CN112175878A - Aniline efficient degradation microbial inoculum, preparation method thereof and application thereof in chemical wastewater - Google Patents
Aniline efficient degradation microbial inoculum, preparation method thereof and application thereof in chemical wastewater Download PDFInfo
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- CN112175878A CN112175878A CN202011117785.2A CN202011117785A CN112175878A CN 112175878 A CN112175878 A CN 112175878A CN 202011117785 A CN202011117785 A CN 202011117785A CN 112175878 A CN112175878 A CN 112175878A
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Abstract
The invention discloses an aniline efficient degradation microbial inoculum, a preparation method and application thereof in chemical wastewater. The aniline degradation microbial inoculum can grow by taking aniline as a unique carbon source and a unique nitrogen source, can degrade aniline containing 1000mg/L to below 50mg/L, and has a removal rate of over 95 percent.
Description
Technical Field
The invention belongs to the field of wastewater treatment by biological methods, and particularly relates to preparation and application of a composite microbial inoculum for treating aniline wastewater.
Background
Aniline is a yellowish or colorless oily liquid with a strong odor, is mainly used for manufacturing medicines, resins, dyes, rubber vulcanization accelerators and the like, and is widely present in waste water of printing and dyeing, rubber, pesticide and other industries, and the concentration of aniline is sometimes even up to thousands of milligrams per liter. However, when the concentration of aniline in wastewater is higher than 100mg/L, serious threat to aquatic organisms and human health can be caused, so aniline or derivatives thereof and the like are listed in a 'preferential pollutant blacklist' by EPA and China environmental protection departments, and are the most important of water pollution control in the environmental protection industry nowadays.
Aniline is a kind of organic matter which is difficult to degrade, and the existing methods for processing the matter are generally divided into chemical methods, physical methods and biological methods. Common physical treatment methods include adsorption, extraction and membrane separation. The adsorption method and the membrane separation method need to frequently replace an adsorption substance or a membrane, and both have higher cost; the aniline and the sewage to be treated are difficult to be completely separated by using the extraction method alone. The chemical method includes a redox method, a chlorine dioxide oxidation method, an ozone oxidation method, and the like. The oxidation method of chlorine dioxide is found in Cao Daoyuan etc. can remove more than 90% aniline; researches such as the navy show that the removal rate of the aniline by the ozone oxidation method reaches 96%, and the removal effect is more obvious when the concentration of the aniline is higher. A plurality of research results show that the biological method also makes a remarkable progress on the degradation of the aniline, a plurality of microorganisms such as bacillus, pseudomonas and the like are separated, and the aniline degradation efficiency is better for low-concentration aniline under aerobic and anaerobic conditions, but the better effect is difficult to obtain in a short time under high-concentration aniline.
Disclosure of Invention
The invention discloses a composite microbial agent for treating high-concentration aniline wastewater, namely an aniline efficient degradation microbial agent, and a preparation method and application thereof in chemical wastewater aiming at the defects in the prior art.
The invention is realized by the following steps:
the preparation method of the aniline efficient degradation microbial inoculum comprises the following sequence and steps:
firstly, respectively inoculating the obtained alcaligenes faecalis, bacteroides gibsonii and bacillus lysinate into an enrichment medium, and performing shake culture to logarithmic phase;
step two, inoculating the seed liquid of each strain in the step one into a seed liquid culture medium according to the inoculation amount of 10% for amplification culture;
step three, inoculating the culture solution obtained in the step two into a fermentation culture medium according to a proportion of 10% for fermentation culture; to obtain a concentration of 1X 109~1×1011CFU/mL fermentation product;
step four, mixing the bacterial agents of the strains obtained in the step three according to the mass ratio of fermentation products of the alcaligenes faecalis, the microbacterium, the bacteroides gibsonii and the bacillus lysinate of 1: 0.5-5 to form a composite bacterial agent;
and step five, adding 20% of protective agent into the obtained microbial inoculum and preparing into dry powder microbial inoculum.
Further, the formula of the enrichment medium in the step one is as follows: 3.0 g/L of beef extract; peptone 10.0 g/L; NaCl 5.0 g/L.
Further, the formula of the seed liquid culture medium in the step two is as follows: 100mg/L of aniline; NaCl 1 g/L; k2HPO4 1 g/L；KH2PO41 g/L; 10 mL of trace element liquid, and pH = 7.0.
