阅读说明：本技术 一种复合菌剂及其对芘-重金属复合污染环境的修复 (Complex microbial inoculant and restoration of pyrene-heavy metal complex polluted environment by complex microbial inoculant ) 是由 刘其友 苏玉华 孙烁 赵朝成 王志伟 于 2021-04-25 设计创作，主要内容包括：本发明属于环境中多环芳烃-重金属复合污染环境生物修复技术领域,具体涉及一种复合菌剂、菌剂的制备方法及其在多环芳烃芘-重金属复合污染环境领域的应用。本发明提供了2株具有重金属和多环芳烃芘修复能力的菌株,1株是假单胞菌(Pseudomonas sp.)YH-1,1株是红球菌(Rhodococcus sp.)YH-3,均从胜利油田长期受石油污染的土壤中筛选而得。本发明所述复合菌剂具有芘降解能力及耐重金属Cr的特性,通过2株菌之间的协同作用,提高多环芳烃芘的降解率的同时,可以吸附转化重金属Cr。2株菌制备成的复合菌剂在水体及土壤多环芳烃-重金属复合污染环境的生物修复方面具有应用前景。(The invention belongs to the technical field of bioremediation of polycyclic aromatic hydrocarbon-heavy metal combined polluted environment in the environment, and particularly relates to a compound microbial inoculum, a preparation method of the microbial inoculum and application of the compound microbial inoculum in the field of polycyclic aromatic hydrocarbon pyrene-heavy metal combined polluted environment. The invention provides 2 strains with heavy metal and polycyclic aromatic hydrocarbon pyrene repairing capability, wherein 1 strain is Pseudomonas sp YH-1, 1 strain is Rhodococcus sp YH-3, and the strains are screened from soil polluted by petroleum in a Shengli oil field for a long time. The composite microbial inoculum has the characteristics of pyrene degradation capability and heavy metal Cr resistance, and can adsorb and convert heavy metal Cr while improving the degradation rate of polycyclic aromatic hydrocarbon pyrene through the synergistic effect of 2 strains of bacteria. The compound microbial inoculum prepared from 2 strains has application prospect in the aspect of bioremediation of polycyclic aromatic hydrocarbon-heavy metal compound polluted environment of water and soil.)

1. The composite microbial inoculum and the restoration of heavy metal-pyrene composite polluted environment are characterized in that the composite microbial inoculum is compounded by Pseudomonas (Pseudomonas sp.) YH-1 and Rhodococcus (Rhodococcus sp.) YH-3, and the strains YH-1 and YH-3 are both preserved in China center for type culture Collection with the addresses: in Wuhan, Wuhan university, the preservation number of the strain YH-1 is M2021160, and the preservation number of the strain YH-3 is M2021161.

2. The complex microbial agent of claim 1, wherein OD of Pseudomonas YH-1 and Rhodococcus YH-3 is₆₀₀The values are all 0.8-1.0, and the compounding volume ratio is 1: 1.

3. The complex microbial inoculum according to claim 2, wherein the preparation method of the complex microbial inoculum comprises the following steps: the pseudomonas YH-1 and the rhodococcus YH-3 of claim 2 are respectively inoculated in a liquid LB culture medium, cultured for 8-10 h at 30 ℃, mixed YH-1 and YH-3 according to a ratio of 1:1(V: V), cultured for 5-8 h at 30 ℃ to obtain a mixed fermentation broth of the compound bacteria, the mixed fermentation broth is centrifuged, supernatant is removed, the mixed fermentation broth is washed for 3 times by phosphate buffer solution, bacteria are collected, and finally the collected bacteria are pre-frozen in a refrigerator at-20 ℃ overnight and then subjected to vacuum freeze drying to obtain the compound bacteria agent.

4. The application of the complex microbial inoculum according to claim 3, which is used for bioremediation of polycyclic aromatic hydrocarbon and heavy metal complex polluted environment, and especially has wide application prospect in bioremediation of polycyclic aromatic hydrocarbon-heavy metal complex polluted environment of water and soil.

Technical Field

The invention belongs to the technical field of bioremediation of polycyclic aromatic hydrocarbon-heavy metal composite polluted environment in environment, and relates to a composite microbial agent consisting of Pseudomonas (Pseudomonas sp.) YH-1 and Rhodococcus (Rhodococcus sp.) YH-3, in particular to a preparation method of the composite microbial agent and application thereof in the field of bioremediation of polycyclic aromatic hydrocarbon pyrene-heavy metal composite polluted environment.

Background

With the continuous acceleration of the industrialized development process, the environmental pollution problem becomes more complex and serious. Polycyclic aromatic hydrocarbon and heavy metal are two typical persistent pollutants in the environment, often exist in the environment at the same time, and interact to form composite pollution, which causes serious influence on the ecological environment and also constitutes great harm to human health.

