Method for acclimating phthalic acid ester-degrading functional microbial consortium

Abstract

The present invention relates to the technical field of biological treatment of environmental pollutants, and specifically to a method for acclimating a phthalic acid ester (PAE)-degrading functional microbial consortium, comprising the following steps: S1. enrichment and acclimation of a S microbial consortium; S2. gradient acclimation of the S microbial consortium; S3. preparation of a S microbial consortium suspension; S4. obtaining of a PAE-polluted crop; S5. enrichment and acclimation of a Y microbial consortium; S6. preparation of a Y microbial consortium suspension; and S7. mixing: mixing the S microbial consortium suspension obtained in step S3 with the Y microbial consortium suspension obtained in step S6 in a mass ratio of 3:1 to obtain an enhanced functional microbial consortium suspension. By recycling activated sludge and using DMP, DEP, and DBP as a unique carbon source and energy source for growth and reproduction to obtain the S microbial consortium suspension, mixed PAEs having an initial concentration of 10 mg / L can be almost completely degraded within 36 h, the degradation rates all exceed 95%, and the present invention has great application potential when applied to biodegradation of toxic and organic pollutants.

Description

Method for domesticating a group of phthalate-degrading functional bacteria TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial treatment, in particular to a method for domesticating a group of phthalate-degrading functional bacteria. BACKGROUND

[0002] Phthalic acid esters (PAEs) are widely used in plastic product production to enhance plasticity, flexibility and multifunctionality. PAEs are non-chemically bonded to polymers and are easily released during the manufacturing, storage and disposal of plastic products. Through biogeochemical cycling, PAEs further migrate to environmental media such as soil, sediment, water and atmosphere, becoming widely detected environmental hormone pollutants. Among them, dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP) and other six PAEs compounds have been listed as priority controlled pollutants and endocrine disruptors. TECHNICAL PROBLEM

[0003] PAEs can enter the human body through food intake, particulate matter inhalation, skin contact and other routes. Related human exposure risk studies have shown that soil ingestion and vegetable consumption are the main contact pathways of PAEs. High doses of PAEs are detected in plastic film-covered greenhouse vegetables in many regions. PAEs are identified as toxic and harmful organic substances and potential carcinogens due to their adverse effects on the endocrine system, red blood cell function and reproductive system of living organisms. PAEs can exert estrogen-like effects after entering the human body, leading to symptoms of testicular dysgenesis syndrome in males. In addition, studies have found that PAEs can also pose certain hazards to the respiratory system, cardiovascular system (coronary heart disease, atherosclerosis, blood pressure problems) of the human body. Therefore, it is urgent to develop effective ways to control the pollution risk of PAEs. TECHNICAL SOLUTION

[0004] To solve the above problems, the present application provides a method for domesticating a group of phthalate-degrading functional bacteria, comprising the following steps:

[0005] S1, S bacterial community enrichment and domestication: 1-1.5 parts of activated sludge by weight are placed in 2-3 parts of inorganic salt medium, a PAEs mixed solution is added to the inorganic salt medium, so that the mass concentration of PAEs in the inorganic salt medium is 6-14 mg / L, and domestication culture is carried out under room temperature conditions for 3 weeks with continuous stirring, to obtain a primary bacterial suspension;

[0006] S2, gradient domestication of S bacterial community:

[0007] S2-1, subculturing the primary bacterial suspension: 1-1.5 portions of the primary bacterial suspension are taken and added to 2-3 portions of the inorganic salt culture medium, a PAEs mixed solution is added to the inorganic salt culture medium, and domestication culture is continuously stirred at room temperature for 3 weeks to obtain a secondary bacterial suspension;

[0008] S2-2, the subculturing operation of step S2-1 is repeated five times, and the mass concentration of PAEs in the inorganic salt culture medium after each subculturing is 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L and 120 mg / L, respectively; after the last subculturing is completed, domestication culture is continued for 3 weeks to obtain a sixth bacterial suspension containing S bacterial flora; 1-1.5 portions of the sixth bacterial suspension are taken, mixed with 30-40% (volume fraction) glycerol at a volume ratio of 1:1, and stored under ultralow temperature conditions to obtain a freeze-dried glycerol containing S bacterial flora for standby;

[0009] S3, preparation of S bacterial flora bacterial suspension: 1-1.5 portions of the freeze-dried glycerol containing S bacterial flora obtained in step S2 are scraped with an inoculation loop and inoculated into 25-30 portions of sterilized LB culture medium; activation culture is continuously stirred at room temperature for 24 h to obtain a preliminarily activated bacterial suspension; the preliminarily activated bacterial suspension is centrifuged to discard the supernatant and obtain a bacterial body liquid; the bacterial body liquid is washed with an inorganic salt solution; the centrifugation and inorganic salt solution washing steps are repeated three times; the bacterial body liquid is placed in 18-20 portions of an inorganic salt culture medium, and the OD600 value of the bacterial body liquid is adjusted to 1.0; the bacterial body liquid is allowed to stand for 2-3 h to obtain an S bacterial flora bacterial suspension, which is stored under low temperature conditions for standby.

[0010] Further, the activated sludge is taken from a secondary sedimentation tank of a domestic sewage treatment plant.

[0011] Description: The activated sludge in the secondary sedimentation tank of the domestic sewage treatment plant contains a large amount of microorganisms, and the degradation flora can be domesticated by adding PAEs.

