 53.41%, 48.34% and 45.49%, respectively. The results showed that when the total concentration of petroleum hydrocarbons was 248 mg / L, the degradation effect of the bacterial community on petroleum hydrocarbons was the best.

[0029] 2. Optimization of temperature conditions Add petroleum hydrocarbon mixed standard solution to the conical flask, and adjust the OD of the cultured cadmium-resistant petroleum hydrocarbon-degrading bacteria with MSM medium. 600 =1.0, at a concentration of 248 mg / L and an inoculation volume of 5%, the conical flask was placed in a constant temperature oscillator at 20°C, 25°C, 30°C, 37°C, and 42°C for 7 days, then taken out. The extraction method was based on the extraction method of petroleum hydrocarbons in the above petroleum hydrocarbon degradation culture medium. After passing through a 0.22 µm organic filter membrane, the gas chromatography was used for determination. By comparison, it can be seen that (such as Fig.10 The total degradation rates of petroleum hydrocarbons by the bacterial community under the above temperature conditions were 30.41%, 38.47%, 55.35%, 46.66% and 22.47%, respectively. The results showed that the degradation effect of the bacterial community on petroleum hydrocarbons was best under the temperature of 30°C.

[0030] 3. Optimization of pH value Add petroleum hydrocarbon mixed standard solution to the conical flask, and adjust the OD of the cultured cadmium-resistant petroleum hydrocarbon-degrading bacteria with MSM medium. 600 =1.0, added to MSM medium with pH values ​​of 5.0, 6.0, 7.0, 8.0, and 9.0 respectively at a concentration of 248 mg / L, an inoculation amount of 5%, and a temperature of 30°C, and cultured for 7 days before being taken out. The extraction method was based on the extraction method of petroleum hydrocarbons in the above-mentioned petroleum hydrocarbon degradation medium, and the samples were filtered through a 0.22 µm organic filter membrane and then subjected to gas chromatography for determination. By comparison, it can be seen that (e.g. Fig.11 The total degradation rates of petroleum hydrocarbons by the bacterial community under the above pH conditions were 14.72%, 31.06%, 52.19%, 45.4% and 44.17%, respectively. The results showed that the degradation effect of the bacterial community on petroleum hydrocarbons was best under the condition of pH 7.0.

[0031] 4. Optimization of salinity conditions Add petroleum hydrocarbon mixed standard solution to the conical flask, and adjust the OD of the cultured cadmium-resistant petroleum hydrocarbon-degrading bacteria with MSM medium. 600 =1.0, at a concentration of 248 mg / L, an inoculum size of 5%, and a pH value of 7.0. At a temperature of 30°C, 0, 5, 10, 15, and 20 g / L NaCl were added to the MSM medium. After 7 days of cultivation, the medium was taken out and the extraction method was referred to the extraction method of petroleum hydrocarbons in the above petroleum hydrocarbon degradation medium. After passing through a 0.22 µm organic filter membrane, the gas chromatography was used for determination. By comparison, it can be seen that (such as Fig.12 The total degradation rates of petroleum hydrocarbons by the bacterial consortium under the above salinity conditions were 45.61%, 53.65%, 61.54%, 53.02% and 46.1%, respectively. The results showed that the degradation effect of the bacterial consortium on petroleum hydrocarbons was the best when 10 g / L NaCl was additionally added.

[0032] In summary, the cadmium-resistant petroleum hydrocarbon-degrading bacteria have good tolerance to cadmium. 2+ The maximum tolerable concentration is between 120 mg·L -1 ~160 mg·L -1 between; Pseudomonas oleovorans 2+ The maximum tolerable concentration is between 0mg· L-1 ~80 mg·L -1 Compared with the degradation ability of single bacteria, the degradation rate of petroleum hydrocarbons was 54% after the two strains were combined 7 days later. Single factor experiments were carried out on the environmental factors affecting the functional bacteria, and the optimal environmental conditions for the degradation of petroleum hydrocarbons by the bacteria were determined to be: external salinity of 10 g / L, pH value = 7, temperature of 30°C, and petroleum hydrocarbon concentration of 248 mg / L.

[0033] The present application also provides an application of a group of cadmium-resistant petroleum hydrocarbon-degrading bacterial flora, which is used to degrade petroleum hydrocarbons based on the above-mentioned cadmium-resistant petroleum hydrocarbon-degrading bacterial flora.

[0034] In one implementation, the cadmium-resistant petroleum hydrocarbon-degrading bacterial community is added to wastewater or soil containing petroleum hydrocarbons to degrade the petroleum hydrocarbons.

