CN113174350B

CN113174350B - Alicyclobacillus for degrading anionic surfactant and application thereof

Info

Publication number: CN113174350B
Application number: CN202110629110.4A
Authority: CN
Inventors: 李阳
Original assignee: Shanghai Zhongjing Huanneng Environmental Protection Co ltd
Current assignee: Zhongzhong Energy (Nantong) New Energy Co.,Ltd.
Priority date: 2021-06-07
Filing date: 2021-06-07
Publication date: 2022-04-29
Anticipated expiration: 2041-06-07
Also published as: CN113174350A

Abstract

The invention discloses a Alicyclobacillus for degrading anionic surfactant, which is Alicyclobacillus A-YW and is preserved in China Center for Type Culture Collection (CCTCC) at 25 months 4 and 2021, wherein the strain preservation number is CCTCC M2021451, and the preservation addresses are as follows: eight-path Lojia mountain in Wuchang region of Wuhan city, Hubei province. The invention obtains the alicyclic acid bacillus (Alicyclobacillus aeris) A-YW by separating sludge collected from a sewage outlet of a detergent manufacturer, and degradation tests of an anionic surfactant show that the strain has good degradation effect on the anionic surfactant, is an environment-friendly strain with potential significance in sewage treatment, has the characteristics of high degradation efficiency, high degradation rate and the like, and provides help for environmental protection in China.

Description

Alicyclobacillus for degrading anionic surfactant and application thereof

Technical Field

The invention belongs to the technical field of biology, relates to a strain for environmental protection, and particularly relates to a Alicyclobacillus A-YW strain (Alicyclobacillus aeris) and application thereof in degrading anionic surfactant in domestic sewage.

Background

The surfactant in the domestic sewage mainly comprises anionic surfactants such as sodium stearate, sodium dodecyl benzene sulfonate, octylphenol ethylene oxide polymer and the like in soap for washing clothes, washing powder, washing cleanness for washing bowls and chopsticks and the like. In recent years, with the continuous improvement of living standard and the continuous increase of population, a large amount of domestic sewage is discharged into a water body by different methods, so that river water sources are polluted to different degrees, wherein the pollution of an anionic surfactant in the sewage to the river environment has attracted general attention of people. The influence of anionic surfactants on rivers and the surrounding environment is mainly reflected in the following aspects: (1) after a large amount of anionic surfactant is discharged into a river, a large amount of foam is generated on the water surface of the river, which is not only an obstacle to the sight, but also obstructs the exchange of oxygen in the water surface and the air and influences the self-cleaning rate of the water body. (2) Anionic surfactants can be harmful to microorganisms, algae, aquatic weeds, fish, etc. in water. When the water contains 1mg/L of the anionic surfactant, the growth of green plants can be blocked, and when the water contains 6-7 mg/L of the anionic surfactant, fish poisoning can be caused. (3) The anionic surfactant can enter human body through drinking water, washing or other channels, and forms new complex with protein in human tissue to induce canceration. Along with the increase of detergent consumption of people, the pollution degree of the detergent to river environment is more and more serious, so that the removal of the detergent pollution in the river and the surrounding environment becomes a major task and a research topic of vast environment protection workers.