[0012] Further, steps S4-S7 are further included:

[0013] S4, obtaining PAEs-contaminated crops: PAEs-contaminated soil is taken, the S bacterial flora bacterial suspension prepared in step S3 is added thereto, and the mass ratio of the PAEs-contaminated soil to the S bacterial flora bacterial suspension is 100:1; after the PAEs-contaminated soil and the S bacterial flora bacterial suspension are uniformly mixed, they are allowed to stand for 48 h, and then plant planting is started on the remediated PAEs-contaminated soil; PAEs-contaminated crops are harvested after one planting cycle;

[0014] S5, Y bacterial group enrichment and domestication: the PAEs contaminated crop is dried and ground through a 200 mesh screen to obtain PAEs contaminated crop powder, 1-1.5 parts of the PAEs contaminated crop powder is placed in 2-3 parts of an inorganic salt culture medium, a PAEs mixed solution is added to the inorganic salt culture medium, so that the mass concentration of PAEs in the inorganic salt culture medium is 6-14 mg / L, and domestication culture is carried out under room temperature for 3 weeks under continuous stirring to obtain a first reinforced bacteria suspension, the first reinforced bacteria suspension is transferred according to the transfer method in step S2 and repeated for five times to obtain a six-time reinforced bacteria suspension containing Y bacterial group, which is mixed with 30% (volume fraction) glycerol at a volume ratio of 1:1, and stored under ultra-low temperature conditions to obtain a freeze-dried glycerol containing Y bacterial group for standby;

[0015] S6, Y bacterial group bacteria suspension preparation: 1-1.5 parts of the freeze-dried glycerol containing Y bacterial group obtained in step S5 is scraped with an inoculation loop and inoculated into 25-30 parts of sterilized LB culture medium, and activated culture is carried out under room temperature for 24 h under continuous stirring to obtain a preliminary activated reinforced bacteria suspension, the preliminary activated reinforced bacteria suspension is centrifuged in a centrifuge, and the supernatant is discarded to obtain a reinforced bacteria body liquid, the reinforced bacteria body liquid is washed with an inorganic salt solution, and the centrifugation and inorganic salt solution washing steps are repeated three times, then the reinforced bacteria body liquid is placed in 18-20 parts of an inorganic salt culture medium, and the OD600 value of the reinforced bacteria body liquid is adjusted to 1.0, and the Y bacterial group bacteria suspension is obtained after standing for 2-3 h, and is stored under low temperature conditions for standby;

[0016] S7, mixing: the S bacterial group bacteria suspension obtained in step S3 is mixed with the Y bacterial group bacteria suspension obtained in step S6 at a mass ratio of 3:1 to obtain a reinforced functional bacteria group bacteria suspension.

[0017] Description: the S bacterial group bacteria suspension prepared is used for soil treatment and planting plants on the soil, and then the plants are recovered to prepare the bacteria suspension again, and the Y bacterial group bacteria suspension complementary to the S bacterial group bacteria suspension can be obtained, and the two bacteria suspensions are mixed at a certain ratio to further improve the degradation effect on various PAEs pollution in the soil.

[0018] Further, the plants are one or more of water spinach, Shanghai green and lettuce.

[0019] Description: the above plants are common plants and easy to plant and recover, the planting cycle is short, and the prepared bacteria suspension has good PAEs treatment effect on the soil.

[0020] Further, the inorganic salt culture medium comprises 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl, 0.2 g / L of MgSO4·7H2O and 1.5 g / L of agar powder, and the solvent is ultrapure water, and the pH of the inorganic salt culture medium is 7.0; and the inorganic salt solution comprises 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl and 0.2 g / L of MgSO4·7H2O, and the solvent is ultrapure water, and the pH of the inorganic salt solution is 7.0.

[0021] Description: The inorganic salt culture medium with the above components and the ratio has the advantages of simple acquisition and low cost, and can complete the culture of the bacterial suspension.

[0022] Further, the mass ratio of the phthalate DMP, diethyl phthalate DEP and dibutyl phthalate DBP in the PAEs mixed solution is 1:1:1.

[0023] Description: By combining the above three PAEs and taking them as the only carbon source and energy source for growth and reproduction, the bacterial suspension capable of degrading multiple PAEs can be obtained.

[0024] Further, the temperature of the room temperature is 30 DEG C, the stirring speed is 150 rpm, the temperature of the ultralow temperature is -80 DEG C, and the temperature of the low temperature is 4 DEG C.

[0025] Description: By strictly controlling the temperature and the stirring condition, the negative influence on the bacterial population can be avoided.

[0026] Further, the LB culture medium comprises 5 g / L of yeast extract, 10 g / L of tryptone, 10 g / L of sodium chloride and 1.5 g / L of agar powder, and the solvent is ultrapure water, and the pH of the LB culture medium is 7.0, and the sterilization temperature is 120-125 DEG C, and the sterilization time is 20-30 min.

[0027] Further, the centrifugal speed of the centrifugal treatment is 5000 rpm, and the centrifugal treatment time is 5 min. Beneficial effects

[0028] Compared with the prior art, the beneficial effects of the present application are:

[0029] (1) The application utilizes the activated sludge to grow and reproduce by taking dimethyl phthalate, diethyl phthalate and dibutyl phthalate as the only carbon source and energy source, and then carries out gradient acclimation in the inorganic salt culture medium and activation in the LB culture medium to obtain the S bacterial group bacterial suspension, which can almost completely degrade the mixed PAEs (containing 10 mg / L of DMP, DEP and DBP respectively) with an initial concentration of 10 mg / L within 36 hours, and the degradation rate is more than 95%, so that the application has great application potential in the biodegradation of toxic organic pollutants;