In the polluted environment caused by natural processes such as volcanic eruption, forest fire and the like and artificial activities such as wastewater irrigation, petroleum or coal production and processing and the like, polycyclic aromatic hydrocarbons and some heavy metals such as lead, copper, chromium and the like can be detected at the same time. The interaction between the polycyclic aromatic hydrocarbon and the heavy metal in the composite polluted environment increases the difficulty of restoration, so that the requirement of restoration of the polluted environment cannot be met only by restoring a single pollutant.

The restoration method of polycyclic aromatic hydrocarbon and heavy metal mainly comprises a physical method, a chemical method and a biological method. Physical and chemical repair methods have the disadvantages of high repair cost, easy generation of secondary pollution and the like. In recent years, bioremediation technology has become a hotspot of environmental field research due to the advantages of environmental friendliness, strong operability, low cost and the like. Microorganisms are abundant and sustainable resources in the environment, and have great potential in the aspect of environmental pollution remediation, and many microorganisms can degrade polycyclic aromatic hydrocarbons or have heavy metal resistance, however, at present, few reports are made on microorganisms capable of remedying the polycyclic aromatic hydrocarbons-heavy metal combined polluted environment, and one strain can only degrade one pollutant generally, and has poor environmental adaptability and undesirable degradation effect. Therefore, for the remediation of the polycyclic aromatic hydrocarbon-heavy metal combined polluted environment, the remediation effect of a single strain is not ideal or can not be realized.

Therefore, the research of the compound microbial agent with low cost, environmental friendliness and high efficiency has important significance for the remediation of the polycyclic aromatic hydrocarbon-heavy metal compound polluted environment.

Disclosure of Invention

Aiming at the research background, the invention provides a compound microbial agent which is composed of Pseudomonas (Pseudomonas sp.) YH-1 and Rhodococcus (Rhodococcus sp.) YH-3, has the characteristics of degrading pyrene and resisting heavy metal chromium, and can be well applied to the restoration of heavy metal-polycyclic aromatic hydrocarbon compound polluted environment.

The invention adopts the following technical scheme:

the composite microbial inoculum provided by the invention is compounded by Pseudomonas YH-1(Pseudomonas sp.YH-1) and Rhodococcus YH-3(Rhodococcus sp.YH-3), wherein 2 strains of bacteria are all preserved in China center for type culture Collection (address: China, Wuhan university), and the preservation numbers are respectively as follows: CCTCC NO: M2021160; CCTCC NO: M2021161, preservation date is 2021 year, 1 month and 27 days.

The two strains YH-1(Pseudomonas sp.) and YH-3(Rhodococcus sp.) are obtained by screening from soil polluted by petroleum in the Shengli oil field for a long time, and researches show that the compound bacteria can effectively improve the degradation rate of polycyclic aromatic hydrocarbon pyrene and have good resistance and adsorption conversion effect on heavy metal chromium.

The preparation method of the compound microbial inoculum comprises the following steps:

YH-1 and YH-3 with the inoculum size of 1% are respectively and independently inoculated into a liquid LB culture medium, the liquid LB culture medium is subjected to shake culture at 30 ℃ and 160rpm for 8-10 h, YH-1 and YH-3 are mixed according to the ratio of 1:1(V: V), then the mixed fermentation liquid is cultured at 30 ℃ and 160rpm for 5-8 h to obtain a compound bacterium mixed fermentation liquid, the mixed fermentation liquid is centrifuged, supernatant is removed, the mixed fermentation liquid is washed for 3 times by phosphate buffer solution, the bacterium is collected, and finally the collected bacterium is pre-frozen in a refrigerator at-20 ℃ overnight and then subjected to vacuum freeze drying to obtain the compound bacterium agent.

The liquid LB culture medium used in the activation and fermentation processes comprises the following components:

5.0g of yeast powder, 10.0g of peptone, 10.0g of NaCl and 1000ml of distilled water;

the pH of the activation medium is adjusted to 7.0-7.5 before use, and the activation medium is used after being sterilized for 30min at 121 ℃.

The beneficial effects of the invention mainly comprise the following aspects:

1. the Pseudomonas strain (Pseudomonas sp.) YH-1 and the Rhodococcus (Rhodococcus sp.) YH-3 have good synergistic performance, form an efficient metabolic degradation system, and can obviously improve the degradation effect of pyrene compared with a single strain;

2. the compound bacteria have good resistance and adsorption conversion effect on heavy metal chromium while degrading pyrene;

3. the method for treating the polycyclic aromatic hydrocarbon-heavy metal combined polluted environment by using the compound bacteria has the advantages of simple and easy operation, low cost, environmental friendliness and wide application prospect.

