CN111908620A - Method for enhancing degradation of aniline by rhodococcus through environmental stress - Google Patents

Method for enhancing degradation of aniline by rhodococcus through environmental stress Download PDF

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CN111908620A
CN111908620A CN202010695689.XA CN202010695689A CN111908620A CN 111908620 A CN111908620 A CN 111908620A CN 202010695689 A CN202010695689 A CN 202010695689A CN 111908620 A CN111908620 A CN 111908620A
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aniline
stress
phenol
rhodococcus
nacl
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CN111908620B (en
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谭周亮
王臣
陈杨武
周后珍
谢翼飞
李旭东
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Chengdu Institute of Biology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/02—Aerobic processes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/34—Organic compounds containing oxygen
    • C02F2101/345—Phenols
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/38—Organic compounds containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F2103/06—Contaminated groundwater or leachate
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    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

Abstract

The invention discloses a method for enhancing Rhodococcus sp degradation aniline by environmental stress, belonging to the field of biological enhancement treatment of environmental pollution. The method of the invention is mainly based on the principle that the responses of Rhodococcus sp to phenol, NaCl, aniline and low temperature (15-25 ℃) stress are the same or similar, and the Rhodococcus sp is pretreated by using phenol, NaCl and low temperature, so that the degradation capability of Rhodococcus sp to aniline under abiotic environmental stress is improved. The method provided by the invention has the advantages of obvious effect of improving the aniline treatment capacity, simple and convenient process, low cost and no secondary pollution, and can be widely applied to industrial wastewater treatment, soil remediation, underground water remediation and other scenes.

Description

Method for enhancing degradation of aniline by rhodococcus through environmental stress
Technical Field
The invention belongs to the field of biological enhancement treatment of environmental pollution, and particularly relates to a method for enhancing rhodococcus to degrade aniline by utilizing environmental stress.
Background
Aniline (also called anilin, anilin oil and aminobenzene) is used as an important chemical raw material, can be used as a black dye, and a derivative methyl orange can be used as an indicator for acid-base titration. Meanwhile, aniline is an important intermediate product of more than 300 chemical substances, is widely applied to industries such as printing and dyeing, plastics, paint, agriculture, medicine and the like, and is one of the most produced industrial chemicals in the world. Aniline was also used as a reference compound for determining the microbial degradation activity during wastewater treatment.
When the environment polluted by aniline is bioremediation, abiotic environment stress such as multi-component solvent, high salt and the like is often suffered, and meanwhile, low-temperature abiotic environment stress is caused by four-season changes of environmental temperature. These abiotic environmental stresses affect intracellular enzymatic activity, membrane fluidity, and the destruction of DNA and protein structures, thereby inhibiting the growth of microorganisms and reducing metabolic activity of microorganisms. This not only results in poor contaminant treatment, but also limits the practical use of microorganisms in bioremediation processes.
Rhodococcus sp is considered as one of the best candidate strains for bioremediation of environmental pollution due to the advantages of degrading various refractory organic pollutants and having high tolerance to extreme environments, and therefore Rhodococcus sp is widely applied to bioremediation. Such as 4-nitrophenol-degrading Rhodococcus sp.BUPNP1 strain, quinoline-degrading Rhodococcus sp.ATCC 49988 strain, heavy metal-reducing Rhodococcus erythropolis SBEL05 strain, biodesulfurizing Rhodococcus erythropolis SX-12 strain. Rhodococcus sp cannot overcome the weakening of aniline-processing ability by various abiotic environmental stresses.
Disclosure of Invention
The invention aims to solve the problems that: improving the aniline processing capacity of the bacteria under the abiotic environmental stress.
The inventor finds that Rhodococcus sp responds to phenol, NaCl, aniline and low-temperature (15-25 ℃) stress on physical and chemical indexes or responds to transcriptome similarly. According to this finding, the inventors pretreated Rhodococcus sp at a low temperature using phenol, NaCl, thereby improving the ability of Rhodococcus sp to treat aniline under abiotic environmental stress.
The technical scheme of the invention is as follows:
a method of enhancing bacterial degradation of aniline using environmental stress, the method comprising:
(1) taking bacteria capable of degrading aniline, and carrying out aerobic culture for 18-30 h under the stress of 150-400 mg/L phenol, 20-45 g/L NaCl or 15-25 ℃;
(2) adding bacteria into wastewater containing 500-2000 mg/L aniline, and degrading aniline.