[0030] (2) The application applies the S bacterial group bacterial suspension to the treatment of the PAEs contaminated soil, plants are planted on the soil after the treatment, and then the plants are recovered to prepare the bacterial suspension again, so that the Y bacterial group bacterial suspension which is complementary to the S bacterial group bacterial suspension can be obtained, and the degradation effect of the two kinds of bacterial suspensions on the soil contaminated by multiple PAEs can be further improved after the two kinds of bacterial suspensions are mixed in a certain proportion, which further enriches the practicability of the bacterial suspension, so that the bacterial suspension is no longer limited to the indoor test stage, but is combined with the secondary treatment, combined with the in-situ soil treatment and the ex-situ soil treatment, and the treatment effect of the toxic organic pollutants can be further improved. BRIEF DESCRIPTION OF DRAWINGS

[0031] Fig. 1 is a set of acclimation method flowcharts of the functional bacterial group for degrading phthalate esters according to the application;

[0032] Fig. 2 is a scanning electron microscope image of the S bacterial group bacterial suspension according to the application in Example 1, with a magnification of 5000 times;

[0033] Fig. 3 is a scanning electron microscope image of the S bacterial group bacterial suspension according to the application in Example 1, with a magnification of 20000 times;

[0034] Fig. 4 is a schematic diagram of the bacterial community structure composition of the indigenous bacterial group S of the S bacterial group bacterial suspension according to the application in Example 1;

[0035] Fig. 5 is a degradation effect diagram of the bacterial group S on the PAEs in the inorganic salt culture medium according to the application in Example 1;

[0036] Fig. 6 is a degradation effect diagram of the S bacterial group and the S bacterial group+Y bacterial group on three kinds of PAEs in the actual soil according to the application in Example 2. BEST MODE FOR CARRYING OUT THE INVENTION

[0037] Example 2: The content described in this embodiment is a set of acclimation method of the functional bacterial group for degrading phthalate esters, which connects the step S3 of Example 1, and further includes steps S4-S7:

[0038] S4, obtaining PAEs contaminated crops: taking the PAEs contaminated soil, adding the S bacterial community bacterial suspension prepared in step S3, the mass ratio of the PAEs contaminated soil to the S bacterial community bacterial suspension was 100:1, after the PAEs contaminated soil and the S bacterial community bacterial suspension were uniformly mixed, they were statically placed for 48 h, then the plant was planted on the remediated PAEs contaminated soil, and the PAEs contaminated crops were harvested after one planting cycle, the plant was spinach;

[0039] S5, Y bacterial community enrichment and domestication: the PAEs contaminated crops were dried and ground through a 200 mesh sieve to obtain PAEs contaminated crop ground powder, 1 part of the PAEs contaminated crop ground powder was placed in 2 parts of inorganic salt medium, the PAEs contaminated crop ground powder was obtained by drying and grinding the PAEs contaminated crops through a 200 mesh sieve, PAEs mixed solution was added to the inorganic salt medium so that the mass concentration of PAEs in the inorganic salt medium was 10 mg / L, domestication culture was carried out at 30°C for 3 weeks under continuous stirring, and a first reinforced bacterial suspension was obtained, the first reinforced bacterial suspension was transferred repeatedly for five times according to the transfer method in step S2, and a six-time reinforced bacterial suspension containing Y bacterial community was obtained, which was mixed with 30% glycerol by volume ratio 1:1, and stored at-80°C to obtain a freeze-dried glycerol containing Y bacterial community for standby;

[0040] The mass ratio of phthalate DMP, diethyl phthalate DEP and dibutyl phthalate DBP in the PAEs mixed solution was 1:1:1;

[0041] S6, Y bacterial community bacterial suspension preparation: 1 part of the freeze-dried glycerol containing Y bacterial community obtained in step S5 was scraped with an inoculation loop and inoculated into 27 parts of sterilized LB medium, the sterilization temperature was 123°C, the sterilization time was 27 min, the preliminary activation culture was carried out at 30°C for 24 h under continuous stirring, and the preliminary activated reinforced bacterial suspension was obtained, the preliminary activated reinforced bacterial suspension was centrifuged in a centrifuge at a centrifugal speed of 5000 rpm for 5 min, the supernatant was discarded to obtain the reinforced bacterial body liquid, the reinforced bacterial body liquid was washed with inorganic salt solution, and after the centrifugation and inorganic salt solution washing steps were repeated three times, the reinforced bacterial body liquid was placed in 20 parts of inorganic salt medium, and the OD600 value of the reinforced bacterial body liquid was adjusted to 1.0, and Y bacterial community bacterial suspension was obtained after static placement for 2 h, which was stored at 4°C for standby;

[0042] S7, mixing: the S bacterial community bacterial suspension obtained in step S3 and the Y bacterial community bacterial suspension obtained in step S6 were mixed in a mass ratio of 3:1 to obtain a reinforced functional bacterial community bacterial suspension.

[0043] The components and mass concentrations of the inorganic salt medium are as follows: 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, 1.5 g / L of agar powder, and the solvent is ultrapure water, and the pH of the inorganic salt medium is 7.0.

[0044] The components and mass concentrations of the inorganic salt solution are as follows: 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, and the solvent is ultrapure water, and the pH of the inorganic salt solution is 7.0.

[0045] The stirring speed is 150 rpm.

[0046] The components and mass concentrations of the LB medium are as follows: 5 g / L of yeast extract, 10 g / L of tryptone, 10 g / L of sodium chloride, 1.5 g / L of agar powder, and the solvent is ultrapure water, and the pH of the LB medium is 7.0. Embodiment of the application

[0047] In order to further illustrate the manner of carrying out the application and the effects achieved, the technical solutions of the application will be described clearly and completely below with reference to experiments.