Further, the formula of the fermentation medium in the third step is as follows: the concentration of glucose in the aqueous solution is controlled,10g/L；K2HPO4 1 g/L；KH2PO41 g/L; 10g/L of yeast extract; 5mL of trace element liquid; 0.2 percent of defoaming agent; 1000mL of distilled water; the pH was adjusted to 7.0.
Further, the fermentation culture conditions in the third step are as follows: the tank temperature is 37 ℃, the tank pressure is 0.01-0.1 MPa, the ventilation volume is 1: 0.6-1.2, the stirring speed is 150rpm, and the fermentation time is 48-72 h.
Further, the formula of the protective agent in the five steps is as follows: 35% of yeast powder; 35% of fish protein; 30 percent of trace elements.
Further, the formula of the trace element liquid in the invention is as follows: MgSO (MgSO)4 0.5 g/L；MnSO4 0.1 g/L；H3BO30.1 g/L；ZnSO4·7H2O 0.1 g/L；FeSO4·7H2O 0.3 g/L；pH=7。
The application of the aniline efficient degradation microbial inoculum is realized by the following modes:
adding the aniline efficient degradation microbial inoculum produced in the step into an inorganic salt culture medium containing 1000mg/L aniline according to 0.1-10 per mill, and reducing the aniline concentration to below 50mg/L with HRT for 72h, wherein the removal rate is over 95%;
adding the aniline efficient degradation microbial inoculum produced in the step into certain pesticide wastewater containing 1000mg/L aniline according to 0.1-10 per mill, and reducing the aniline concentration to below 50mg/L with HRT for 72h, wherein the removal rate is over 95%;
compared with the prior art, the invention has the beneficial effects that:
1) according to the invention, a plurality of functional strains with aniline degradation effect are mixed according to a certain proportion, so that the degradation performance of the compound microorganism on aniline can be obviously improved;
2) the strain product adopts a special fermentation process, the viable count of the product exceeds 10^9, and the product can be directly added to improve the better degradation performance of aniline;
3) the strain product aniline has high tolerance capacity, can reach 3000mg/L and 1000mg/L aniline at most, and HRT72h can be degraded to more than 95%.
Drawings
FIG. 1 is a graph showing the degradation of various aniline concentrations over time in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and effects of the present invention more clear, the present invention is further described in detail by the following examples. It should be noted that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The main components of the aniline efficient degrading microbial agent are alcaligenes faecalis, bacteroides gibsonii and bacillus lysinate. The preparation method of the aniline efficient degradation microbial inoculum comprises the following sequence and steps:
firstly, respectively inoculating the obtained alcaligenes faecalis, bacteroides gibsonii and bacillus lysinate into an enrichment medium, and performing shake culture to logarithmic phase; the formula of the enrichment medium is as follows: 3.0 g/L of beef extract; peptone 10.0 g/L; NaCl 5.0 g/L.
Step two, inoculating the seed liquid of each strain in the step one into a seed liquid culture medium according to the inoculation amount of 10% for amplification culture; the formula of the seed liquid culture medium is as follows: 100mg/L of aniline; NaCl 1 g/L; k2HPO4 1 g/L；KH2PO41 g/L; 10 mL of trace element liquid, and pH = 7.0.
Step three, inoculating the culture solution obtained in the step two into a fermentation culture medium according to a proportion of 10% for fermentation culture; to obtain a concentration of 1X 109~1×1011CFU/mL fermentation product; the formula of the fermentation medium is as follows: glucose, 10 g/L; k2HPO4 1 g/L；KH2PO41 g/L; 10g/L of yeast extract; 5mL of trace element liquid; 0.2 percent of defoaming agent; 1000mL of distilled water; the pH was adjusted to 7.0. The fermentation culture conditions are as follows: the tank temperature is 37 ℃, the tank pressure is 0.01-0.1 MPa, the ventilation volume is 1: 0.6-1.2, the stirring speed is 150rpm, and the fermentation time is 48-72 h.