As mentioned above, the bacterium is Rhodococcus (Rhodococcus sp.), preferably Rhodococcus sp.an-P1 deposited in china general microbiological culture collection center (CGMCC) under the following accession number: CGMCC No. 2783.
The method as described above, comprising the steps of:
1) taking bacteria capable of degrading aniline, and activating the bacteria by using a culture medium containing 500-2000 mg/L aniline;
2) carrying out aerobic culture on the bacteria for 18-30 h under the stress of 150-400 mg/L phenol, 20-45 g/L NaCl and/or 15-25 ℃;
3) taking wastewater to be subjected to aniline degradation, inoculating the bacteria obtained after the culture in the step 2) into the wastewater with the aniline concentration of 500-2000 mg/L, and degrading aniline.
In the method, the wastewater in step 3) further contains 150-400 mg/L phenol, or further contains 20-45 g/L NaCl, or is at a temperature of 15-25 ℃.
According to the method, the stress type of the step 2) is 150-400 mg/L phenol stress, and the wastewater in the step 3) contains 150-400 mg/L phenol, 20-45 g/L NaCl or is at the temperature of 15-25 ℃;
or the stress type of the step 2) is 20-45 g/L NaCl stress, and the wastewater in the step 3) contains 150-400 mg/L phenol and 20-45 g/L NaCl or is at the temperature of 15-25 ℃;
or the stress type in the step 2) is 15-25 ℃ temperature stress, and the wastewater in the step 3) contains 150-400 mg/L phenol and 20-45 g/L NaCl or is at 15-25 ℃.
The method is as described above, and the concentration of the aniline in the step 1) and/or the step 3) is 1500 mg/L; preferably, the concentration is 1000 mg/L.
As in the previous method, the concentration of phenol in step 2) was 250 mg/L.
As in the previous method, the NaCl concentration in step 2) was 35 g/L.
As in the previous method, the temperature in step 2) is 20 ℃.
As the method, step 3) is: the bacteria were formulated as OD600After the value is 1.00 +/-0.05, inoculating the strain into the wastewater according to the inoculation amount of 5-10% v/v.
The invention has the main beneficial effects that:
1. effectively improve the resistance of Rhodococcus sp to abiotic environmental stress and the degradation capability of aniline under the stress. For example: after the Rhodococcus sp is pretreated at low temperature (20 ℃), the tolerance of the Rhodococcus sp to phenol stress can be improved, the degradation rate of the Rhodococcus sp to aniline in phenol-containing environment is further improved, and the increase reaches 32.88% within 18 h.
2. The method has the advantages of simple and convenient process, low cost and no secondary pollution, and can improve the growth condition of cells under other stress conditions and the degradation condition of aniline by only collecting stressed Rhodococcus sp.AN-P1 thalli.
3. The method has wide application range and is suitable for industrial wastewater treatment, soil remediation, groundwater remediation and other scenes.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: AN-P1 gene expression changes under 250mg/L phenol stress condition; nosig, no significant difference; up, up regulation; down, down regulation.
FIG. 2: the gene expression of AN-P1 is changed under the condition of 2500mg/L aniline stress; nosig, no significant difference; up, up regulation; down, down regulation.
FIG. 3: AN-P1 gene expression changes under 35g/L NaCl stress condition; nosig, no significant difference; up, up regulation; down, down regulation.
FIG. 4: AN-P1 gene expression change under 20 ℃ low temperature stress condition; nosig, no significant difference; up, up regulation; down, down regulation.
Detailed Description
Example 1 method for enhancing degradation ability of Rhodococcus sp. by phenol stress
First, culture medium formula
The LB culture medium formula is: 10g of peptone, 5g of yeast powder, 10g of NaCl, 18-20 g of agar, pH 7.0 and supplementing water to 1000 mL; sterilizing at 121 deg.C under humid heat and high temperature for 20 min;
the formula of the inorganic salt solid culture medium is as follows: na (Na)2HPO4·12H2O 2g，KH2PO4 0.5g，MgSO4·7H20.3g of O, 5mL of trace element solution, 18-20 g of agar, pH 7.0 and supplementing water to 1000 mL; sterilizing at 121 deg.C under humid heat and high temperature for 20 min;
liquid culture with inorganic saltsThe base formula is as follows: na (Na)2HPO4·12H2O 2g，KH2PO4 0.5g，MgSO4·7H20.3g of O, 5mL of trace element solution, pH 7.0 and water to 1000 mL; sterilizing at 121 deg.C under humid heat and high temperature for 20 min;
the formula of the trace element solution comprises the following components: EDTA0.5g/L, ZnSO4·7H2O 0.22g/L，CaCl2 0.055g/L，MnCl2·4H2O 0.051g/L，FeSO4·7H2O 0.049g/L，(NH4)2Mo7O24·4H2O 0.011g/L，CuSO4·5H2O 0.0157g/L，CoCl2·6H2O0.016 g/L, pH 6.0 adjusted with KOH; filtering and sterilizing with 0.22um filter membrane.
