CN116254195A - Liquid microbial agent for degrading TNT (TNT) polluted wastewater as well as preparation method and application thereof - Google Patents
Liquid microbial agent for degrading TNT (TNT) polluted wastewater as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a liquid microbial agent for degrading TNT polluted wastewater, and a preparation method and application thereof. The liquid microbial agent comprises: pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and Bacillus cereus TC08, 8; the bacterial content of Pseudomonas aeruginosa TC01 is (0.6-1.5) x 10 10 CFU/mL, bacillus thuringiensis TC05 with a bacterial content of (1.0-2.0). Times.10 9 CFU/mL, bacillus thuringiensis T C has a bacterial content of (1.5-2.0). Times.10 9 CFU/mL. Seed solutions of three strains are prepared according to a proportion of 3:4:3, culturing after mixing the materials according to the volume ratio, centrifuging and concentrating, and then adding sodium alginate and glycerol to obtain the liquid microbial agent. The liquid microbial agent for degrading TNT polluted wastewater is prepared by utilizing the three strains, and the three strains can cooperate to obviously improve the rapid degradation of TNT.
Description
Technical Field
The invention relates to the technical field of environmental remediation, in particular to a liquid microbial agent for degrading TNT (TNT) polluted wastewater, and a preparation method and application thereof.
Background
Nitroorganic explosives, such as 2, 4, 6-trinitrotoluene (TNT) and hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX), are commonly used in military operations, construction demolition, and mining explosions. Because of the wide manufacturing of these compounds, their use in ammunition testing and retirement activities, a large number of these compounds have contaminated the soil and waters around the world. The TNT particles dissolve and spread over time, causing contamination of soil and groundwater. The wastewater containing TNT is easy to pollute water sources, and nitro compounds in the wastewater are easy to accumulate in soil, so that the soil is polluted. The soil has strong adsorption effect on TNT, TNT can quickly infiltrate into the ground from the surface, so that groundwater is polluted, and TNT is easily absorbed and stored by roots of plants and finally influences human health through a food chain. At present, researchers at home and abroad conduct a great deal of research on TNT wastewater degradation. The technology for treating TNT wastewater comprises physical adsorption, electric flocculation, extraction, fenton method, anaerobic and aerobic biological methods and the like. Although the physicochemical method degrades the surface of the material, a plurality of byproduct pollutants are generated, secondary pollution is caused, the research cost is too high, and the practical application is limited greatly. The bioremediation has wide prospect in practical application because of environmental protection, no secondary pollution and low degradation cost.
At present, the use of microbial agents to reduce and eliminate environmental pollutants and to relieve environmental pressure is one of the important means for environmental protection, and has been widely used in the field of environmental protection. Microbial agents are classified into liquid, powder, granule and the like according to different classification methods. The liquid microbial agent has the characteristics of simple preparation process, short activation time, low cost and the like, and has great development potential and application prospect in practice. Pseudomonas aeruginosa (P.aeromonas) is originally called pseudomonas aeruginosa, is widely distributed in nature, is one of the most common bacteria existing in soil, has a certain TNT degradation capability, can play an important role in the bioremediation process of TNT-polluted soil or TNT-containing wastewater, and for example, the patent with the application number of CN201510563062.8 discloses Pseudomonas aeruginosa, a microbial inoculum containing the pseudomonas aeruginosa and a method for degrading TNT by using the pseudomonas aeruginosa, but the degradation speed is low, only about 56% of TNT is degraded in 48 hours, the efficient degradation of TNT is realized in a biostimulation and biofragmentation environment, and the complex degradation cost is high. He Yajing and the like research on degrading TNT wastewater by rhodopseudomonas palustris, but the degradation of TNT by the strain depends on the addition amount of an N source, and the highest degradation rate in the later period of 5d degradation is only 81%. For the mixed bacterial degradation of multiple strains, the breeding of TNT degrading bacteria and the treatment effect thereof under the condition of co-matrix (Xue Xiangdong, etc., xinjiang environmental protection 2002, 27 (1): 41-45) uses 2 pseudomonas and 1 bacillus, three strains come from degrading activated sludge in an SBR ammunition destruction wastewater treatment system, and the activated sludge is domesticated and screened to degrade TNT in the wastewater by simulating the treatment of the wastewater: the degradation rate is 80-90% within 10d, and the degradation efficiency is still to be improved. Therefore, the development of the efficient degradation strain of TNT and the preparation of the composite microbial agent with practical production and application values are effective ways for TNT wastewater treatment.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a liquid microbial agent for degrading TNT polluted wastewater, and a preparation method and application thereof. According to the invention, pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and bacillus cereus TC08 are utilized to prepare the liquid microbial agent for degrading TNT polluted wastewater, and three strains can cooperate to obviously improve the rapid degradation of TNT. The preparation cost is low for the pollution of the energetic compound TNT in the technical field of environmental protection, the application is convenient, the liquid microbial agent is easy to store, and the TNT content can be rapidly reduced after the liquid microbial agent is used.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a liquid microbial agent for degrading TNT contaminated wastewater, the liquid microbial agent comprising: pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and Bacillus cereus TC08;
the bacterial content of the pseudomonas aeruginosa TC01 in the liquid microbial agent is (0.6-1.5) multiplied by 10 10 CFU/mL, bacillus thuringiensis TC05 with a bacterial content of (1.0-2.0). Times.10 9 CFU/mL, bacillus thuringiensis T C with a bacterial content of (1.5-2.0). Times.10 9 CFU/mL。
Preferably, the pseudomonas aeruginosa TC01 has a deposit number of: CGMCC No.1.868; the bacillus thuringiensis TC05 has a deposit number of: CGMCC No.1.6894; the preservation number of the bacillus cereus TC08 is as follows: CGMCC No.1.7914.