[0048] Example 1: The content described in this example is a set of phthalate degradation functional bacterial population domestication methods, as shown in Figure 1, including the following steps:

[0049] S1, S bacterial population enrichment and domestication: 1 part of activated sludge is placed in 2 parts of inorganic salt medium, the activated sludge is taken from the secondary sedimentation tank of a domestic sewage treatment plant, a PAEs mixed solution is added to the inorganic salt medium, so that the mass concentration of PAEs in the inorganic salt medium is 10 mg / L, and domestication culture is carried out under the condition of continuous stirring at 30 DEG C for 3 weeks, to obtain a primary bacterial suspension;

[0050] S2, gradient domestication of S bacterial population:

[0051] S2-1, the primary bacterial suspension is transferred: 1 part of the primary bacterial suspension is taken and added to 2 parts of inorganic salt medium, a PAEs mixed solution is added to the inorganic salt medium, and domestication culture is carried out under the condition of continuous stirring at 30 DEG C for 3 weeks, to obtain a secondary bacterial suspension;

[0052] S2-2, repeat the transfer operation of step S2-1 five times, and make the mass concentration of PAEs in the inorganic salt medium after each transfer correspond to 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L respectively, continue to acclimate and culture for 3 weeks after the last transfer is completed, obtain six bacterial suspensions containing S flora, take out one of the six bacterial suspensions, mix it with glycerol with a volume fraction of 30% at a volume ratio of 1:1, and store it at-80°C to obtain freeze-dried glycerol containing S flora for standby;

[0053] The mass ratio of phthalate DMP, diethyl phthalate DEP and dibutyl phthalate DBP in the PAEs mixed solution is 1:1:1;

[0054] S3, S flora bacterial suspension preparation: take 1 part of the freeze-dried glycerol containing S flora obtained in step S2 with an inoculation loop, inoculate it into 28 parts of LB medium sterilized at 121°C, the sterilization time is 25 min, continuously stir at 30°C for 24 h to obtain a preliminarily activated bacterial suspension, centrifuge the preliminarily activated bacterial suspension in a centrifuge at a centrifugal speed of 5000 rpm for 5 min, discard the supernatant to obtain a bacterial liquid, wash the bacterial liquid with an inorganic salt solution, repeat the centrifugation and inorganic salt solution washing steps three times, then place the bacterial liquid in 20 parts of an inorganic salt medium, adjust the OD600 value of the bacterial liquid to 1.0, and stand for 2 h to obtain an S flora bacterial suspension, which is stored at 4°C for standby.

[0055] The components and mass concentrations of the inorganic salt medium are as follows: 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, 1.5 g / L of agar powder, the solvent is ultrapure water, and the pH of the inorganic salt medium is 7.0;

[0056] The components and mass concentrations of the inorganic salt solution are as follows: 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, the solvent is ultrapure water, and the pH of the inorganic salt solution is 7.0;

[0057] The stirring speed is 150 rpm,

[0058] The components and mass concentrations of the LB medium are as follows: 5 g / L of yeast extract, 10 g / L of tryptone, 10 g / L of sodium chloride, 1.5 g / L of agar powder, the solvent is ultrapure water, and the pH of the LB medium is 7.0.

[0059] Embodiment 2: The content described in this embodiment is a set of phthalate degradation functional bacterial population domestication methods, which links to step S3 of embodiment 1, and further includes steps S4-S7:

[0060] S4, obtaining PAEs contaminated crops: taking PAEs contaminated soil, adding S bacterial population bacterial suspension prepared in step S3 to it, the mass ratio of PAEs contaminated soil to S bacterial population bacterial suspension is 100:1, after mixing the PAEs contaminated soil and the S bacterial population bacterial suspension uniformly, standing for 48h, then starting to plant plants on the remediated PAEs contaminated soil, harvesting PAEs contaminated crops after one planting cycle, the plant is water spinach;

[0061] S5, Y bacterial population enrichment and domestication: drying the PAEs contaminated crops and grinding them through a 200 mesh sieve to obtain PAEs contaminated crop grinding powder, taking 1 part of the PAEs contaminated crop grinding powder and placing it in 2 parts of inorganic salt medium, the PAEs contaminated crop grinding powder is obtained by drying the PAEs contaminated crops and grinding them through a 200 mesh sieve, adding a PAEs mixed solution to the inorganic salt medium so that the mass concentration of PAEs in the inorganic salt medium is 10mg / L, continuously stirring and domesticating and culturing at 30℃ for 3 weeks to obtain a first reinforced bacterial suspension, repeating the transfer of the first reinforced bacterial suspension according to the transfer method in step S2 for five times to obtain a six-time reinforced bacterial suspension containing Y bacterial population, mixing it with 30% (volume fraction) glycerol at a volume ratio of 1:1, and storing it at -80℃ to obtain a freeze-dried glycerol containing Y bacterial population for standby;

[0062] The mass ratio of phthalates DMP, diethyl phthalate DEP, dibutyl phthalate DBP in the PAEs mixed solution is 1:1:1;

[0063] S6, preparation of Y bacterial population bacterial suspension: taking 1 part of the freeze-dried glycerol containing Y bacterial population obtained in step S5 with a inoculating loop, inoculating it into 27 parts of sterilized LB medium, sterilizing at a temperature of 123℃ for 27min, continuously stirring and activating culturing at 30℃ for 24h to obtain a preliminarily activated reinforced bacterial suspension, centrifuging the preliminarily activated reinforced bacterial suspension in a centrifuge at a centrifugal speed of 5000rpm for 5min, discarding the supernatant to obtain a reinforced bacterial body liquid, washing the reinforced bacterial body liquid with inorganic salt solution, repeating the centrifugation and inorganic salt solution washing steps three times, then placing the reinforced bacterial body liquid in 20 parts of inorganic salt medium and adjusting the OD600 value of the reinforced bacterial body liquid to 1.0, standing for 2h to obtain a Y bacterial population bacterial suspension, which is stored at 4℃ for standby;

[0064] S7, mixing: the S flora bacteria suspension obtained in step S3 is mixed with the Y flora bacteria suspension obtained in step S6 at a mass ratio of 3:1 to obtain a functional flora bacteria suspension.