Drawings

FIG. 1 (a) shows the growth status of screened Pseudomonas (Pseudomonas sp.) YH-1 on solid LB medium;

FIG. 1 (b) shows the growth status of screened Rhodococcus (Rhodococcus sp.) YH-3 on solid LB medium;

FIG. 2 is a graph showing the effect of strain YH-1 and strain YH-3 on pyrene degradation after combination;

in FIG. 3, (a) and (b) are energy spectra of the complex bacteria when heavy metal chromium is added;

FIGS. 3 (c) and (d) are mapping graphs of the elements on the cell surface of the compound bacteria when heavy metal chromium is added;

FIGS. 4 (a) and (b) are TEM images of the recompounded bacteria with the addition of chromium as a heavy metal;

FIG. 4 (c) is a TEM energy spectrum of the complex bacteria when heavy metal chromium is added;

FIG. 5 is an FTIR chart before and after heavy metal chromium is adsorbed by the complex bacteria;

FIG. 6 (a) shows that the heavy metal Cr is adsorbed by the compound bacteria⁶⁺The XPS picture later, (b) is that the compound bacteria adsorbs heavy metal Cr⁶⁺The XRD pattern after the reaction.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description will be made on the specific technical solution of the present application with reference to the specific examples. It should be noted that the embodiments described are not all embodiments of the present application. The experimental materials used in the following examples are all conventional products commercially available unless otherwise specified.

Example 1 Strain selection

Materials and methods

Soil sample: the method can be used for overcoming the soil of the oil field polluted by petroleum for a long time.

Screening Medium

Inorganic salt culture medium: (NH)₄)₂SO₄1.0 g，K₂HPO₄1.0 g，KH₂PO₄1.0 g, anhydrous CaCl₂0.01 g，MgSO₄·7H₂O 0.2 g，NaCl 8.0g，FeCl₃Trace amount of 1000ml of distilled water, pH value of 7.2-7.5, and sterilization at 121 ℃ for 30 min.

Screening culture medium for pyrene degrading bacteria: adding a certain amount of pyrene-acetone solution on the basis of an inorganic salt culture medium.

Heavy metal screening culture medium: and adding 20g/L agar powder into the LB liquid culture medium to prepare an LB solid culture medium, and then adding heavy metal chromium solutions with different concentrations.

Strain screening

Weighing a certain amount of crude oil contaminated soil, adding the crude oil contaminated soil into a sterilized LB liquid culture medium, and carrying out enrichment culture for 48 h. Adding a certain amount of pyrene-acetone solution into an inorganic salt culture medium, shaking overnight, inoculating 5mL of enrichment culture solution after acetone is completely volatilized, shaking for culture for 7d, then adding 5mL of culture solution into a fresh culture medium, and performing transfer culture for 5 times by adopting the method, wherein the concentration of pyrene is sequentially increased (20, 30, 50, 80 and 100 mg/L). And (3) diluting the final domestication culture solution, coating the diluted final domestication culture solution on LB solid culture media containing pyrene with different concentrations, culturing for 2-3d, selecting colonies with better pyrene tolerance, repeatedly streaking and separating until a single pyrene degrading bacterium is obtained, streaking the obtained pyrene degrading bacterium on an LB solid flat plate containing heavy metal chromium with different concentrations, and culturing for 2-3d in a 37-DEG C constant temperature incubator to obtain the pyrene degrading bacterium with better heavy metal chromium tolerance.

Identification of strains

And (3) determining the screened strains by adopting a 16S rRNA molecular biology identification technology, determining the genus of the strains according to a sequencing result, and constructing a systemic developmental tree.

Screening results

As shown in Table 1, 2 pyrene-degrading bacteria YH-1 and YH-3 resistant to heavy metal chromium were obtained in total by screening.

The strains YH-1 and YH-3 have better tolerance to heavy metal chromium.

Example 2 Strain compounding

Materials and methods

The strain is as follows: 2 strains YH-1 and YH-3 which have higher pyrene degradation rate and heavy metal chromium resistance are obtained by screening.

Culture medium

LB liquid culture medium, pyrene-inorganic salt culture medium

The determination method comprises the following steps: preparing a pyrene-inorganic salt culture medium, adding heavy metal chromium (the concentration of pyrene in the culture medium is 50 mg/L; the content of heavy metal chromium is 30mg/L), and sterilizing for later use;

4 treatment groups were set up and inoculated with 10% (v: v) of the corresponding strain broth, respectively, and the experimental treatments were as follows:

treatment 1: only inoculating the strain YH-1;

and (3) treatment 2: only inoculating the strain YH-3;

and (3) treatment: inoculation of strains YH-1 and YH-3(1: 1);

and (4) treatment: pyrene-inorganic salt medium without inoculating strain is blank control group.

All treatments were incubated at 30 ℃ at 160r/min for 5d with shaking.