In a second aspect of the present invention, there is provided a method of preparing a liquid microbial agent comprising the steps of:
(1) Respectively inoculating pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and bacillus cereus TC08 into a first LB culture medium, and respectively obtaining pseudomonas aeruginosa TC01 seed bacterial liquid, bacillus thuringiensis TC05 seed bacterial liquid and bacillus cereus TC08 seed bacterial liquid after shake culture;
(2) The pseudomonas aeruginosa TC01 seed bacterial liquid, the bacillus thuringiensis TC05 seed bacterial liquid and the bacillus cereus TC08 seed bacterial liquid are mixed according to the volume ratio of 3:4:3, uniformly mixing to obtain mixed strain seed liquid, inoculating the mixed strain seed liquid into a second LB culture medium, shake-culturing for 24-36 h at 150-180 rpm at 30-37 ℃, centrifuging, and removing supernatant to obtain concentrated solution;
(3) And (3) adding sodium alginate and glycerol into the concentrated solution obtained in the step (2) to obtain the liquid microbial agent.
Preferably, in step (1), the composition of the first LB medium is: 10.0g/L tryptone, 10.0g/L NaCl, 5.0g/L yeast powder and H 2 O1L; the pH of the first LB medium was 7.0.
Preferably, in the step (1), the shake culture conditions are as follows: shake frequency is 150r/min, shake culture is carried out for 12h at 37 ℃; the inoculation amounts of pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and bacillus cereus TC08 are all 2.0wt%.
Preferably, in step (2), the composition of the second LB medium is: 10.0g/L tryptone, 10.0g/L NaCl, 5.0g/L yeast powder and H 2 O1L; the pH of the second LB medium was 7.0.
Preferably, in the step (2), the inoculation amount of the mixed bacteria seed liquid is 10.0wt%; the volume of the concentrated solution is 1/10-1/5 times of the volume before concentration.
Preferably, in the step (2), the addition amounts of the sodium alginate and the glycerol respectively account for 0.50% -1.00% of the total mass of the mixed solution.
In a third aspect of the invention, the application of the liquid microbial agent in degrading TNT polluted wastewater is provided.
Preferably, 10 to 20 weight percent of liquid microbial agent is added into TNT polluted wastewater and uniformly mixed, the temperature is 140 to 180r/min, the shaking culture is carried out for 4 to 6 days at 37 ℃, and the degradation of TNT polluted wastewater can be realized.
The invention has the beneficial effects that:
(1) According to the invention, pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and bacillus cereus TC08 are adopted to prepare the liquid microbial agent for degrading TNT polluted wastewater, and the three strains can cooperate to obviously improve the rapid degradation of TNT. And sodium alginate and glycerol are added, so that the shelf life of the liquid microbial agent is prolonged, and the utilization rate of the liquid microbial agent is further enhanced.
(2) The invention relates to a liquid microbial agent which is an energetic compound TNT pollution preparation cost in the technical field of environmental protection, is convenient to apply and easy to store, and the TNT content can be rapidly reduced after the liquid microbial agent is used.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, pseudomonas aeruginosa has the ability to degrade TNT, but has a slow degradation rate and requires degradation in a biostimulating and bio-enhancing environment.
Based on the above, the invention aims to provide a liquid microbial agent for degrading TNT polluted wastewater, and a preparation method and application thereof. According to the invention, bacillus thuringiensis TC05 and bacillus cereus TC08 which have no TNT degradation effect are compounded with pseudomonas aeruginosa TC01 which has TNT degradation effect, and three strains produce synergistic effect, so that the rapid degradation of TNT is obviously improved.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and are commercially available.