[0065] The components and mass concentrations of the inorganic salt solution are as follows: 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, and the solvent is ultrapure water, and the pH of the inorganic salt solution is 7.0.

[0066] The components and mass concentrations of the inorganic salt solution are as follows: 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, and the solvent is ultrapure water, and the pH of the inorganic salt solution is 7.0.

[0067] The stirring speed is 150 rpm.

[0068] The components and mass concentrations of the LB medium are as follows: 5 g / L of yeast extract, 10 g / L of tryptone, 10 g / L of sodium chloride, and 1.5 g / L of agar powder, and the solvent is ultrapure water, and the pH of the LB medium is 7.0.

[0069] Example 3: The difference between this example and example 1 is that:

[0070] S1, S flora enrichment and domestication: 1.2 parts of activated sludge is placed in 2.4 parts of inorganic salt medium, the activated sludge is taken from the secondary sedimentation tank of a domestic sewage treatment plant, and a PAEs mixed solution is added to the inorganic salt medium, so that the mass concentration of PAEs in the inorganic salt medium is 6 mg / L, and the domestication culture is continuously stirred at 30°C for 3 weeks to obtain a primary bacteria suspension;

[0071] S2, S flora gradient domestication:

[0072] S2-1, the primary bacteria suspension is transferred: 1.2 parts of the primary bacteria suspension is added to 2.4 parts of inorganic salt medium, and a PAEs mixed solution is added to the inorganic salt medium, and the domestication culture is continuously stirred at 30°C for 3 weeks to obtain a secondary bacteria suspension;

[0073] S2-2, repeat the transfer operation of step S2-1 five times, and make the mass concentration of PAEs in the inorganic salt medium after each transfer correspond to 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L respectively, continue to acclimate and culture for 3 weeks after the last transfer is completed, obtain six bacterial suspensions containing S bacterial community, take out 1.2 parts of the six bacterial suspensions, mix them with 35% (volume fraction) glycerol at a volume ratio of 1:1, and store them at -80°C to obtain freeze-dried glycerol containing S bacterial community for standby;

[0074] S3, S bacterial community bacterial suspension preparation: take 1.2 parts of the freeze-dried glycerol containing S bacterial community obtained in step S2 with a inoculation loop, inoculate into 25 parts of LB medium sterilized at high temperature of 120°C, the sterilization time is 30 min, place the bacterial liquid in 18 parts of inorganic salt medium, and adjust the OD600 value of the bacterial liquid to 1.0, stand for 2.5 h to obtain S bacterial community bacterial suspension, and store it at 4°C for standby.

[0075] Example 4: The difference between this example and example 1 is:

[0076] S1, S bacterial community enrichment and acclimation: take 1.5 parts of activated sludge by weight and place it in 3 parts of inorganic salt medium, the activated sludge is taken from the secondary sedimentation tank of a domestic sewage treatment plant, add PAEs mixed solution to the inorganic salt medium, so that the mass concentration of PAEs in the inorganic salt medium is 14 mg / L, continuously stir and acclimate and culture at 30°C for 3 weeks to obtain a primary bacterial suspension;

[0077] S2, S bacterial community gradient acclimation:

[0078] S2-1, transfer the primary bacterial suspension: take 1.5 parts of the primary bacterial suspension and add it to 3 parts of inorganic salt medium, add PAEs mixed solution to the inorganic salt medium, continuously stir and acclimate and culture at 30°C for 3 weeks to obtain a secondary bacterial suspension;

[0079] S2-2, repeat the transfer operation of step S2-1 five times, and make the mass concentration of PAEs in the inorganic salt medium after each transfer correspond to 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L respectively, continue to acclimate and culture for 3 weeks after the last transfer is completed, obtain six bacterial suspensions containing S bacterial community, take out 1.5 parts of the six bacterial suspensions, mix them with 40% (volume fraction) glycerol at a volume ratio of 1:1, and store them at -80°C to obtain freeze-dried glycerol containing S bacterial community for standby;

[0080] S3, S group bacteria suspension preparation: 1.5 parts of the S group bacteria-containing freeze-dried glycerol obtained in step S2 were scraped with an inoculation loop, inoculated into 30 parts of LB medium sterilized at high temperature of 125°C for 20 min, and the OD600 value of the bacterial liquid was adjusted to 1.0. The bacterial liquid was placed in 19 parts of inorganic salt medium and statically placed for 3 h to obtain an S group bacteria suspension, which was stored at 4°C for standby.