Step four, mixing the bacterial agents of the strains obtained in the step three according to the mass ratio of fermentation products of the alcaligenes faecalis, the microbacterium, the bacteroides gibsonii and the bacillus lysinate of 1: 0.5-5 to form a composite bacterial agent;
and step five, adding 20% of protective agent into the obtained microbial inoculum and preparing into dry powder microbial inoculum. The formula of the protective agent is as follows: 35% of yeast powder; 35% of fish protein; 30 percent of trace elements. The formula of the trace element liquid is as follows: MgSO (MgSO)4 0.5 g/L；MnSO4 0.1 g/L；H3BO3 0.1 g/L；ZnSO4·7H2O 0.1 g/L；FeSO4·7H2O 0.3 g/L；pH=7。
Example 1
The produced high-efficiency aniline degradation microbial inoculum is respectively added into inorganic salt culture media containing 200mg/L, 400mg/L, 600mg/L, 800mg/L and 1000mg/L according to the addition amount of 1 per mill, shaking table culture is carried out at 37 ℃, sampling is carried out at different time periods, and the content of the residual aniline is measured. The result is shown in figure 1, under the aniline concentration of 200mg/L, the concentration can be reduced to below 25mg/L within 24h, and the aniline can be completely degraded within 48 h; under the concentration of 400-1000 mg/L aniline, 72 hours can degrade to below 50mg/L aniline.
Example 2
The produced high-efficiency aniline-degrading microbial inoculum is added into 1000mg/L pesticide wastewater according to the addition amount of 1 per mill, shaking culture is carried out at 37 ℃, samples are taken at different time periods, and the content of the residual aniline is determined. The result shows that the aniline concentration of 72h can be degraded to below 50mg/L, and the removal rate is more than 95%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.
Claims (10)
1. A preparation method of an aniline efficient degradation microbial inoculum is characterized by comprising the following steps:
firstly, respectively inoculating the obtained alcaligenes faecalis, microbacterium, bacteroides gibsonii and bacillus lysinate into an enrichment medium, and performing shake culture to logarithmic phase;
step two, inoculating the seed liquid of each strain in the step one into a seed liquid culture medium according to the inoculation amount of 10% for amplification culture;
step three, the step twoThe culture solution is inoculated in a fermentation culture medium according to the proportion of 10 percent for fermentation culture; to obtain a concentration of 1X 109~1×1011CFU/mL fermentation product;
step four, mixing the bacterial agents of the strains obtained in the step three according to the mass ratio of fermentation products of the alcaligenes faecalis, the microbacterium, the bacteroides gibsonii and the bacillus lysinate of 1: 0.5-5 to form a composite bacterial agent;
and step five, adding 20% of protective agent into the obtained microbial inoculum and preparing into dry powder microbial inoculum.
2. The method for preparing the aniline efficient degradation microbial inoculum according to claim 1, wherein the formula of the enrichment medium in the first step is as follows: 3.0 g/L of beef extract; peptone 10.0 g/L; NaCl 5.0 g/L.
3. The method for preparing the aniline efficient degradation microbial inoculum according to claim 1, wherein the formula of the seed liquid culture medium in the second step is as follows: 100mg/L of aniline; NaCl 1 g/L; k2HPO4 1 g/L；KH2PO41 g/L; 10 mL of trace element liquid, and pH = 7.0.
4. The preparation method of the aniline efficient degradation microbial inoculum according to claim 1, wherein the formula of the fermentation medium in the third step is as follows: glucose, 10 g/L; k2HPO4 1 g/L；KH2PO41 g/L; 10g/L of yeast extract; 5mL of trace element liquid; 0.2 percent of defoaming agent; 1000mL of distilled water; the pH was adjusted to 7.0.
5. The method for preparing the aniline efficient degradation microbial inoculum according to claim 1, wherein the fermentation culture conditions in the third step are as follows: the tank temperature is 37 ℃, the tank pressure is 0.01-0.1 MPa, the ventilation volume is 1: 0.6-1.2, the stirring speed is 150rpm, and the fermentation time is 48-72 h.
6. The preparation method of the aniline efficient degradation microbial inoculum according to claim 1, wherein the formula of the protective agent in the fourth step is as follows: 35% of yeast powder; 35% of fish protein; 30 percent of trace elements.
7. The preparation method of the aniline efficient degradation microbial inoculum according to any one of claims 3 to 6, wherein the formula of the trace element liquid is as follows: MgSO (MgSO)4 0.5 g/L；MnSO4 0.1 g/L；H3BO3 0.1 g/L；ZnSO4·7H2O 0.1 g/L；FeSO4·7H2O 0.3 g/L；pH=7。
8. The aniline efficient degradation microbial inoculum prepared by the preparation method of the aniline efficient degradation microbial inoculum according to claim 1.
