CN115786191B - Citrobacter freundii and application thereof in pesticide production wastewater treatment - Google Patents
Citrobacter freundii and application thereof in pesticide production wastewater treatment Download PDFInfo
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Abstract
The invention discloses a Citrobacter freundii and application thereof in pesticide production wastewater treatment, wherein the Citrobacter freundii (Citrobacter freundii) is named as GH-2 and is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation date of 2022, 9 months and 9 days and the preservation number of CGMCC No.25671. The citrobacter freundii provided by the invention can efficiently degrade organic pollutants in pesticide production wastewater, and the strain has a wide application range on salt concentration and temperature, provides efficient microbial germplasm resources for intermediate degradation in pesticide wastewater, and has a good application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to citrobacter freundii and application thereof in treatment of pesticide production wastewater.
Background
Pesticides are an important field of fine chemical engineering. To date, the number of pesticide varieties developed globally reaches hundreds of thousands, more than 600 pesticides are commercialized, and more than ten thousands of related intermediates. Due to the complex production process of the pesticide, low utilization rate of raw materials and more synthetic intermediates, wastewater containing raw pesticide, main raw materials for production and intermediates is generated in the production process. Compared with industrial sewage generated by other industries, the pesticide production wastewater contains a large amount of toxic substances due to the growth inhibition effect of targeting a pesticide target to a specific organism, has higher biotoxicity, is difficult to degrade in the traditional biochemical treatment mode of a municipal sewage treatment plant, and causes serious harm to the surrounding ecological environment if the treatment is not good. Meanwhile, because of the technological processes of condensation, elution and the like in the production process of the pesticide, the finally produced wastewater has higher salt content, and most of high-salt components are CI - 、SO 4 2- 、Na + 、Ca 2+ And the like.
The allyl alcohol ketone is one of important intermediates required in the synthesis process of the allethrin, is mainly prepared from 5-methylfurfural and furfuryl alcohol, and can discharge waste water containing high-concentration 5-methylfurfural and furfuryl alcohol in the preparation process. The traditional pure physical process can not effectively degrade organic matters, and needs to be added with post-treatment, while the chemical method has complex process flow and higher operation and maintenance cost, and most of the physical and chemical methods can only aim at one pollutant composition, so the effect of treating the actual wastewater containing various substances is not ideal. Therefore, the technical process which is economic, reasonable and good in comprehensive effect is adopted for carrying out appropriate effective treatment on the pesticide production wastewater, and the problem which needs to be solved in the pesticide industry of China at present is solved.
Compared with the traditional treatment mode, the microbial degradation method has the advantages of simultaneously removing various pollutants, reducing the cost, reducing secondary pollution and the like; different kinds of microorganisms correspond to the wastewater with different compositions, or one microorganism can degrade multiple pollutants simultaneously. The final products obtained after the microbial metabolism treatment are low-carbon products which are low in toxicity and non-toxicity, or carbon dioxide and water. In view of the above, the technical problem to be solved by the invention is to find a high-efficiency microorganism capable of realizing rapid degradation of the microorganism, and to enhance the removal of pollutants in a water body through a microorganism dominant bacterial strain.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the Citrobacter freundii GH-2 which can efficiently degrade organic pollutants in pesticide production wastewater under the conditions of low temperature and high salt.
In order to achieve the above object, the present invention adopts the following technical solutions:
a strain of Citrobacter freundii, named GH-2, has been preserved in China general microbiological culture Collection center (CGMCC), with a preservation date of 2022, 9 months and 9 days, and a preservation number of CGMCC No.25671.
An application of Citrobacter freundii in degrading organic pollutants in pesticide production wastewater under low-temperature and high-salt conditions, wherein the organic pollutants are one or more of allethrin, allyl alcohol ketone, 5-methylfurfural or furfuryl alcohol, and the content of the organic pollutants is 0.1-1g/L; the salt concentration in the pesticide production wastewater is 1 to 20g/L, and the temperature is 10 to 30 ℃.
A microbial inoculum prepared from a strain of citric acid Freund's bacillus.
An application of a microbial inoculum in degrading organic pollutants in pesticide production wastewater under low-temperature and high-salt conditions, wherein the organic pollutants are one or more of allethrin, allylketol, 5-methylfurfural or furfuryl alcohol, and the content of the organic pollutants is 0.1-1g/L; the salt concentration in the pesticide production wastewater is 1 to 20g/L, and the temperature is 10 to 30 ℃.