Description: pseudomonas aeruginosa TC01 accession number: CGMCC No.1.868, which is purchased from China general microbiological culture collection center;
the bacillus thuringiensis TC05 has a deposit number of: CGMCC No.1.6894, which is purchased from China general microbiological culture collection center;
the preservation number of the bacillus cereus TC08 is as follows: CGMCC No.1.7914, which is purchased from China general microbiological culture Collection center.
Example 1
Inoculating Pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and Bacillus cereus TC08 into tryptone 10.0g/L, naCl 10.0g/L, yeast powder 5.0g/L and H 2 LB at O1L, pH 7.0In the culture medium, the inoculation amount is 2.0wt percent, 150r/min and the culture medium is subjected to shake culture at 37 ℃ for 12 hours to respectively obtain pseudomonas aeruginosa TC01 seed bacterial liquid, bacillus thuringiensis TC05 seed bacterial liquid and bacillus cereus TC08 seed bacterial liquid.
And (3) preparing a microbial liquid degradation microbial agent: firstly, mixing raw liquid: pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and bacillus cereus TC08 seed bacterial solutions are mixed according to a volume ratio of 3:4:3, fully and uniformly mixing the materials according to the proportion to obtain the mixed strain seed liquid. Second, inoculating: inoculating 10.0wt% of mixed bacteria seed solution into 10.0g/L of tryptone, 10.0g/L of NaCl, 5.0g/L of yeast powder and H 2 O1L, LB medium at pH 7.0. Thirdly, culturing: shake culturing at 34 deg.C and 150rpm for 30 hr until the content of Pseudomonas aeruginosa TC01 in the culture solution is not lower than 6X10 9 CFU/mL, bacillus thuringiensis TC05 content is not less than 1.0X10 9 CFU/mL, bacillus cereus TC08 content is not less than 1.5X10 9 CFU/mL. Part of the supernatant is removed by centrifugation, the concentration of bacteria is increased, and the mixed solution of the compound microorganisms is concentrated to 1/10 volume times. Fourthly, preparing a microbial inoculum: and adding 0.50 weight percent of sodium alginate serving as a protective agent and 0.50 weight percent of glycerol into the concentrated mixed solution to obtain the TNT-polluted wastewater liquid microbial degradation microbial inoculum.
Example 2
The difference from example 1 is that:
thirdly, culturing: shake culturing at 30deg.C and 180rpm for 36 hr until the content of Pseudomonas aeruginosa TC01 in the culture solution is not less than 6X10 9 CFU/mL, bacillus thuringiensis TC05 content is not less than 1.0X10 9 CFU/mL, bacillus cereus TC08 content is not less than 1.5X10 9 CFU/mL. Part of the supernatant is removed by centrifugation, the concentration of bacteria is increased, and the mixed solution of the compound microorganisms is concentrated to 1/7.5 volume times. Fourthly, preparing a microbial inoculum: and adding 0.75 weight percent of sodium alginate serving as a protective agent and 0.75 weight percent of glycerol into the concentrated mixed solution to obtain the TNT-polluted wastewater liquid microbial degradation microbial inoculum.
Example 3
The difference from example 1 is that:
thirdly, culturing: shake culturing at 37deg.C and 165rpm for 24 hr until the content of Pseudomonas aeruginosa TC01 in the culture solution is not lower than 6X10 9 CFU/mL, bacillus thuringiensis TC05 content is not less than 1.5X10 9 CFU/mL, bacillus cereus TC08 content is not less than 1.0X10 9 CFU/mL. Part of the supernatant is removed by centrifugation, the concentration of bacteria is increased, and the mixed solution of the compound microorganisms is concentrated to 1/5 volume times. Fourthly, preparing a microbial inoculum: and adding 1.0 weight percent of sodium alginate serving as a protective agent and 1.0 weight percent of glycerol into the concentrated mixed solution to obtain the TNT-polluted wastewater liquid microbial degradation microbial inoculum.
Comparative example 1
The difference from example 1 is that: and (3) adding no bacillus thuringiensis TC05 and bacillus cereus TC08, and finally preparing the pseudomonas aeruginosa liquid degradation microbial inoculum.
Comparative example 2
The difference from example 1 is that: and (3) adding no pseudomonas aeruginosa TC01 and bacillus cereus TC08 to finally prepare the bacillus thuringiensis liquid degradation microbial inoculum.
Comparative example 3
The difference from example 1 is that: and (3) adding no pseudomonas aeruginosa TC01 and bacillus thuringiensis TC05 to finally prepare the bacillus cereus liquid degradation microbial inoculum.
Test examples
Taking TNT polluted wastewater in a ammunition destruction field, testing the components of the taken wastewater, taking 1mL of wastewater, adding an equal volume of acetonitrile for extraction, filtering the extract by a microporous membrane of 0.22 mu m, transferring the filtered extract into a chromatographic bottle, and carrying out liquid chromatography analysis, wherein the TNT content is 180mg/L.