Second, method
1. Inoculating the preserved Rhodococcus sp.AN-P1 into a slant LB culture medium, and culturing at 30 ℃ for 36-48 h to obtain a T1 generation strain of Rhodococcus sp.AN-P1; transferring the T1 generation strain to a slant inorganic salt solid culture medium containing 1000mg/L aniline, and culturing at 30 deg.C for 36-48 h to obtain activated Rhodococcus sp.AN-P1 strain;
2. inoculating the activated Rhodococcus sp.AN-P1 into an inorganic salt liquid culture medium containing 1000mg/L aniline, and culturing for 18-24 h at 30 ℃ under aerobic conditions to obtain Rhodococcus sp.AN-P1 seed liquid capable of efficiently degrading aniline;
3. respectively applying 150-400 mg/L phenol stress to the seed liquid in the step (2), and carrying out aerobic culture for 18-30 h under the stress condition to obtain a strengthened strain;
4. collecting the reinforced strain obtained in (3), and collecting the bacterial liquid OD600The value of (3) is adjusted to 1.0 +/-0.05;
5. will adjust the OD600The bacterial liquid with the bacterial concentration is obtained by respectively treating the synthetic wastewater containing aniline under the stress of phenol, NaCl and the temperature of 15-25 ℃ by using the bacterial liquid with the inoculum concentration of 5-15% v/v.
Example 2 method for enhancing degradation ability of Rhodococcus sp. by NaCl stress
First, culture medium formula
The same as in example 1.
Second, method
1. Inoculating the preserved Rhodococcus sp.AN-P1 into a slant LB culture medium, and culturing at 30 ℃ for 36-48 h to obtain a T1 generation strain of Rhodococcus sp.AN-P1; transferring the T1 generation strain to a slant inorganic salt solid culture medium containing 1000mg/L aniline, and culturing at 30 deg.C for 36-48 h to obtain activated Rhodococcus sp.AN-P1 strain;
2. inoculating the activated Rhodococcus sp.AN-P1 into an inorganic salt liquid culture medium containing 1000mg/L aniline, and culturing for 18-24 h at 30 ℃ under aerobic conditions to obtain Rhodococcus sp.AN-P1 seed liquid capable of efficiently degrading aniline;
3. respectively applying 20-45 g/L NaCl stress to the seed liquid in the step (2), and carrying out aerobic culture for 18-30 h under the stress condition to obtain an enhanced strain;
4. collecting the reinforced strain obtained in (3), and collecting the bacterial liquid OD600The value of (3) is adjusted to 1.0 +/-0.05;
5. will adjust the OD600The bacterial liquid with the bacterial concentration is obtained by respectively treating the synthetic wastewater containing aniline under the stress of phenol, NaCl and the temperature of 15-25 ℃ by using the bacterial liquid with the inoculum concentration of 5-15% v/v.
Example 3 method for enhancing degradation ability of Rhodococcus sp. by Low temperature stress
First, culture medium formula
The same as in example 1.