[0081] Example 5: The difference between this example and example 2 is that:

[0082] The plant in S4 is Shanghai green;

[0083] S5, Y group bacteria enrichment and domestication: 1.2 parts of PAEs-polluted crop grinding powder were placed in 2.4 parts of inorganic salt medium, the PAEs-polluted crop grinding powder was obtained by grinding the dried PAEs-polluted crop through a 200-mesh screen, and a PAEs mixed solution was added to the inorganic salt medium to make the mass concentration of PAEs in the inorganic salt medium 6 mg / L. Domestication culture was carried out at 30°C for 3 weeks under continuous stirring to obtain a first enrichment bacteria suspension. The first enrichment bacteria suspension was transferred repeatedly for five times according to the transfer method in step S2 to obtain a six-time enrichment bacteria suspension containing Y group bacteria. The six-time enrichment bacteria suspension containing Y group bacteria was mixed with 30% (volume fraction) glycerol at a volume ratio of 1:1, and stored at -80°C to obtain freeze-dried glycerol containing Y group bacteria for standby;

[0084] S6, Y group bacteria suspension preparation: 1.2 parts of the Y group bacteria-containing freeze-dried glycerol obtained in step S5 were scraped with an inoculation loop, inoculated into 25 parts of sterilized LB medium, the sterilization temperature was 120°C, the sterilization time was 30 min, the enrichment bacteria liquid was placed in 18 parts of inorganic salt medium, and the OD600 value of the enrichment bacteria liquid was adjusted to 1.0. Static placement for 2.5 h obtained Y group bacteria suspension, stored at 4°C for standby.

[0085] Example 6: The difference between this example and example 2 is that:

[0086] The plant in S4 is lettuce;

[0087] S5, Y bacterial group enrichment and domestication: the PAEs contaminated crop is dried and ground through a 200 mesh screen to obtain PAEs contaminated crop ground powder, 1.5 parts of the PAEs contaminated crop ground powder is placed in 3 parts of inorganic salt medium, the PAEs contaminated crop ground powder is obtained by drying and grinding the PAEs contaminated crop through a 200 mesh screen, a PAEs mixed solution is added to the inorganic salt medium, so that the mass concentration of PAEs in the inorganic salt medium is 14 mg / L, and domestication culture is carried out at 30°C under continuous stirring for 3 weeks to obtain a first reinforced bacterial suspension, the first reinforced bacterial suspension is transferred repeatedly for five times according to the transfer method in step S2 to obtain a six-time reinforced bacterial suspension containing Y bacterial group, which is mixed with 30% glycerol by volume ratio of 1:1 and stored at -80°C to obtain a freeze-dried glycerol containing Y bacterial group for standby;

[0088] S6, Y bacterial group bacterial suspension preparation: 1.5 parts of the freeze-dried glycerol containing Y bacterial group obtained in step S5 is scraped with an inoculation loop and inoculated into 30 parts of sterilized LB medium, the sterilization temperature is 125°C, and the sterilization time is 20 min, the reinforced bacterial liquid is placed in 19 parts of inorganic salt medium, and the OD600 value of the reinforced bacterial liquid is adjusted to 1.0, and Y bacterial group bacterial suspension is obtained after standing for 3 h and is stored at 4°C for standby.

[0089] Description: When higher temperature is selected for sterilization, the sterilization time can be appropriately reduced, and any one set of parameters in Example 1 or Examples 3-6 can achieve good sterilization conditions.

[0090] Experimental Example 1: The description of this experimental example is based on the recorded scheme in Example 1, and is intended to illustrate the indoor experimental effect of the functional bacterial group S bacterial group bacterial suspension prepared by the present application on the biodegradation of toxic organic pollutants.

[0091] First, we observe the morphology of the S bacterial group bacterial suspension by scanning electron microscopy, the LB culture is sterilized at 121°C for 30 min, and after cooling at room temperature, the S bacterial group bacterial suspension is added and shaken for 24 h (30°C, 150 rpm) for activation, 1 ml of bacterial liquid is taken and placed in a 1.5 ml centrifuge tube for centrifugation (5000 rpm, 5 min), the supernatant is discarded, and bean-sized bacterial bodies are obtained, 2.5% glutaraldehyde solution is added to the centrifuge tube and the tube is filled (so that the S bacterial group bacterial suspension is completely immersed in the fixing solution), and is stored at 4°C, and is detected after being stored for 12 h. The biological scanning electron micrograph of the S bacterial group bacterial suspension is shown in Figures 2 and 3, as can be seen from the figures (magnification 5000 times and 20000 times), most of the bacteria in the bacterial group S are long rod-shaped.

[0092] Subsequently, we explored the strain composition of the S group bacterial suspension. 1 mL of the S group bacterial suspension cultured for 24 h was added to a 1.5 mL centrifuge tube, centrifuged at 4°C and 5000 r / min for 2 min, the supernatant was discarded, and the bacterial cells were collected. The bacterial genomic DNA was extracted using a bacterial DNA extraction kit (Omega, USA) according to the operating instructions. The extracted DNA was subjected to 1% agarose gel electrophoresis and quality testing by Nano Drop 2000 (Thermo Scientific, USA). The above samples were subjected to 16S rRNA gene high-throughput sequencing by Shanghai Shenguo Biological Engineering Co., Ltd. The extracted DNA was used as a template to amplify the V3-V4 variable region of the bacterial 16S rRNA gene using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and primers 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The PCR product was recovered using 2% agarose gel, and the NEXTFLEX Rapid DNA-Seq Kit was used to construct a library. The ILLUMINA PE300 / PE250 platform was used for sequencing. Thus, the bacterial community structure composition of the indigenous bacterial group S was obtained, and the results are shown in FIG. 4.