The liquid microbial degradation microbial agents prepared in examples 1 to 3 and comparative examples 1 to 3 were added respectively and mixed uniformly (the addition amount of the liquid microbial degradation microbial agent prepared in examples 1 and comparative examples 1 to 3 was 10wt% and the liquid microbial degradation microbial agent prepared in example 2 was 15wt% and the liquid microbial degradation microbial agent prepared in example 3 was 20 wt%), 150r/min, shaking culture was performed at 37℃for 24 hours, the content or concentration of TNT in the contaminated wastewater was again detected, and the degradation rate of TNT was calculated, and the obtained results are shown in Table 1.
TABLE 1
| Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | |
| Degradation rate% | 97.6 | 96.9 | 98.2 | 50.7 | 21.3 | 23.2 |
As can be seen from Table 1, the degradation rates of TNT in wastewater by the liquid microbial degradation agents prepared in examples 1 to 3 are higher than the sum of the degradation rates of TNT in comparative examples 1 to 3, which indicates that the three strains of Pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and Bacillus cereus TC08 produce a synergistic effect, and the rapid degradation rate of TNT is improved. When the liquid microbial degradation microbial inoculum prepared by the invention is used for degrading TNT, other nutrient substances such as glucose and the like are not required to be added, the degradation cost is low, and the degradation speed is high.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (10)
1. A liquid microbial agent for degrading TNT contaminated wastewater, the liquid microbial agent comprising: pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and Bacillus cereus TC08;
the bacterial content of the pseudomonas aeruginosa TC01 in the liquid microbial agent is (0.6-1.5) multiplied by 10 10 CFU/mL, bacillus thuringiensis TC05 with a bacterial content of (1.0-2.0). Times.10 9 CFU/mL, bacillus thuringiensis T C has a bacterial content of (1.5-2.0). Times.10 9 CFU/mL。
2. The liquid microbial agent of claim 1, wherein pseudomonas aeruginosa TC01 has the accession number: CGMCC No.1.868; the bacillus thuringiensis TC05 has a deposit number of: CGMCC No.1.6894; the preservation number of the bacillus cereus TC08 is as follows: CGMCC No.1.7914.
3. The method for preparing the liquid microbial agent according to claim 1 or 2, characterized by comprising the following steps:
(1) Respectively inoculating pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and bacillus cereus TC08 into a first LB culture medium, and respectively obtaining pseudomonas aeruginosa TC01 seed bacterial liquid, bacillus thuringiensis TC05 seed bacterial liquid and bacillus cereus TC08 seed bacterial liquid after shake culture;
(2) The pseudomonas aeruginosa TC01 seed bacterial liquid, the bacillus thuringiensis TC05 seed bacterial liquid and the bacillus cereus TC08 seed bacterial liquid are mixed according to the volume ratio of 3:4:3, uniformly mixing to obtain mixed strain seed liquid, inoculating the mixed strain seed liquid into a second LB culture medium, shake-culturing for 24-36 h at 150-180 rpm at 30-37 ℃, centrifuging, and removing supernatant to obtain concentrated solution;
(3) And (3) adding sodium alginate and glycerol into the concentrated solution obtained in the step (2) to obtain the liquid microbial agent.
4. The liquid microbial agent according to claim 3, wherein in the step (1), the first LB medium has the composition: 10.0g/L tryptone, 10.0g/L NaCl, 5.0g/L yeast powder and H 2 O1L; the pH of the first LB medium was 7.0.
5. The liquid microbial agent according to claim 3, wherein in the step (1), the shake culture conditions are as follows: shake frequency is 150r/min, shake culture is carried out for 12h at 37 ℃; the inoculation amounts of pseudomonas aeruginosa TC01, bacillus thuringiensis TC05 and bacillus cereus TC08 are all 2.0wt%.
6. The method according to claim 3, wherein in the step (2), the composition of the second LB medium is: 10.0g/L tryptone, 10.0g/L NaCl, 5.0g/L yeast powder and H 2 O1L; the pH of the second LB medium was 7.0.
7. The method according to claim 3, wherein in the step (2), the inoculation amount of the mixed bacterial seed liquid is 10.0wt%; the volume of the concentrated solution is 1/10-1/5 times of the volume before concentration.
8. The preparation method of claim 3, wherein in the step (2), the addition amount of the sodium alginate and the glycerol is 0.50% -1.00% of the total mass of the mixed solution.
9. Use of the liquid microbial agent of claim 1 or 2 in degrading TNT contaminated wastewater.
10. The use according to claim 9, wherein 10-20 wt% of the liquid microbial agent according to claim 1 or 2 is added to the TNT contaminated wastewater and mixed uniformly, and the degradation of the TNT contaminated wastewater is achieved by shake cultivation for 1-3 d at 140-180 r/min and 37 ℃.
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