CN112694986A - Aniline efficient degrading bacterium and application thereof - Google Patents
Aniline efficient degrading bacterium and application thereof Download PDFInfo
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Abstract
The invention provides an aniline efficient degrading bacterium and application thereof, wherein the strain is bacillus albus (bacillus cereus)Bacillus albus) Compared with other aniline degrading bacteria, the bacteria can tolerate higher aniline concentration and can realize complete degradation of aniline at high concentration. Under the condition that the initial concentration of the aniline is 5g/L, the removal rate of the aniline can reach 100% within 48h, and the degradation effect is very obvious. The invention can effectively solve the problem of poor biochemical effect of the high-concentration aniline wastewater and provides a new effective way for biochemical treatment of the high-aniline wastewater.
Description
Technical Field
The invention relates to the field of environmental microorganisms, in particular to an aniline efficient degrading bacterium and application thereof.
Background
Aniline is one of the important chemical raw materials and intermediates in the pesticide, dye, plastic and pharmaceutical industries, and the chemical products and intermediates produced and processed from aniline are hundreds of. Can be used for preparing acid ink blue G, acid medium BS, acid bright yellow, direct orange S, direct pink, indigo blue, disperse yellow brown, etc. in dye industry; the organic pigment is used for manufacturing golden bright red, scarlet powder, phenol cyanine red, oil-soluble black and the like. In the printing and dyeing industry for the dye nigrosine; in the agrochemical industry for the production of many insecticides, fungicides such as DDV, aclonifen, propachlor and the like; aniline is an important raw material of rubber auxiliaries and is used for preparing an anti-aging agent A, an anti-aging agent D, an anti-aging agent 4010, accelerators M, 808, D, CA and the like; it can also be used as raw material of sulfa drug, aniline is important raw material for producing pesticide, and can be used as intermediate of bactericide such as sodium diformate, fenfluramine, triazophos, pyridaphenthion, quinalphos, herbicide such as alachlor, hexazinone, and imidazoquinolinic acid.
Aniline is widely existed in production wastewater of pesticides, dyes and the like, and aniline has certain toxicity to common microorganisms, so that the existing biochemical treatment effect of aniline-containing wastewater is not ideal, and therefore, aniline-containing wastewater is treated by some physical and chemical methods in practice, the cost is high, and secondary pollution is easily caused. Therefore, the screening of the bacterial strain capable of efficiently degrading aniline is very important for solving the problem that aniline wastewater is difficult to treat.
Various bacteria have been reported to have the effect of degrading aniline, including various types of microorganisms such as Bacillus, Pseudomonas, Acinetobacter, Micrococcus, etc. Because aniline is toxic to general microorganisms, most of the reported aniline degrading bacteria have a less than ideal degrading effect and tolerate too low an aniline concentration. Such as pseudomonas ZD-13 and bacillus SA-9, can achieve 100% degradation of aniline within 500 mg/L, but with increasing aniline concentration, the degradation rate decreases significantly. The degradation rate of 2000 mg/L aniline at 48h is only 40% (nutmeg, 2007). Therefore, the technical problem to be solved by the invention is to find a microorganism which can not only tolerate high-concentration aniline but also rapidly realize the degradation of the aniline. In addition, the invention provides a fermentation process for quickly culturing the aniline high-efficiency degrading bacteria.
Disclosure of Invention
Aiming at the problem that aniline wastewater is difficult to treat, the invention screens and separates an aniline efficient degrading bacterium, can effectively and quickly degrade aniline in sewage, and simultaneously provides a fermentation culture process of the aniline efficient degrading bacterium, so that the strains can be quickly enriched and expanded in a short time.
The invention provides an aniline efficient degrading bacterium BA-7 which is classified and named as white bacillus (B)Bacillus albus) The strain is preserved in China general microbiological culture Collection center at 22 months 10 in 2020, address: no. 3 Xilu No. 1 Beijing, Chaoyang, and the preservation number is CGMCC NO. 20936.
The physiological characteristics are: the shape of the thallus is rod-shaped, spores are produced, the gelatin is liquefied, heterotrophic aerobic bacteria are produced, and gram staining is positive. Obvious colonies appeared after 24h of agar solid plate culture, and the colonies were round or oval, neat in edge, white, large and flat, opaque, moist and matt.
The aniline degrading bacterium BA-7 provided by the invention can grow by taking aniline as a unique carbon source. BA-7 achieved 100% completion of the initial concentration of 5g/L aniline within 48h under laboratory shake culture conditions. Meanwhile, the actual treatment effect of the aniline efficient degrading bacteria BA-7 on the aniline-containing wastewater is verified. The bacterial agent obtained by BA-7 fermentation is added into the aniline wastewater containing 2g/L according to the volume ratio of 10 percent, the degradation rate of the aniline can reach 98 percent after 24 hours of aeration treatment, and the treatment effect is very ideal.