[0035] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0036] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A compounding method for a group of cadmium-resistant petroleum hydrocarbon-degrading bacteria. It is characterized in that include: activating Pseudomonas oleovorans and Pseudomonas mongrowingii, and preparing Pseudomonas oleovorans liquid and Pseudomonas mongrowingii liquid, wherein the 16S rRNA sequence of the Pseudomonas oleovorans is shown as SEQ ID NO: 1, and the 16S rRNA sequence of the Pseudomonas mongrowingii is shown as SEQ ID NO: 2; inoculating the oleovorable Pseudomonas liquid and the Pseudomonas mongrowingii liquid into a petroleum hydrocarbon degradation medium, and culturing the oleovorable Pseudomonas and the Pseudomonas mongrowingii using the petroleum hydrocarbon degradation medium to obtain cultured Pseudomonas oleovorable Pseudomonas and cultured Pseudomonas mongrowingii; Respectively performing cadmium resistance acclimation on the cultured Pseudomonas oleovorans and the cultured Pseudomonas monteri, to obtain an acclimated Pseudomonas oleovorans liquid and an acclimated Pseudomonas monteri liquid; The cadmium-resistant petroleum hydrocarbon-degrading bacterial community was obtained by compounding the domesticated Pseudomonas oleivorans solution and the domesticated Pseudomonas monteridium solution.

2. The method according to claim 1, It is characterized in that The method of activating Pseudomonas oleovorans and Pseudomonas mongrowingii and preparing Pseudomonas oleovorans liquid and Pseudomonas mongrowingii liquid comprises: The oleovorable Pseudomonas and the monterodendron Pseudomonas are inoculated into LB liquid culture medium respectively, and cultured to the logarithmic growth phase; After centrifuging the Pseudomonas oleovorans grown to the logarithmic phase and the Pseudomonas montereyii grown to the logarithmic phase for multiple times, the Pseudomonas oleovorans cells and the Pseudomonas montereyii cells were collected; The OD values ​​of the Pseudomonas oleovorans cells and the Pseudomonas monteridium cells are respectively adjusted by using MSM liquid culture medium. 600 to 1.0, to obtain the Pseudomonas oleovorans liquid and the Pseudomonas monteroni liquid.

3. The method according to claim 1, It is characterized in that The oleovorable Pseudomonas liquid and the Pseudomonas monteri liquid are inoculated into a petroleum hydrocarbon degradation medium, and the oleovorable Pseudomonas liquid and the Pseudomonas monteri are cultured using the petroleum hydrocarbon degradation medium, comprising: The Pseudomonas oleovorans liquid and the Pseudomonas montereyii liquid are added to a petroleum hydrocarbon degradation culture medium at a volume ratio of 1:25 to 1:10, and the cultured Pseudomonas oleovorans and the cultured Pseudomonas montereyii are extracted on the 3rd to 6th day.

4. The method according to claim 1, It is characterized in that The preparation method of the petroleum hydrocarbon degradation culture medium is: MSM liquid culture medium was added to petroleum hydrocarbon at a ratio of 50ul:9.95ml to obtain the petroleum hydrocarbon degradation culture medium.

5. The method according to claim 1, It is characterized in that The cultured Pseudomonas oleovorans and Pseudomonas monteri are respectively subjected to cadmium resistance acclimation, comprising: The OD values ​​of the cultured Pseudomonas oleovorans and the cultured Pseudomonas monteri respectively were adjusted by using MSM liquid culture medium. 600 to 1.0, to obtain a cultured Pseudomonas oleovorans liquid and a cultured Pseudomonas monteridium liquid; Add 0 ml, 0.4 ml, 0.8 ml, 1.2 ml, 1.6 ml and 2 ml of 1 g / L Cd to 10 ml of LB liquid medium respectively. 2+ solution, and obtain Cd 2+ LB liquid medium containing cadmium at gradient concentrations of 0 mg / L, 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L and 200 mg / L; Then, the cultured Pseudomonas oleifera solution and the cultured Pseudomonas monteridium solution were inoculated into the Cd 2+ In LB liquid medium containing cadmium at gradient concentrations of 0 mg / L, 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L and 200 mg / L; The growth of Pseudomonas oleovorans and Pseudomonas monnieri in LB liquid culture medium containing cadmium was observed, and the cadmium-resistant Pseudomonas oleovorans and Pseudomonas monnieri with the best growth were extracted respectively.

6. The method according to claim 5, It is characterized in that The cadmium-resistant petroleum hydrocarbon-degrading bacterial community is obtained by compounding the domesticated Pseudomonas oleifera solution and the domesticated Pseudomonas monteridium solution, including: The OD values ​​of the cadmium-resistant oil-eating Pseudomonas and the cadmium-resistant Pseudomonas monteri respectively were adjusted by using MSM liquid culture medium. 600 to 1.0, to obtain cadmium-resistant oil-eating Pseudomonas solution and cadmium-resistant Pseudomonas monteridium solution; The cadmium-resistant oil-eating Pseudomonas liquid and the cadmium-resistant Pseudomonas monteritus liquid are mixed in a volume ratio of 1:0.8 to 1:1.2 to obtain a cadmium-resistant petroleum hydrocarbon-degrading bacterial community.

7. Use of a group of cadmium-resistant petroleum hydrocarbon-degrading bacteria obtained by compounding according to any one of claims 1 to 6, It is characterized in that Used to degrade petroleum hydrocarbons.

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