Second, method
1. Inoculating the preserved Rhodococcus sp.AN-P1 into a slant LB culture medium, and culturing at 30 ℃ for 36-48 h to obtain a T1 generation strain of Rhodococcus sp.AN-P1; transferring the T1 generation strain to a slant inorganic salt solid culture medium containing 1000mg/L aniline, and culturing at 30 deg.C for 36-48 h to obtain activated Rhodococcus sp.AN-P1 strain;
2. inoculating the activated Rhodococcus sp.AN-P1 into an inorganic salt liquid culture medium containing 1000mg/L aniline, and culturing for 18-24 h at 30 ℃ under aerobic conditions to obtain Rhodococcus sp.AN-P1 seed liquid capable of efficiently degrading aniline;
3. respectively applying low temperature stress of 15-25 ℃ to the seed liquid in the step (2), and carrying out aerobic culture for 18-30 h under the stress condition to obtain a strengthened strain;
4. collecting the reinforced strain obtained in (3), and collecting the bacterial liquid OD600The value of (3) is adjusted to 1.0 +/-0.05;
5. will adjust the OD600The bacterial liquid with the bacterial concentration is obtained by respectively treating the synthetic wastewater containing aniline under the stress of phenol, NaCl and the temperature of 15-25 ℃ by using the bacterial liquid with the inoculum concentration of 5-15% v/v.
The advantageous effects of the present invention are further illustrated in the form of experimental examples. The formulation of the medium used in the experimental examples is not specifically described, and is the same as in example 1.
Experimental example 1: degradation of phenol
Method and device
Inoculating the preserved Rhodococcus sp.AN-P1 into LB slant culture medium, and culturing at 30 deg.C for 36-48 h to obtain T1 generation strain of Rhodococcus sp.AN-P1; transferring the T1 generation strain to an inorganic salt slant solid culture medium containing 1000mg/L aniline, and culturing at 30 ℃ for 36-48 h to obtain an activated Rhodococcus sp.AN-P1 strain;
the above strains were inoculated into synthetic media containing 50mg/L, 100mg/L, 200mg/L, and 250mg/L phenol, respectively, and cultured aerobically at 30 ℃ and sampled every 3 hours to detect the phenol content (as shown in Table 1). The determination result shows that AN-P1 can degrade phenol which is the only carbon source in the synthetic medium and utilize the phenol for growth and propagation.
The formula of the synthetic culture medium is as follows: na (Na)2HPO4 2g，KH2PO4 0.5g，(NH4)2SO4 0.3g，MgSO4·7H20.3g of O, 5mL of trace element solution, pH 7.0 and water to 1000 mL.
Second, result in
As shown in Table 1, AN-P1 has a phenol degrading effect.
TABLE 1 degradation Effect of AN-P1 on phenol of different concentrations
Figure BDA0002590170320000051
Phenol is an important organic chemical raw material, and has important application in the industries of chemical raw materials, alkylphenol, synthetic fiber, plastics, synthetic rubber, medicines, pesticides, spices, dyes, coatings, oil refining and the like, and phenol pollution is very serious due to the wide application of phenol. The experimental example proves that AN-P1 has the function of degrading phenol, and lays the foundation for AN-P1 to treat phenol pollution.
Experimental example 2: gene expression response
Method and device
The method comprises the steps of collecting thalli from seed liquid stressed by 250mg/L phenol, 2500mg/L aniline, 35g/L NaCl and 20 ℃, extracting mRNA, carrying out sequencing by an Illumina sequencing platform, and carrying out comparative analysis by taking Rhodococcus pyrivorans strain GF3(https:// www.ncbi.nlm.nih.gov/nucore/NZ _ CP022915.1) as a reference genome to obtain the significant condition of gene expression change, wherein the significant condition is sequentially shown in figures 1-4.
Second, result in
Rhodococcus sp.AN-P1 under stress conditions of 250mg/L phenol, 2500mg/L aniline, 35g/L NaCl and 20 ℃ had 670, 949, 820 and 963 differential genes for significant response. The number of the genes with the common significant difference between every two stresses is shown in table 2, and the number of the genes which respond to AN-P1 together under the conditions of high salt stress and high-concentration aniline stress is the largest, 95 expression genes are significantly up-regulated together, and 356 expression genes are significantly down-regulated together.
In terms of the functions of the genes with significant differences, the genes are mainly distributed in the aspects of five metabolic systems, namely an amino acid metabolic system, an ABC transport system, a fatty acid synthesis and degradation system, an antioxidant system and an energy metabolic system. For example, in the aspect of fatty acid synthesis and degradation systems, the synthesis and degradation genes of phenol, high-concentration aniline, NaCl and fatty acid under the stress condition of 20 ℃ are obviously down-regulated and expressed to different degrees. In the aspect of energy metabolism, AN-P1 shows that related genes related to energy metabolism are obviously down-regulated and expressed when suffering from phenol, high-concentration aniline, NaCl and 20 ℃ stress, such as genes of a pyruvate dehydrogenase system, succinate dehydrogenase and the like in a TCA cycle, and genes of a complex I, a complex II, a complex IV and the like in AN oxidative phosphorylation process.