[0093] Next, we began to set up the indoor S group bacterial suspension degradation experiment. 0.1 g of DMP, 0.1 g of DEP, and 0.1 g of DBP were weighed into a 100 ml volumetric flask, and methanol was added to make up the volume to obtain a 1 g / L PAEs mixed solution (containing 1 g / L DMP, 1 g / L DEP, and 1 g / L DBP). 19 ml of inorganic salt medium was added to a 100 ml sterilized conical flask, 1 ml of bacterial liquid was added according to a 5% dosage, 200 μL of a 1 g / L PAEs mixed solution was added, the initial concentrations of DMP, DEP, and DBP were all 10 mg / L, a PAEs-containing inorganic salt medium solution without bacteria was set up as a control group, the pH was adjusted to 7.0, each group had three replicates, and samples were taken at 4 h, 12 h, 36 h, and 48 h. 2 ml of supernatant was drawn into a syringe, filtered through a 0.22 μm organic phase filter, and stored in a 2 mL brown liquid phase vial. High-performance liquid chromatography was used for detection.

[0094] The detection conditions of PAEs by high performance liquid chromatography are as follows: an LC-20AT high performance liquid chromatograph (equipped with an SPD-2A ultraviolet detector) is used, the detection time is 40 min, the injection volume of the injection system is 20 μL, the initial flow rate of the separation system is 1.0 mL / min, the PAEs are separated by gradient elution using acetonitrile-water as the mobile phase, the chromatographic column is a Φ4.6*250 mm Inertsil ODS-P liquid chromatographic column, the column temperature is 40°C; the detection system is detected by an ultraviolet detector, and the dual-wavelength detection mode is opened, and the wavelengths are 205 nm and 225 nm, respectively.

[0095] Finally, we obtain the degradation efficiency of the S group: the S group has good degradation effect on DEP, DMP and DBP in the inorganic salt medium. In the initial stage of degradation, the S group degrades DMP faster due to the shorter alkyl side chain and smaller molecular weight of DMP. The degradation rates of DMP, DEP and DBP are 39.05%, 16.06% and 10.90%, respectively. After 36 h of culture, DMP, DEP and DBP are almost completely degraded, and the degradation rates are 99.85%, 99.85% and 95.85%, respectively. The above results show that the S group has excellent application potential in the field of biodegradation of DMP, DEP and DBP.

[0096] Experimental Example 2: The description of this experimental example is based on the description in Example 2, and is intended to illustrate the actual use effect of the strong functional bacterial suspension prepared by the present application on the biodegradation of toxic organic pollutants.

[0097] We repair the PAEs contaminated soil according to the process in step S4, collect the contaminated soil to the laboratory for repair (ex situ soil remediation), and detect the comprehensive removal rate of the three PAEs in the soil. The results are shown in Figure 6. ∑PAEs represents the total degradation rate of the three PAEs. It can be seen that the comprehensive removal rate of the three PAEs in the soil is 68%. In actual application, the degradation effect is different from that of indoor test. This may be because the composition of pollutants in actual soil is complex and there is certain interference. It may also be because the bacterial community is affected by environmental conditions (such as temperature and pH value), and there may be certain competition with indigenous bacterial community in the soil.

[0098] Subsequently, we prepare the strong functional bacterial suspension according to the processes in steps S5-S7, and apply the strong functional bacterial suspension to the soil remediation in the contaminated area again. This time, we backfill the soil remediated in step S4 to the original place, and directly add the strong functional bacterial suspension to the contaminated soil (in situ soil remediation). The application amount is 2.5 L / m 2After 3 weeks, the soil samples were taken to detect the comprehensive removal rate of PAEs, and the results are shown in Figure 6. It can be seen that the comprehensive removal rate of PAEs in the soil sample at this time is 85%; in summary, it is shown that the functional bacteria group suspension prepared by mixing the S bacteria group suspension obtained in step S3 and the Y bacteria group suspension obtained in step S6 in a mass ratio of 3:1 has stronger biodegradability, and the difference between the two can be more directly observed in combination with Figure 6, which makes up for the possible interference of the single S bacteria group suspension in the actual soil and improves the degradation completeness of DBP, and the combination of in-situ soil treatment and ex-situ soil treatment can further improve the treatment effect of toxic organic pollutants. Industrial applicability

[0099] The functional bacteria group for degrading phthalate esters in the application can almost completely degrade mixed PAEs (containing 10 mg / L of DMP, DEP and DBP, respectively) with an initial concentration of 10 mg / L within 36 h, and the degradation rate is more than 95%, which has great application potential in the biodegradation of toxic organic pollutants; and the combination of actual soil treatment and secondary treatment, the combination of in-situ soil treatment and ex-situ soil treatment can further improve the treatment effect of toxic organic pollutants.