The preparation method of the microbial inoculum comprises the following specific steps:
s1, selecting a single colony of Citrobacter freundii GH-2, inoculating the single colony in an LB liquid culture medium, oscillating in a shaking table, and culturing to a logarithmic phase to obtain a bacterial liquid;
s2, inoculating the bacterial liquid to a seed tank, and culturing to logarithmic phase to obtain a seed liquid;
s3, inoculating the seed liquid into a fermentation tank for fermentation culture, and obtaining the microbial inoculum after fermentation is finished.
Preferably, in the step S1, the LB liquid medium comprises: 5g/L of yeast extract powder, 10g/L of peptone, 10g/L of sodium chloride and 7.0 to 7.5 of pH; the culture conditions were: the temperature is 30 to 35 ℃, and the time is 12 to 24h.
Preferably, in the foregoing steps S2 and S3, the components of the fermenter medium and the seed tank medium are the same and are both: glucose 8g/L, yeast extract 5g/L, K 2 HPO 4 1g/L、NaCl 5g/L、CaCO 3 2g/L、MgSO 4 •7H 2 0.2g/L of O, 0.1% (v/v) of soybean oil and 7.0 to 7.5 of pH.
Preferably, in the steps S2 and S3, the culture conditions of the seed tank and the fermentation tank are the same, the volume ratio of the ventilation volume of the sterile air to the volume of the fermentation medium in the culture process is 1.6-1.2, the stirring speed is 180-240 rpm, the culture temperature is 30-35 ℃, and the whole-process culture time is 48-96h.
Preferably, in the step S2, the inoculation amount of the bacterial liquid is 5% -10% by volume; in the step S3, the inoculation amount of the seed liquid is 1-8% by volume ratio.
Preferably, in step S3, the number of cells in the fermentation broth after the fermentation is finished reaches 10 8 More than one/mL.
The invention has the advantages that: the invention provides a Citrobacter freundii GH-2 capable of degrading organic pollutants in pesticide production wastewater, wherein the degradation rate of the strain GH-2 to allethrin is over 88% in 24 hours, and the degradation rates to allylenone, 5-methylfurfural and furfuryl alcohol are over 96%; the salt concentration of the pesticide production wastewater is within the range of 1-20g/L, the degradation rates of the strain GH-2 to the allyl alcohol ketone, the 5-methylfurfural and the furfuryl alcohol are all more than 96%, and the degradation rates of the strain GH-2 to the allyl alcohol ketone, the 5-methylfurfural and the furfuryl alcohol are all more than 93% within the temperature range of 10-30 ℃, so that the strain can efficiently degrade organic pollutants in the pesticide production wastewater under the conditions of low temperature and high salt, and has important significance in the aspects of biologically strengthening treatment of the pesticide production wastewater, protection of ecological environment and the like.
Drawings
FIG. 1 is a colony morphology of Citrobacter freundii GH-2;
FIG. 2 is a graph showing the degradation effect of Citrobacter freundii GH-2 on organic pollutants in pesticide production wastewater;
FIG. 3 is a graph showing the effect of salt concentration on the degradation effect of Citrobacter freundii GH-2;
FIG. 4 is a graph showing the effect of temperature on the degradation effect of Citrobacter freundii GH-2;
FIG. 5 shows the degradation effect of strain GH-2 on organic pollutants with different concentrations;
FIG. 6 is a graph showing the effect of the microbial inoculum on the degradation of organic pollutants in wastewater from the actual production of pesticides.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
Example 1
Isolation/purification and identification of the strains:
taking aerobic activated sludge in a biochemical tank of a sewage treatment plant of a certain pesticide production enterprise, standing, removing supernatant, adding clear water, mixing uniformly, standing, removing supernatant, and repeating the steps for 2 times. Adding 20mL of the sludge into an inorganic salt culture medium with the initial concentrations of allethrin and allyl alcohol ketone of 100mg/L and the initial concentrations of 5-methylfurfural and furfuryl alcohol of 500mg/L, performing acclimatization culture at 30 ℃ and 160rpm for 7 days, inoculating the mixture into a fresh inorganic salt culture medium according to the inoculation amount of 6 percent after the culture for 7 days, continuing the culture for 7 days, increasing the concentration of organic pollutants in the transfer culture medium by 100mg/L each time, and continuously enriching and transferring for 5 times to ensure that the concentrations of allethrin and allyl alcohol ketone in the inorganic salt culture medium reach 500mg/L and the concentrations of 5-methylfurfural and furfuryl alcohol reach 1000mg/L to obtain an enrichment solution.