TABLE 2 number of significantly different genes shared by AN-P1 under different stress conditions
Figure BDA0002590170320000061
Note: two stresses share significant difference gene number (share significant up-regulated difference gene number/share significant down-regulated difference gene number)
And (4) conclusion: the gene expression is the most critical link of biological adaptation environment, and the same or similar response mechanisms exist in multiple aspects of gene expression among phenol, high-concentration aniline, NaCl and 20 ℃ stress, so that the improvement of the aniline degrading capability of Rhodococcus sp by phenol, NaCl and low-temperature pretreatment becomes possible.
Experimental example 3: physical and chemical index change of stress response
Method and device
The surface hydrophobicity change, the self-aggregation change and the fatty acid content change are respectively detected by an adhering hydrocarbon method, a sedimentation method and a gas chromatography.
Second, result in
As shown in Table 3, the changes of surface hydrophobicity of AN-P1 under stress conditions of 250mg/L phenol, 2500mg/L aniline, 35g/L NaCl and 20 ℃ respectively are shown. The hydrophobicity responses under phenol, high-concentration aniline and high-salt stress are similar, and are respectively improved by 13.86%, 8.67% and 5.17% compared with a control group. This may be beneficial for the phenol or high salt stressed strains to increase their ability to handle phenol, high concentrations of aniline and high salt stressed aniline.
TABLE 3 hydrophobicity Change under different stress conditions
Control group Phenol and its preparation High concentration of aniline High salt 20℃
Hydrophobicity% 48.79 62.65 57.46 53.94 15.22
Change in hydrophobicity% +13.86 +8.67 +5.15 -33.57
Note: + denotes an increase; -represents a decrease.
As shown in Table 4, the self-aggregative changes of AN-P1 under stress conditions of 250mg/L phenol, 2500mg/L aniline, 35g/L NaCl and 20 ℃ are shown. The self-aggregation change is similar under the stress of phenol and high-concentration aniline, and is respectively increased by 16.14 percent and 8.4 percent in 4 hours and 12.82 percent and 6.69 percent in 8 hours compared with a control group. The change of self-aggregation property under high salt and 20 ℃ aniline stress is similar, the change is respectively reduced by 6.86 percent and 1.15 percent in the comparison group, and the change is respectively reduced by 8.27 percent and 3.14 percent in 8 hours in comparison with the comparison group. The strain stressed by phenol can be beneficial to improving the capability of the strain stressed by phenol and aniline under the stress of high-concentration aniline; is beneficial to improving the capability of the strain stressed at 20 ℃ for treating aniline with high salt content and under 20 ℃ stress.
TABLE 4 Segregation changes under different stress conditions
Control group Phenol and its preparation High concentration of aniline High salt 20℃
Self-agglutination for 4 h% 27.87 44.01 36.27 21.01 26.72
Self-agglutination for 8 h% 45.19 58.01 51.88 36.92 42.05
4h self-aggregative Change% +16.14 +8.4 -6.86 -1.15
Self-aggregative change after 8 h% +12.82 +6.69 -8.27 -3.14
Note: + denotes an increase; -represents a decrease.
Table 5 shows the changes of the fatty acid content of AN-P1 under 250mg/L phenol, 2500mg/L aniline, 35g/L NaCl and 20 ℃ stress conditions, respectively. Responses under phenol, high salt and 20 ℃ stress of C18:0, C16:1 and C18:1 are similar, wherein C18:0 is reduced by 1.85%, 2.17% and 2.40% respectively relative to a control group, C16:1 is increased by 2.47%, 0.79% and 9.08% respectively relative to the control group, and C18:1 is increased by 6.47%, 15.05% and 9.52% respectively relative to the control group. This may be beneficial for the ability of the strain to handle phenol, high salt, 20 ℃ stressed, and high concentrations of aniline at 20 ℃. In addition, the response of C16:0 is similar under high salt and 20 ℃ stress, and is respectively reduced by 6.13 percent and 7.94 percent; similar to phenol and high-concentration aniline under stress, the phenol and the aniline are respectively increased by 0.64 percent and 7.17 percent. The degradation of the strain to the aniline under the stress of high-concentration aniline is improved after phenol stress; is beneficial to improving the degradation of the aniline under the high salt stress of the strain after the stress of 20 ℃.