And measuring the residual amount of the cultured pyrene by adopting an ultrasonic extraction-ultraviolet spectrophotometry method and a gas chromatography-mass spectrometry method, and calculating the degradation rate of the pyrene.

Results of the experiment

The concentration of the pyrene in the treated 1 is 35.19mg/L, and the degradation rate of the pyrene is 29.63%;

the concentration of the processed 2 pyrene is 39.34mg/L, and the degradation rate of the pyrene is 21.32%;

the concentration of the processed 3 pyrene is 28.67mg/L, and the degradation rate of the pyrene is 42.67%;

and (4) conclusion:

from the above results, it can be seen that, through a degradation experiment of 5d, the degradation rate of YH-1 on pyrene is 29.63%, the degradation rate of YH-3 on pyrene is 21.32%, the degradation rate of YH-1 and YH-3 on pyrene after being compounded in a ratio of 1:1 is 42.67%, and the degradation rate of the compound bacteria on pyrene is higher than that of the single bacteria, so that YH-1 and YH-3 have a synergistic effect in the degradation process of pyrene, and the degradation rate of pyrene after being compounded by the 2 bacteria is obviously improved.

Example 3 adsorption conversion of heavy chromium Metal by Complex bacteria

And (3) experimental setting:

preparing a pyrene-inorganic salt culture medium, adding heavy metal chromium (the content of the heavy metal chromium in the culture medium is 30mg/L), and sterilizing for later use;

10% (v: v) of mixed fermentation liquor of compound bacteria is inoculated into a prepared pyrene-inorganic salt culture medium containing heavy metal chromium.

All treatments were incubated at 30 ℃ at 160r/min for 5 d.

Centrifuging the cultured culture medium, separating the thallus and culture medium, and measuring total chromium and Cr respectively⁶⁺The content of (a).

The content of total chromium in heavy metal is measured by Inductively Coupled Plasma (ICP) spectrometry, and Cr is⁶⁺The content of (a) was determined by diphenylcarbodihydrazide spectrophotometry.

The experimental results are as follows:

the concentration of pyrene is 50mg/L, heavy metal Cr⁶⁺The concentration of (A) is 30 mg/L;

as a result:

TABLE 1 Cr⁶⁺Adsorption conversion effect of

And (4) conclusion:

as can be seen from the table above, the complex bacteria have the function of adsorbing and converting heavy metal chromium in the presence of pyrene. Cr (chromium) component⁶⁺The initial concentration of the culture medium is 30mg/L, the temperature is 30 ℃, 160r/min, after the shaking culture is carried out for 5d, the total chromium content in the supernatant is 9.41mg/L, and the Cr content is 9.41mg/L⁶⁺The content of (A) is 7.00mg/L, which shows that the compound bacteria can adsorb Cr⁶⁺And part of Cr with high toxicity⁶⁺Conversion to Cr of low toxicity³⁺Composite bacteria pair Cr⁶⁺The adsorption conversion rate of (A) was 68.63%, and it was found that the complex bacteria showed Cr adsorption⁶⁺Has good repairing effect.

Example 4 adsorption conversion mechanism of heavy metal chromium by built-up bacteria

Materials and methods

Strain: compound bacterium

Culture medium

Pyrene-inorganic salt culture medium

The method comprises the following steps: SEM-EDS, TEM, FTIR, XRD and XPS assay

Preparing a pyrene-inorganic salt culture medium, adding heavy metal chromium (the content of the heavy metal chromium in the culture medium is 30mg/L), and sterilizing for later use;

preparing a pyrene-inorganic salt culture medium without adding heavy metals, and sterilizing for later use;

10% (v: v) of mixed fermentation broth of complex bacteria is inoculated into the treated culture medium respectively.

All treatments were incubated at 30 ℃ at 160r/min for 5 d.

Culturing for 5 days to obtain Cr containing heavy metal⁶⁺The pyrene-inorganic salt culture medium is centrifuged to collect thalli, washed for 3 times by Phosphate Buffer Solution (PBS), supernatant fluid is discarded to collect thalli, one part of the thalli is fixed by 2.5% glutaraldehyde and is put in a refrigerator at 4 ℃ overnight and then is sent to a compass company to be measured by SEM-EDS, TEM and Netherlands, the other part of the thalli is frozen and dried in vacuum to prepare bacterial powder and is sent to the compass company to be measured and analyzed by FTIR, XRD and XPS.

And (4) conclusion:

the SEM-EDS measurement result shows that the heavy metal Cr is adsorbed by the bacterial cells of the compound bacteria⁶⁺(ii) a TEM andthe energy spectrum measurement result shows that a small amount of heavy metal chromium exists in the bacterial strain cell, which indicates that metal ions can enter the bacterial strain cell through a membrane; XRD and XPS determination results analysis of compound bacteria can convert Cr⁶⁺Reduction to Cr³⁺The reduction product is Cr (OH)₃。