9. The application of the aniline efficient degrading microbial inoculum according to claim 8 in chemical wastewater.
10. The application of the aniline efficient degradation microbial inoculum in chemical wastewater according to claim 9, wherein the aniline efficient degradation microbial inoculum is added into an inorganic salt culture medium containing 1000mg/L aniline according to the proportion of 0.5-5 per thousand, the aniline can be reduced to below 50mg/L within 72 hours, and the removal rate exceeds 95%; the aniline efficient degradation microbial inoculum is added into certain pesticide wastewater containing 1000mg/L aniline according to the proportion of 0.5-5 per mill, the aniline can be reduced to below 50mg/L within 72 hours, and the removal rate exceeds 95%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113755379A (en) * | 2021-09-23 | 2021-12-07 | 江苏蓝必盛化工环保股份有限公司 | Efficient dimethylformamide degrading microbial inoculum, preparation and application thereof |
| WO2024084197A1 (en) * | 2022-10-17 | 2024-04-25 | Cambridge Enterprise Limited | Methods for reducing the level of xenobiotics in an environment |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831391A (en) * | 2009-03-10 | 2010-09-15 | 中国科学院成都生物研究所 | Effective phenylamine degrading strain as well as use and use method thereof |
| CN103910435A (en) * | 2014-01-14 | 2014-07-09 | 暨南大学 | Use of Lysinibacillus sp. FS1 in dye degradation |
| CN104371948A (en) * | 2014-07-02 | 2015-02-25 | 中国中化股份有限公司 | Microbacterium sp. strain and application thereof |
| CN106520611A (en) * | 2016-11-08 | 2017-03-22 | 中海油天津化工研究设计院有限公司 | High-efficiency composite biological agent for processing high-salt degradation-resistant organic wastewater and application of high-efficiency composite biological agent |
| CN106591180A (en) * | 2016-12-05 | 2017-04-26 | 盐城工学院 | Efficient chlorobenzene degradation obligate bacterium, composite microbial agent and preparation method and application of composite microbial agent |
| CN110643548A (en) * | 2019-11-11 | 2020-01-03 | 黄河三角洲京博化工研究院有限公司 | Microbacterium flavum for efficiently degrading aniline and application thereof |
-
2020
- 2020-10-19 CN CN202011117785.2A patent/CN112175878A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831391A (en) * | 2009-03-10 | 2010-09-15 | 中国科学院成都生物研究所 | Effective phenylamine degrading strain as well as use and use method thereof |
| CN103910435A (en) * | 2014-01-14 | 2014-07-09 | 暨南大学 | Use of Lysinibacillus sp. FS1 in dye degradation |
| CN104371948A (en) * | 2014-07-02 | 2015-02-25 | 中国中化股份有限公司 | Microbacterium sp. strain and application thereof |
| CN106520611A (en) * | 2016-11-08 | 2017-03-22 | 中海油天津化工研究设计院有限公司 | High-efficiency composite biological agent for processing high-salt degradation-resistant organic wastewater and application of high-efficiency composite biological agent |
| CN106591180A (en) * | 2016-12-05 | 2017-04-26 | 盐城工学院 | Efficient chlorobenzene degradation obligate bacterium, composite microbial agent and preparation method and application of composite microbial agent |
| CN110643548A (en) * | 2019-11-11 | 2020-01-03 | 黄河三角洲京博化工研究院有限公司 | Microbacterium flavum for efficiently degrading aniline and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 张悦周等: "硝基苯废水处理的微生物及工艺", 《化学与生物工程》 * |
| 曾国驱等: "微生物降解苯胺的研究现状", 《中国医学生物技术应用杂志》 * |
| 王镔等: "苯胺高效降解菌的筛选及共代谢机制", 《环境保护科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113755379A (en) * | 2021-09-23 | 2021-12-07 | 江苏蓝必盛化工环保股份有限公司 | Efficient dimethylformamide degrading microbial inoculum, preparation and application thereof |
| WO2024084197A1 (en) * | 2022-10-17 | 2024-04-25 | Cambridge Enterprise Limited | Methods for reducing the level of xenobiotics in an environment |
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