TABLE 5 fatty acid content Change under different stress conditions
Figure BDA0002590170320000071
Figure BDA0002590170320000081
Note: + denotes an increase; -represents a decrease.
This experimental example reveals the physiological basis of Rhodococcus anococcus AN-P1 for resistance to phenol, high concentrations of aniline, high salt, 20 degrees Celsius (low temperature).
Experimental example 4: phenol stress increases the ability of Rhodococcus to adapt to other stresses
Method and device
Inoculating Rhodococcus sp.AN-P1 to LB slant culture medium, and culturing at 30 deg.C for 36-48 h to obtain T1 generation strain of Rhodococcus sp.AN-P1; transferring the T1 generation strain to an inorganic salt slant solid culture medium containing 1000mg/L aniline, and culturing at 30 ℃ for 36-48 h to obtain an activated Rhodococcus sp.AN-P1 strain; inoculating the activated Rhodococcus sp.AN-P1 strain to an inorganic salt liquid culture medium containing 1000mg/L aniline, and culturing at 30 ℃ for 18-24 h under aerobic conditions to obtain the Rhodococcus sp.AN-P1 seed liquid capable of efficiently degrading aniline.
Collecting the bacteria, inoculating the bacteria into an inorganic salt culture medium containing 250mg/L phenol and 1000mg/L aniline according to the inoculation amount of 5%, and culturing for 18-24 h under aerobic conditions at 30 ℃ to obtain the phenol-stressed Rhodococcus sp.AN-P1 seed solution. Adjusting OD of bacterial suspension after phenol stress600The value was 1.00. + -. 0.05. Respectively degrading 250mg/L phenol, 2500mg/L high-concentration aniline and 35g/L aniline stressed by NaCl according to the inoculum size of 10% at 30 ℃ under aerobic conditions. Meanwhile, the bacterial suspension stressed and regulated by phenol is directly degraded at the temperature of 20 ℃ under aerobic condition by 1000mg/L aniline according to the inoculation amount of 10%. After 18h and 24h, sampling to detect the aniline concentration. Meanwhile, non-stressed cells were used as a control group to compare aniline reductionAnd (4) solving rate.
Second, result in
As shown in Table 6, when 250mg/L phenol-stressed AN-P1 bacterial suspension is subjected to 250mg/L phenol and 1000mg/L aniline, the degradation rate of aniline at 18h is 30.64% higher than that of aniline at 24h, and the degradation rate of aniline at 24h is 41.29% higher than that of aniline at 24 h; under the stress of 2500mg/L high-concentration aniline, the degradation rate of aniline is increased by 8.33% at 18h compared with that without phenol stress, and the degradation rate of aniline is increased by 11.64% at 24h compared with that without phenol stress; under the conditions of 35g/L NaCl and 1000mg/L aniline stress, the degradation rate of aniline is improved by 6.74% in 18h and 7.13% in 24h compared with that of aniline; under the condition of meeting the aniline condition of 20 ℃ and 1000mg/L, the degradation rate of aniline is improved by 5.22% in 18h compared with that without phenol stress, and the degradation rate of aniline is improved by 6.50% in 24h compared with that without phenol stress.
TABLE 6 enhancement of aniline degradation by phenol stress
Figure BDA0002590170320000091
Third, conclusion
Rhodococcus sp, previously stressed with phenol, is more adaptable to phenol, high concentrations of aniline, high salt and 20 ℃ stress, and increases the degradation rate of aniline in the presence of the aforementioned stress.
Experimental example 5: 20 ℃ stress improves the capability of rhodococcus to adapt to other stresses
Method and device
Inoculating Rhodococcus sp.AN-P1 to LB slant culture medium, and culturing at 30 deg.C for 36-48 h to obtain T1 generation strain of Rhodococcus sp.AN-P1; transferring the T1 generation strain to an inorganic salt slant solid culture medium containing 1000mg/L aniline, and culturing at 30 ℃ for 36-48 h to obtain an activated Rhodococcus sp.AN-P1 strain; inoculating the activated Rhodococcus sp.AN-P1 strain to an inorganic salt liquid culture medium containing 1000mg/L aniline, and culturing at 30 ℃ for 18-24 h under aerobic conditions to obtain the Rhodococcus sp.AN-P1 seed liquid capable of efficiently degrading aniline.