Diluting the obtained enrichment solution with sterile water to different concentration gradients (10) -3 ~10 -7 ) And taking the diluted bacterial liquid, coating the diluted bacterial liquid on an LB solid culture medium, and selecting single bacterial colonies with different forms for streak separation and purification after the bacterial colonies grow out. And transferring the purified strain into an inorganic salt liquid culture medium again, carrying out shake culture at 30 ℃ and 160rpm, verifying the degradation effect of different strains, and screening to obtain the dominant strains capable of efficiently degrading the pesticide and the intermediate thereof. Warp beamSeparating and purifying to obtain a strain which can efficiently degrade the pesticide and the intermediate thereof at the same time and is named as GH-2.
The inorganic salt culture medium comprises the following components: naCl 1.0g/L, (NH) 4 ) 2 SO 4 1.0g/L、K 2 HPO 4 1.5 g/L、KH 2 PO 4 0.5g/L、MgSO 4 •7H 2 O0.2 g/L and water 1L, pH 7.0, and the medium was sterilized at 121 ℃ for 20min.
The LB solid medium comprises the following components: 5g/L yeast extract powder, 10g/L peptone, 10g/L sodium chloride, pH 7.0, adding 2% agar into solid culture medium, and sterilizing the culture medium at 121 deg.C for 20min.
The strain GH-2 is mainly characterized by gram staining negativity, rod-shaped thalli, smooth, low-convexity, mild, semi-transparent or opaque colonies on an LB solid culture medium, glossy surface, neat edges, and 2-4 mm of colony diameter, can grow by taking a pesticide intermediate as a unique carbon source, and has the colony morphology shown in figure 1.
Example 2
Preparation of the microbial inoculum:
selecting a single colony of a strain GH-2, inoculating the single colony in an LB liquid culture medium, culturing at the temperature of 30 ℃ for 12 hours, and carrying out shake culture until the logarithmic phase is reached; inoculating the cultured bacterial liquid into a seeding tank according to the inoculation amount of 10%, and culturing to logarithmic phase; inoculating the seed solution into a fermentation tank according to the inoculation amount of 5% for culture, wherein the culture medium used by the fermentation tank is the same as that of the seed tank; the ventilation quantity of sterile air in the culture process of the seeding tank and the production tank is 1.0, the stirring speed is 240 rpm, the culture temperature is 35 ℃, the whole-process culture time is 96h, and the thallus number reaches 10 after the fermentation is finished 8 More than one per mL, directly taking the fermentation broth out of the tank after fermentation and subpackaging the fermentation broth into liquid formulations by using a plastic packaging barrel or a packaging bottle.
The LB liquid medium comprises the following components: 5g/L of yeast extract powder, 10g/L of peptone, 10g/L of sodium chloride and pH 7.0.
The culture medium components of the seeding tank and the fermentation tank are as follows: glucose 8g/L, yeast extract 5g/L, K 2 HPO 4 1g/L、NaCl 5g/L、CaCO 3 2g/L、MgSO 4 0.2g/L, 0.1% (v/v) of soybean oil,the pH value is 7.0.
Example 3
The strain GH-2 has the following degradation effect on organic pollutants in pesticide production wastewater:
inoculating Citrobacter freundii GH-2 into an LB culture medium to be cultured to a logarithmic phase, wherein the LB liquid culture medium comprises the following components: 10g/L of peptone, 5g/L of yeast extract powder and 10g/L of sodium chloride, collecting the culture solution, centrifuging at 4 ℃ and 8000r/min for 5 min, discarding supernatant, collecting thalli, washing and resuspending the thalli with sterile water, centrifuging again to remove the supernatant, repeating the steps for 3 times to obtain the washed thalli, and resuspending thalli cells with sterile water in equal volume to obtain seed solution.
Respectively inoculating the seed liquid into an inorganic salt culture medium containing allethrin, allyl alcohol ketone, 5-methylfurfural and furfuryl alcohol in an inoculation amount of 5%, wherein the inorganic salt culture medium comprises the following components: (NH) 4 ) 2 SO 4 0.5g/L、KH 2 PO 4 1g/L、Na 2 HPO 4 1g/L、MgCl 2 0.02g/L、CaCl 2 0.03g/L, pH 7.0, initial concentrations of both allethrin and allylketol were 500mg/L, initial concentrations of both 5-methylfurfural and furfuryl alcohol were 1000mg/L, shaking culture was performed at 30 ℃ and 160rpm, and the concentrations were measured after 24 hours, as shown in FIG. 2.