The invention provides a simple and rapid culture and fermentation process for aniline efficient degrading bacteria, which comprises the steps of activation, transfer, culture expansion and the like, and the specific process flow is as follows:
(1) and (3) activation: selecting a single colony of the aniline efficient degrading bacteria from the solid plate, transferring the single colony into an LB liquid culture medium containing aniline, and carrying out shake culture at 37 ℃ for 3-4 h until the logarithmic phase at the rotation speed of 150 rpm. The LB culture medium comprises the following components: 1g/L aniline, 5g/L yeast extract, 10g/L peptone and 10g/L sodium chloride, and the pH value is kept between 7.2 and 7.5.
(2) Transferring: transferring the aniline degradation bacterial liquid activated in the logarithmic phase in the flow 1 to a 50L seeding tank according to the inoculation amount of 5% for culturing, controlling the temperature of the seeding tank to be maintained at 30-37 ℃, the rotating speed to be maintained at 250rpm of 220-. The culture medium of the seeding tank comprises the following components in percentage by mass: aniline 0.2%, glucose 0.6%, NH40.2% of Cl, 0.1% of KH2PO40.1%, 0.05% of MgSO40.05%, 0.02% of NaCl, 0.3% of CaCO, 0.01% of yeast extract and the balance of water. The pH is maintained between 7.2 and 7.5.
(3) Expanding culture: transferring the aniline-degrading bacterial liquid cultured in the seeding tank of the process 2 into a 1000L fermentation tank for amplification culture according to the inoculation amount of 2-5%, wherein the components of a culture medium of the fermentation tank are the same as those of the seeding tank, and the indexes of physical and chemical parameters are as follows: the temperature is 37 ℃, the rotating speed is 250rpm, the dissolved oxygen is 8 mg/L, and the fermentation time is 48-60 h. After the fermentation is finished, the effective viable count of the tank body bacterial liquid can reach 109More than one/ml, the fermentation culture solution is taken out of the tank and then packaged by a plastic barrel to obtain the aniline efficient degradation bacterium agent.
The aniline efficient degrading bacteria is applied to degrading aniline.
Mixing Bacillus albus (A), (B)Bacillus albus) BA-7 is added into wastewater containing aniline to be treated to degrade aniline after strain culture. The strain can tolerate high-concentration aniline wastewater and has high degradation rate.
The aniline efficient degrading bacterium provided by the invention can realize the fast and efficient degradation of aniline, and provides an effective solution for the problem of difficult biochemical treatment of aniline-containing wastewater. Has important practical significance for protecting ecological environment and human health. Meanwhile, the fermentation culture process of the aniline efficient degrading bacteria is simple, convenient and easy to operate, greatly reduces the bacterial contamination risk of an intermediate link, is low in production cost, short in fermentation period time and rapid in propagation, and has wide application prospect in the field of biological fermentation.
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FIG. 1 is a photograph of crystal violet staining of the cells of Aniline-degrading bacteria BA-7 under an oil microscope of 1000 times x;
FIG. 2 is a plot of the initial amount of aniline at 5g/L as a function of time for strain BA-7 under laboratory shake flask conditions and the growth of strain BA-7 under these conditions as a function of time.
FIG. 3 is the curve of aniline concentration and degradation rate with time in the experiment of strain BA-7 fermentation liquid in treating practical aniline-containing waste water.
Detailed Description
The following examples are suitable for the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
EXAMPLE 1 isolation and purification of bacterial species
The strain BA-7 separation and purification steps are as follows:
(1) domestication and enrichment
50ml of activated sludge is added into a 1L domestication device containing a basic culture solution taking aniline as a unique carbon source, and the activated sludge comes from a biochemical pond of a sewage treatment plant for treating aniline wastewater for a long time in Jiangsu. The basic culture solution formula is as follows: aniline 0.5 g/L, NH4Cl 0.5g/L、KH2PO4 1g/L、Na2HPO4 1g/L、MgCl2 0.02g/L、CaCl20.03 g/L. Introducing air to acclimatize and culture for about 15-20 days, and periodically monitoring aniline concentration and sludge growth condition. And (3) after complete degradation of aniline is detected, timely supplementing aniline, and gradually increasing the aniline concentration to 5g/L to obtain the aniline degrading bacteria enriched liquid.