Collecting the bacteria, inoculating the bacteria into an inorganic salt culture medium containing 1000mg/L aniline according to the inoculation amount of 5%, and culturing for 27-30 h under the conditions of stress at 20 ℃ and aerobiosis to obtain the phenol-stressed Rhodococcus sp.AN-P1 seed solution. Adjusting OD of bacterial suspension after phenol stress600The value was 1.00. + -. 0.05. Respectively degrading 250mg/L phenol, 2500mg/L high-concentration aniline and 35g/L aniline stressed by NaCl according to the inoculum size of 10% at 30 ℃ under aerobic conditions. Meanwhile, the bacterial suspension stressed and regulated at the temperature of 20 ℃ is directly degraded by 1000mg/L aniline at the temperature of 20 ℃ under aerobic conditions according to the inoculation amount of 10%. After 18h and 24h, sampling to detect the aniline concentration. Meanwhile, the cells which are not stressed are used as a control group, and the degradation rate of the aniline is compared with that of an experimental group.
Second, result in
As shown in Table 7, the AN-P1 bacterial suspension stressed at 20 ℃ can improve the degradation rate of aniline at 18h by 32.88% compared with that without stress at 20 ℃ and improve the degradation rate of aniline at 24h by 39.45% compared with that without stress at 20 ℃ under the conditions of 250mg/L phenol and 1000mg/L aniline; under the stress of 2500mg/L high-concentration aniline, the degradation rate of aniline is improved by 6.12% in 18h compared with that without the stress of 20 ℃, and the degradation rate of aniline is improved by 8.56% in 24h compared with that without the stress of 20 ℃; under the conditions of 35g/L NaCl and 1000mg/L aniline stress, the degradation rate of aniline is improved by 13.35% in 18h and 13.69% in 24h compared with that without 20 ℃ stress; under the condition of meeting the 20 ℃ and 1000mg/L of aniline, the degradation rate of aniline in 18 hours is increased by 19.29 percent compared with that without 20 ℃ stress, and the degradation rate of aniline in 24 hours is increased by 26.44 percent compared with that without 20 ℃ stress.
TABLE 720 ℃ first stress-enhanced degradation of aniline
Figure BDA0002590170320000101
Third, conclusion
Rhodococcus sp, previously stressed at 20 ℃ is more adaptable to phenol, high concentrations of aniline, high salt and 20 ℃ stress, and increases the degradation rate of aniline in the presence of the aforementioned stress.
Experimental example 6: utilization of NaCl stress
Method and device
Inoculating Rhodococcus sp.AN-P1 to LB slant culture medium, and culturing at 30 deg.C for 36-48 h to obtain T1 generation strain of Rhodococcus sp.AN-P1; transferring the T1 generation strain to an inorganic salt slant solid culture medium containing 1000mg/L aniline, and culturing at 30 ℃ for 36-48 h to obtain an activated Rhodococcus sp.AN-P1 strain; inoculating the activated Rhodococcus sp.AN-P1 strain to an inorganic salt liquid culture medium containing 1000mg/L aniline, and culturing at 30 ℃ for 18-24 h under aerobic conditions to obtain the Rhodococcus sp.AN-P1 seed liquid capable of efficiently degrading aniline.
Collecting the bacteria, inoculating the bacteria into an inorganic salt culture medium containing 35g/L NaCl and 1000mg/L aniline according to the inoculation amount of 5%, and culturing for 18-24 h under aerobic conditions at 30 ℃ to obtain a NaCl-stressed Rhodococcus sp.AN-P1 seed solution. Adjusting OD of bacterial suspension after phenol stress600The value was 1.00. + -. 0.05. Respectively degrading 250mg/L phenol, 2500mg/L high-concentration aniline and 35g/L aniline stressed by NaCl according to the inoculum size of 10% at 30 ℃ under aerobic conditions. Meanwhile, the bacterial suspension stressed and regulated by NaCl is directly degraded at the temperature of 20 ℃ under aerobic condition by 1000mg/L aniline according to the inoculation amount of 10%. After 18h and 24h, sampling to detect the aniline concentration. Meanwhile, cells without stress are used as a control group, and the degradation rate of the aniline is compared.