As can be seen from FIG. 2, the strain GH-2 has the degradation rates of 88.2%, 96.3%, 96.8% and 97.5% on allethrin, naloxone, 5-methylfurfural and furfuryl alcohol, respectively, and has a good degradation effect.
Example 4
Effect of salt concentration on the efficiency of GH-2 degradation by the strain:
the seed liquid obtained in example 3 was inoculated into an inorganic salt medium containing allyl alcohol ketone, 5-methylfurfural and furfuryl alcohol at an inoculum size of 5%, and the inorganic salt medium had the following composition: (NH) 4 ) 2 SO 4 0.5g/L、KH 2 PO 4 1g/L、Na 2 HPO 4 1g/L、MgCl 2 0.02g/L、CaCl 2 0.03g/L, pH 7.0 and initial concentration of organic pollutants in the pesticide production wastewater of 0.5g/L. Adjusting the NaCl concentration in the inorganic salt culture medium to be 1.0g/L,5g/L, 10g/L, 15 g/L, 20g/L, were shake cultured at 30 ℃ in a shaker at 160rpm, and the content was measured after 24 hours, as shown in FIG. 3.
As can be seen from FIG. 3, the salt concentration in the pesticide production wastewater is within the range of 1-20g/L, and the degradation rates of the strain GH-2 to allyl alcohol ketone, 5-methylfurfural and furfuryl alcohol are all over 96%, which shows that the strain has a wide application range to the salt concentration and has high tolerance to the wastewater salt concentration.
Example 5
Effect of temperature on the degradation efficiency of strain GH-2:
the seed liquid obtained in example 3 was inoculated into an inorganic salt medium containing allyl alcohol ketone, 5-methylfurfural and furfuryl alcohol at an inoculum size of 5%, and the inorganic salt medium had the following composition: (NH) 4 ) 2 SO 4 0.5g/L、KH 2 PO 4 1g/L、Na 2 HPO 4 1g/L、MgCl 2 0.02g/L、CaCl 2 0.03g/L, pH 7.0, and initial concentrations of pesticide and intermediate were all 0.5g/L. The cells were cultured in a shaker at 10 ℃ 15 ℃ 20 ℃ 25 ℃ 30 ℃ 160rpm, and the content was measured after 24 hours, and the results are shown in FIG. 4.
As can be seen from FIG. 4, the degradation rates of the strain GH-2 to the allyl alcohol ketone, the 5-methylfurfural and the furfuryl alcohol are all over 93% within the temperature range of 10 to 30 ℃, which shows that the strain has good adaptability to the environmental temperature and can be applied to the degradation of organic pollutants in pesticide production wastewater in a low-temperature environment.
Example 6
The degradation effect of the strain GH-2 on organic pollutants with different concentrations is as follows:
the seed liquid obtained in example 3 was inoculated into inorganic salt medium containing allyl alcohol ketone of different concentrations at an inoculation amount of 5%, and the composition of the inorganic salt medium was: (NH) 4 ) 2 SO 4 0.5g/L、KH 2 PO 4 1g/L、Na 2 HPO 4 1g/L、MgCl 2 0.02g/L、CaCl 2 0.03g/L, pH 7.0, initial concentrations of allyl alcohol ketone are 0.1g/L, 0.3g/L, 0.5g/L, 0.7g/L, 1g/L, shaking culture is carried out at 30 deg.C and 160rpm, after 24hThe concentration was measured, and the results are shown in FIG. 5.
As can be seen from FIG. 5, the degradation rate of the strain GH-2 to the allyl alcohol ketone with the concentration of 0.1-1g/L is more than 95%, which shows that the strain can effectively degrade organic pollutants in the pesticide production wastewater.
Example 7
The degradation application of the microbial inoculum in the actual pesticide production wastewater is as follows:
the used wastewater is derived from the production wastewater of a certain pesticide enterprise, the pH of the wastewater is 7.6, the total salt concentration is 12g/L, the measured concentration of allethrin is 146mg/L, the concentration of allyl alcohol ketone is 412mg/L, the concentration of 5-methylfurfural is 973mg/L, and the concentration of furancarbinol is 1028mg/L. Taking aerobic activated sludge in a biochemical tank of the enterprise sewage treatment plant, standing, removing supernatant, adding clear water, mixing uniformly, standing, removing supernatant, and repeating the operation for 3 times to obtain cleaned sludge. Setting a control group: adding 200mL of the cleaned sludge into a 2L wide-mouth glass bottle containing 1.5L of wastewater, uniformly mixing, and placing an aerator; the experimental group further added the microbial inoculum prepared from the strain GH-2 on the basis of the addition of the activated sludge, the addition amount was 10%, and the concentration of each intermediate was measured after 3 days, and the results are shown in FIG. 6.