Claims

1. A method for domesticating a functional bacterial community for degrading phthalate esters, characterized by, The method comprises the following steps: S1, S bacterial community enrichment and domestication: 1-1.5 parts of activated sludge by weight is placed in 2-3 parts of inorganic salt culture medium, a PAEs mixed solution is added to the inorganic salt culture medium, so that the mass concentration of PAEs in the inorganic salt culture medium is 6-14 mg / L, and domestication culture is continuously stirred at room temperature for 3 weeks to obtain a primary bacterial suspension, and the activated sludge is taken from a secondary sedimentation tank of a domestic sewage treatment plant; S2, gradient domestication of S bacterial community: S2-1, the primary bacterial suspension is transferred: 1-1.5 parts of the primary bacterial suspension is added to 2-3 parts of inorganic salt culture medium, a PAEs mixed solution is added to the inorganic salt culture medium, and domestication culture is continuously stirred at room temperature for 3 weeks to obtain a secondary bacterial suspension; S2-2, the transfer operation of step S2-1 is repeated five times, and the mass concentration of PAEs in the inorganic salt culture medium after each transfer corresponds to 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L and 120 mg / L respectively, and the domestication culture is continued for 3 weeks after the last transfer is completed to obtain a sixth bacterial suspension containing S bacterial community, 1-1.5 parts of the sixth bacterial suspension is taken out, mixed with 30-40% (by volume fraction) glycerol at a volume ratio of 1:1, and stored under ultralow temperature conditions to obtain a freeze-dried glycerol containing S bacterial community for standby; S3, preparation of S bacterial community bacterial suspension: 1-1.5 parts of the freeze-dried glycerol containing S bacterial community obtained in step S2 is scraped with an inoculation loop and inoculated into 25-30 parts of sterilized LB culture medium, and the preliminary activation culture is continuously stirred at room temperature for 24 h to obtain a preliminarily activated bacterial suspension, the preliminarily activated bacterial suspension is centrifuged in a centrifuge, the supernatant is discarded to obtain a bacterial body liquid, the bacterial body liquid is washed with an inorganic salt solution, and the centrifugation and inorganic salt solution washing steps are repeated three times, then the bacterial body liquid is placed in 18-20 parts of inorganic salt culture medium, the OD600 value of the bacterial body liquid is adjusted to 1.0, and the bacterial suspension of S bacterial community is obtained after standing for 2-3 h and is stored under low temperature conditions for standby; S4, obtaining PAEs contaminated crops: PAEs contaminated soil is taken, the S bacterial community bacterial suspension prepared in step S3 is added, the mass ratio of PAEs contaminated soil to S bacterial community bacterial suspension is 100:1, the PAEs contaminated soil and the S bacterial community bacterial suspension are uniformly mixed, and then standing for 48 h, and then plant planting is started on the repaired PAEs contaminated soil, and PAEs contaminated crops are harvested after one planting period. S5, Y bacterial group enrichment and domestication: the PAEs contaminated crops are dried and ground through a 200 mesh screen to obtain PAEs contaminated crop powder, 1-1.5 parts of the PAEs contaminated crop powder is placed in 2-3 parts of inorganic salt culture medium, a PAEs mixed solution is added to the inorganic salt culture medium, so that the mass concentration of PAEs in the inorganic salt culture medium is 6-14 mg / L, and domestication culture is carried out under room temperature for 3 weeks with continuous stirring to obtain a first enhanced bacteria suspension, the first enhanced bacteria suspension is transferred according to the transfer method in step S2 and repeated for five times to obtain a six-time enhanced bacteria suspension containing Y bacterial group, which is mixed with 30% glycerol by volume ratio of 1:1 and stored under ultra-low temperature to obtain a freeze-dried glycerol containing Y bacterial group for standby; S6, Y bacterial group bacteria suspension preparation: 1-1.5 parts of the freeze-dried glycerol containing Y bacterial group obtained in step S5 is scraped with an inoculation loop and inoculated into 25-30 parts of sterilized LB medium, and activated culture is carried out under room temperature for 24 h with continuous stirring to obtain a preliminarily activated enhanced bacteria suspension, the preliminarily activated enhanced bacteria suspension is centrifuged to discard the supernatant to obtain an enhanced bacteria body liquid, the enhanced bacteria body liquid is washed with an inorganic salt solution, and the centrifugation and inorganic salt solution washing steps are repeated three times, then the enhanced bacteria body liquid is placed in 18-20 parts of inorganic salt culture medium, and the OD600 value of the enhanced bacteria body liquid is adjusted to 1.0, and the Y bacterial group bacteria suspension is obtained after standing for 2-3 h and stored under low temperature for standby; S7, mixing: the S bacterial group bacteria suspension obtained in step S3 and the Y bacterial group bacteria suspension obtained in step S6 are mixed in a mass ratio of 3:1 to obtain an enhanced functional bacteria group bacteria suspension.

2. The temperature of the room temperature is 30°C, the stirring speed is 150 rpm, the temperature of the ultra-low temperature is -80°C, and the temperature of the low temperature is 4°C.

3. The method for domesticating a group of functional microbial communities that degrade phthalates as described in claim 1, characterized in that, The plants are one or more of water spinach, Shanghai green and lettuce.

4. The method for domesticating a group of functional microbial communities that degrade phthalates as described in claim 1, characterized in that, The components and mass concentrations of the inorganic salt culture medium are as follows: 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, and 1.5 g / L of agar powder, the solvent is ultrapure water, and the pH of the inorganic salt culture medium is 7.0; the components and mass concentrations of the inorganic salt solution are as follows: 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, and the solvent is ultrapure water, and the pH of the inorganic salt solution is 7.

0.

5. The method for domesticating a group of functional microbial communities that degrade phthalates as described in claim 1, characterized in that, The mass ratio of dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP) in the PAEs mixed solution is 1:1:

1.

6. The method for domesticating a group of functional microbial communities that degrade phthalates as described in claim 1, characterized in that, The components and mass concentrations of the LB culture medium are as follows: 5 g / L of yeast extract, 10 g / L of trypsin peptone, 10 g / L of sodium chloride, and 1.5 g / L of agar powder, the solvent is ultrapure water, the pH of the LB culture medium is 7.0, the sterilization temperature is 120-125 DEG C, and the sterilization time is 20-30 min.

7. The method for domesticating a group of functional microbial communities that degrade phthalates as described in claim 1, characterized in that, The centrifugal speed of the centrifugal treatment is 5000 rpm, and the centrifugal treatment time is 5 min.

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