Second, result in
As shown in Table 8, after 35g/L NaCl stress, the AN-P1 bacterial suspension can be subjected to 250mg/L phenol and 1000mg/L aniline, the aniline degradation rate at 18h is improved by 30.64% compared with that without phenol stress, and the aniline degradation rate at 24h is improved by 41.29% compared with that without phenol stress; under the stress of 2500mg/L high-concentration aniline, the degradation rate of aniline is increased by 8.33% at 18h compared with that without phenol stress, and the degradation rate of aniline is increased by 11.64% at 24h compared with that without phenol stress; under the conditions of 35g/L NaCl and 1000mg/L aniline stress, the degradation rate of aniline is improved by 6.74% in 18h and 7.13% in 24h compared with that of aniline; under the condition of meeting the aniline condition of 20 ℃ and 1000mg/L, the degradation rate of aniline is improved by 5.22% in 18h compared with that without phenol stress, and the degradation rate of aniline is improved by 6.50% in 24h compared with that without phenol stress.
TABLE 8 enhancement of aniline degradation by NaCl stress
Figure BDA0002590170320000111
Third, conclusion
Rhodococcus sp, previously stressed with NaCl, is more adaptable to phenol, high concentrations of aniline, high salt and 20 ℃ stress and increases the degradation rate of aniline in the presence of the aforementioned stress.
In conclusion, the method can effectively improve the degradation capability of Rhodococcus sp on aniline under various abiotic environmental stresses through 150-400 mg/L phenol stress, 20-45 g/L NaCl stress or 15-25 ℃ temperature stress. It should be especially noted that the method of the present invention is also applicable to other strains having aniline degrading ability, and it is within the scope of the present invention to replace Rhodococcus sp.

Claims (10)

1. A method for enhancing bacterial degradation of aniline using environmental stress, the method comprising:
(1) taking bacteria capable of degrading aniline, and carrying out aerobic culture for 18-30 h under the stress of 150-400 mg/L phenol, 20-45 g/L NaCl or 15-25 ℃;
(2) adding bacteria into wastewater containing 500-2000 mg/L aniline, and degrading aniline.
2. The method of claim 1, wherein: the bacterium is Rhodococcus (Rhodococcus sp.), preferably Rhodococcus sp.AN-P1 preserved in China general microbiological culture Collection center (CGMCC) with the preservation number: CGMCC No. 2783.
3. A method according to claim 1 or 2, characterized in that the method comprises the steps of:
1) taking bacteria capable of degrading aniline, and activating the bacteria by using a culture medium containing 500-2000 mg/L aniline;
2) carrying out aerobic culture on the bacteria for 18-30 h under the stress of 150-400 mg/L phenol, 20-45 g/L NaCl and/or 15-25 ℃;
3) taking wastewater to be subjected to aniline degradation, inoculating the bacteria obtained after the culture in the step 2) into the wastewater with the aniline concentration of 500-2000 mg/L, and degrading aniline.
4. The method of claim 3, wherein: the wastewater in the step 3) also contains 150-400 mg/L phenol, or also contains 20-45 g/L NaCl, or is at the temperature of 15-25 ℃.
5. The method of claim 4, wherein:
the stress type of the step 2) is 150-400 mg/L phenol stress, and the wastewater in the step 3) contains 150-400 mg/L phenol and 20-45 g/L NaCl or is at the temperature of 15-25 ℃;
or the stress type of the step 2) is 20-45 g/L NaCl stress, and the wastewater in the step 3) contains 150-400 mg/L phenol and 20-45 g/L NaCl or is at the temperature of 15-25 ℃;
or the stress type in the step 2) is 15-25 ℃ temperature stress, and the wastewater in the step 3) contains 150-400 mg/L phenol and 20-45 g/L NaCl or is at 15-25 ℃.
6. The method of any of claims 3 to 5, wherein:
the concentration of the aniline in the step 1) and/or the step 3) is 1500 mg/L; preferably, the concentration is 1000 mg/L.
7. The method of any of claims 3 to 5, wherein:
the concentration of phenol in step 2) was 250 mg/L.
8. The method of any of claims 3 to 5, wherein:
the NaCl concentration in step 2) was 35 g/L.
9. The method of any of claims 3 to 5, wherein:
the temperature in step 2) was 20 ℃.
10. The method of any of claims 3 to 5, wherein: the step 3) is as follows: the bacteria were formulated as OD600After the value is 1.00 +/-0.05, inoculating the strain into the wastewater according to the inoculation amount of 5-10% v/v.
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