As can be seen from fig. 6, when only activated sludge was added, the degradation rates of allethrin, allylenone, 5-methylfurfural, and furfuryl alcohol after 3 days were 9.5%, 13%, 15.2%, and 21.6%, respectively; after the strain GH-2 is added for reinforcement, the degradation rates of allethrin, allyl alcohol ketone, 5-methylfurfural and furfuryl alcohol in the wastewater respectively reach 94.5%, 96%, 97.4% and 98.1%. The experimental data show that the strain GH-2 has good application prospect in actual pesticide wastewater treatment.
The foregoing shows and describes the general principles, principal features and advantages of the invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalents or equivalent changes fall within the protection scope of the present invention.
Claims (10)
1. A strain of Citrobacter freundii, characterized in thatCitrobacter freundii) Naming ofGH-2, which has been preserved in the China general microbiological culture Collection center (CGMCC), the preservation date is 2022, 9 months and 9 days, and the preservation number is CGMCC No.25671.
2. The application of the citrobacter freundii in degrading organic pollutants in pesticide production wastewater under the low-temperature and high-salt conditions as claimed in claim 1, wherein the organic pollutants are one or more of allethrin, allyl alcohol ketone, 5-methyl furfural or furan methanol, and the content of the organic pollutants is 0.1-1g/L; the salt concentration in the pesticide production wastewater is 1 to 20g/L, and the temperature is 10 to 30 ℃.
3. A microbial preparation produced by using the Corynebacterium freundii strain according to claim 1.
4. The application of the microbial inoculum of claim 3 in degrading organic pollutants in pesticide production wastewater under low-temperature and high-salt conditions, wherein the organic pollutants are one or more of allethrin, allylenone, 5-methylfurfural or furfuryl alcohol, and the content of the organic pollutants is 0.1-1g/L; the salt concentration in the pesticide production wastewater is 1 to 20g/L, and the temperature is 10 to 30 ℃.
5. A preparation method of the microbial inoculum according to claim 3, which comprises the following specific steps:
s1, selecting a single colony of Citrobacter freundii GH-2, inoculating the single colony in an LB liquid culture medium, oscillating in a shaking table, and culturing to a logarithmic phase to obtain a bacterial liquid;
s2, inoculating the bacterial liquid to a seed tank, and culturing to logarithmic phase to obtain a seed liquid;
s3, inoculating the seed liquid into a fermentation tank for fermentation culture, and obtaining the microbial inoculum after fermentation is finished.
6. The method for preparing microbial inoculum according to claim 5, wherein in the step S1, the LB liquid culture medium comprises the following components: 5g/L of yeast extract powder, 10g/L of peptone and 10g/L of sodium chloride, and the pH value is 7.0-7.5; the culture conditions were: the temperature is 30 to 35 ℃, and the time is 12 to 24h.
7. The method for preparing microbial inoculum according to claim 5, wherein in the steps S2 and S3, the components of the fermentation tank culture medium and the seeding tank culture medium are the same and are as follows: glucose 8g/L, yeast extract powder 5g/L, K 2 HPO 4 1g/L、NaCl 5g/L、CaCO 3 2g/L、MgSO 4 ·7H 2 0.2g/L of O, 0.1% (v/v) of soybean oil and 7.0 to 7.5 of pH.
8. The method for preparing the microbial inoculum according to claim 5, wherein in the steps S2 and S3, the culture conditions of a seeding tank and a fermentation tank are the same, the volume ratio of the ventilation volume of sterile air to the fermentation medium in the culture process is 1:0.6 to 1.2, the stirring speed is 180 to 240 rpm, the culture temperature is 30 to 35 ℃, and the whole-process culture time is 48 to 96h.
9. The method for preparing the microbial inoculum according to claim 5, wherein in the step S2, the inoculation amount of the bacterial liquid is 5-10% by volume; in the step S3, the inoculation amount of the seed liquid is 1-8% by volume ratio.
10. The method for preparing microbial inoculum according to claim 5, wherein in step S3, the number of thalli in fermentation broth after fermentation is finished reaches 10 8 More than one/mL.
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| CN113461172A (en) * | 2021-06-08 | 2021-10-01 | 戴威 | Microbial agent for treating high-salt industrial wastewater and preparation method thereof |
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