(2) Separation of
Under the aseptic condition, the domesticated enriched bacterial liquid is transferred to an aseptic fresh basic culture solution taking aniline as a unique carbon source according to the inoculum concentration of 2 percent, and the formula of the basic culture solution is the same as that in the step 1. Shaking table culturing at 37 deg.C to logarithmic phase, diluting and coating the culture medium liquid with aniline as the only carbon source by dilution coating plate method, wherein the solid separation medium contains aniline 1g/L, NH4Cl 0.5g/L、KH2PO4 1g/L、MgCl2 0.02g/L、CaCl20.03g/L and 2% of agar. Culturing in 37 deg.C incubator for 2-3 days.
(3) Purification of
And in a clean bench, selecting a single colony grown in an aniline separation solid culture medium by using an inoculating needle, and further streaking, purifying and culturing the aniline degradation bacteria by using a plate streaking method until the single colony is grown. Repeating the operation for more than 3 times until the colony growing on the plate is single in shape, thus obtaining the purified aniline high-efficiency degrading bacterium.
EXAMPLE 2 identification of the strains
The purified aniline degrading bacteria are subjected to 16s rDNA sequencing, and the sequencing comparison result is bacillus albicans (bacillus subtilis)Bacillus albus) Named BA-7. The main physiological characteristics of the growth regulator are gram-positive and aerobic growth, and aniline can be used as a unique carbon source and energy source for growth. Taking a little bacterial sample to carry out crystal violet staining, observing that the shape of the bacterial is rod-shaped and has spores under an X1000 times oil microscope, carrying out physiological and biochemical identification of the bacterial strain according to a common bacteria system identification manual, and carrying out a microscopic examination photo as shown in an attached figure 1.
EXAMPLE 3 growth Curve of Strain BA-7 and degradation experiment of para-Aniline
Selecting a single bacterial colony of purified aniline degrading bacteria BA-7 in the invention to a shake flask of an inorganic salt culture medium containing 5g/L aniline, wherein the formula of the inorganic salt culture medium comprises: NH (NH)4Cl 0.5g/L、KH2PO4 1g/L、Na2HPO4 1g/L、MgCl2 0.02g/L、CaCl20.03 g/L. The rotation speed of the shaker is 180 rpm, the temperature is 37 ℃, and the shaking culture is carried out. The OD600 of the bacterial cell concentration and the aniline concentration were measured at 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h, 60h and 72h, respectively. OD600 was measured by a visible spectrophotometer, and aniline concentration was measured by liquid chromatography. The data are shown in figure 2, under the condition that the initial concentration of the aniline is 5g/L, the strain BA-7 slowly grows within 12h, so that the aniline degradation is slow, the aniline degradation speed is obviously improved along with the increase of the concentration of the bacteria, and the aniline removal rate reaches 50% after 24 h. CulturingAnd when 48 hours, the aniline achieves the effect of complete degradation, the thalli grow and lose carbon sources along with complete degradation of the aniline, so that the thalli tend to be stable, and the OD600 value is stabilized to be about 1.2. The data show that the strain BA-7 has good degradation effect on the aniline degradation bacteria, and the thallus culture is very convenient and fast.
EXAMPLE 4 fermentation culture Process of Strain BA-7
Selecting a single colony of the aniline efficient degrading bacteria from the solid plate, transferring the single colony into an LB liquid culture medium containing aniline, and carrying out shake culture at 37 ℃ for 3-4 h until the logarithmic phase at the rotation speed of 150 rpm. The LB culture medium comprises the following components: 1g/L aniline, 5g/L yeast extract, 10g/L peptone and 10g/L sodium chloride, and the pH value is kept between 7.2 and 7.5. Then transferring the activated aniline degradation bacterial liquid in the logarithmic phase to a 50L seeding tank according to the inoculation amount of 5 percent for culture, controlling the temperature of the seeding tank to be maintained at 37 ℃, the rotating speed to be maintained at 250rpm of 220-. The culture medium of the seeding tank comprises the following components in percentage by mass: aniline 0.2%, glucose 0.6%, NH40.2% of Cl, 0.1% of KH2PO40.1%, 40.05% of MgSO40.05%, 0.02% of NaCl, 30.3% of CaCO, 0.01% of yeast extract and the balance of water, and the pH value is maintained between 7.2 and 7.5. Finally, transferring the aniline-degrading bacteria liquid cultured in the seeding tank into a 1000L fermentation tank for amplification culture according to the inoculation amount of 2-5%, wherein the components of a culture medium of the fermentation tank are the same as those of the seeding tank, and the indexes of physical and chemical parameters are as follows: the temperature is 37 ℃, the rotating speed is 250rpm, the dissolved oxygen is 8 mg/L, and the fermentation time is 56 h. After the fermentation is finished, the effective viable count of the tank body bacterial liquid can reach 109More than one/ml, the fermentation culture solution is taken out of the tank and then packaged by a plastic barrel to obtain the aniline efficient degradation bacterium agent.
EXAMPLE 5 use of the Strain BA-7 in actual Aniline wastewater
In order to further verify the application effect of the aniline efficient degrading bacteria BA-7 used in the invention in the actual aniline-containing wastewater, the aniline-containing production wastewater is obtained from a sewage station of a chemical plant which takes aniline as a main production raw material. Through determination, the aniline concentration in the wastewater is 2g/L, and the concentration is higher. Aniline degrading bacteria BA-7 fermentation liquor is directly added into aniline production wastewater according to the inoculation amount of 10 percent, and a proper amount of nitrogen is supplementedSource NH4KH Cl and phosphorus source2PO4And the pH value of the system is adjusted to be 7.2 so as to meet the requirement of normal growth of microorganisms. Under the condition of aeration at room temperature, sampling is carried out at the fixed time of 0h, 2h, 6h, 12h, 18h and 24h to monitor the change of aniline in the system. Aniline degradation data are shown in figure 3: aniline in the wastewater is degraded within 2 hours, the aniline concentration is reduced from 2g/L to 1.8g/L, the degradation effect reaches 10%, and after 12 hours, the aniline concentration is reduced to 0.6g/L, and the degradation rate reaches 70%. After 24 hours, the aniline concentration is reduced to 0.04 g/L, and the degradation rate is as high as 98%. The experimental data show that the aniline efficient degrading bacteria BA-7 also have good treatment effect in actual aniline-containing wastewater and have wide application prospect.
Claims (7)
1. An aniline efficient degrading bacterium, which is classified and named as bacillus albicans (Bacillus albus)Bacillus albus) BA-7, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO. 20936.
2. The use of the aniline efficient degrading bacterium according to claim 1 for degrading aniline.
3. Use according to claim 2, characterized in that: mixing Bacillus albus (A), (B)Bacillus albus) BA-7 is added into wastewater containing aniline to be treated to degrade aniline after strain culture.
4. Use according to claim 3, characterized in that: bacillus albicans (B.albicans)Bacillus albus) The strain culture of BA-7 comprises the steps of activation, transfer and expanding culture,
and (3) activation: selecting a single colony of the aniline efficient degrading bacteria from the solid plate, transferring the single colony into an LB liquid culture medium containing aniline, and performing shake culture at the rotating speed of a shaker of 150 rpm and the temperature of 37 ℃ for 3-4 h until a logarithmic phase;
transferring: transferring the aniline degrading bacteria liquid in the logarithmic phase to a seeding tank for culture according to the inoculation amount of 5 percent, controlling the temperature of the seeding tank to be maintained at 30-37 ℃, the rotating speed to be maintained at 250rpm with the dissolved oxygen DO being controlled at 4-6 mg/L, and culturing for 24-48 h;
expanding culture: transferring the aniline-degrading bacteria liquid cultured in the seeding tank into a fermentation tank according to the inoculation amount of 2% -5% for amplification culture, wherein the components of a culture medium of the fermentation tank are the same as those of the seeding tank, and the indexes of physical and chemical parameters are as follows: the temperature is 37 ℃, the rotating speed is 250rpm, the dissolved oxygen is 8 mg/L, and the fermentation time is 48-60 h.
5. Use according to claim 4, characterized in that: the LB liquid culture medium comprises the following components: 1g/L aniline, 5g/L yeast extract, 10g/L peptone and 10g/L sodium chloride, and the pH value is kept between 7.2 and 7.5.
6. Use according to claim 4, characterized in that: the culture medium of the seeding tank comprises the following components in percentage by mass: aniline 0.2%, glucose 0.6%, NH40.2% of Cl, 0.1% of KH2PO40.1%, 0.05% of MgSO40.05%, 0.02% of NaCl, 0.3% of CaCO, 0.01% of yeast extract and pH maintained between 7.2 and 7.5.
7. Use according to claim 4, characterized in that: the effective viable count of the bacterial liquid after the bacterial strain is cultured can reach 109More than one/ml, and packaging the fermentation culture solution after being taken out of the tank to obtain the aniline efficient degradation bacterium agent.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113046261A (en) * | 2021-03-01 | 2021-06-29 | 南京信息工程大学 | Bacillus albus and application thereof |
| CN114262679A (en) * | 2021-12-28 | 2022-04-01 | 江苏南资环保科技有限公司 | Autotrophic denitrifying bacteria and rapid propagation process thereof |
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