The currently common methods for treating the wastewater mainly comprise an activated carbon adsorption method, a foam separation method, a photocatalytic oxidation method, an ultrasonic degradation method, a membrane filtration technology, a chemical flocculation method, a microbiological method and the like. Although these methods have various applications in treating such waste water, they have certain limitations and need further improvement: the active carbon adsorption method has good adsorption effect at normal temperature, but the regeneration energy consumption of the active carbon is high, and the adsorption capacity after regeneration can be reduced to different degrees, thereby limiting the application of the active carbon adsorption method. The foam separation method has simple process, simple and convenient operation and stable operation, but the method can cause the transfer of anionic surfactant and pollute the air in the treatment process, and simultaneously can cause pathogenic bacteria to be diffused together, thereby increasing the risk of epidemic diseases. The photocatalytic oxidation method needs continuous aeration in the treatment process to ensure that sufficient dissolved oxygen is provided, and the operation cost is high. The degradation capability of the ultrasonic degradation method is greatly influenced by pH, and the degradation degree is increased along with the increase of the power and the action time of the ultrasonic action. The membrane filtration technology has high separation efficiency and simple process, but the membrane is expensive and easy to block. The chemical flocculation method has a certain treatment effect on the anionic surfactant, but in the treatment process, calcium oxide is required to be used as a coagulant aid, waste residues and sludge are generated, and secondary pollution is caused due to improper treatment. The microbiological process has low investment and operation cost, but the application conditions and the available microorganisms are limited, the low concentration of the anionic surfactant can cause the proliferation of the microorganisms, and the high concentration of the anionic surfactant can inhibit the growth of the organisms. The biological treatment technology is to decompose organic pollutants into CO by utilizing the metabolism of microorganisms₂And H₂O, or organic acid and alcohol, etc., thereby achieving the purpose of purifying sewage. Article withCompared with physical and chemical methods, the biological treatment method has low cost, and simultaneously, the microorganisms used for sewage treatment have quick propagation, easy culture and strong capability of adapting to different types of wastewater environments. In the technical field of biological treatment, strains for efficiently degrading characteristic pollutants in domestic sewage are usually screened so as to realize biological degradation treatment of sewage. Therefore, the method has important significance for searching the bacterial strain capable of efficiently degrading the anionic surfactant in the domestic sewage.

Disclosure of Invention

The invention aims to provide alicyclobacillus for efficiently degrading an anionic surfactant.

In order to achieve the purpose, the technical scheme of the invention is as follows: provided is a Bacillus alicyclolyticus for degrading an anionic surfactant, wherein: the Alicyclobacillus is Alicyclobacillus A-YW, which is preserved in China Center for Type Culture Collection (CCTCC) at 25/4/2021, the preservation number of the strain is CCTCC M2021451, and the preservation addresses are as follows: eight-path Lojia mountain in Wuchang region of Wuhan city, Hubei province.

Further, the Alicyclobacillus is separated from high-concentration anionic surfactant sewage, and is subjected to morphology, culture traits, conventional physiological biochemistry and DNA sequence identification, the strain is a variety of the Alicyclobacillus, is a new strain of the Alicyclobacillus and is named as Alicyclobacillus (Alicyclobacillus aeris) A-YW.

Further, the basic biological properties of Bacillus alicyclolyticus (Alicyclobacillus aeris) A-YW are:

bacterial colonies formed by the strains on a flat TYS culture medium are all milky white, have tidy edges, are wet, have slightly raised centers, are glossy, have concentric circles, are semitransparent, and are not easy to pick up and are round. The scanning electron microscope photo shows that the bacteria are rod-shaped, most of the bacteria generate spores after being cultured in a culture medium for 3 days, the spores are terminal or secondary, the cells of the bacteria are expanded, the width of the bacteria is 0.3-0.8 um, and the length of the bacteria is 2.0-6.0 um, as shown in figure 1.

The invention also aims to provide the application of the alicyclobacillus for degrading the anionic surfactant in the efficient degradation of the anionic surfactant in the domestic sewage.

Furthermore, the biological agent containing the alicyclobacillus active ingredient can also be applied to efficiently degrading anionic surfactants in domestic sewage. The biological preparation can contain common carriers and auxiliary materials for preparing biological preparations.

Further, inoculating the A-YW strain seed solution cultured to logarithmic phase to domestic sewage for degradation treatment of anionic surfactant.

Further, activated colonies were picked and inoculated in a seed medium, and then A-YW was cultured at 30 ℃ for 3d at 200rpm to grow in the logarithmic phase to prepare a seed solution.

Further, the seed medium contained 0.5g of peptone, 0.5g of sucrose, 0.2g K per liter₂HPO₄、0.1g MgSO₄·7H₂O、0.1g(NH4)₂SO₄、0.001g FeSO₄·7H₂O、0.02g CaCl₂Autoclaving at 121 deg.C for 20min at pH 6.0.

Further, the inoculation amount of the A-YW strain seed liquid is 1-3%.

Further, the inoculation amount of the A-YW strain seed solution is 1%.

The degradation effect of the bacillus cycloaliphatic (Alicyclobacillus aeris) A-YW on the anionic surfactant is detected by directly inoculating the cultured bacillus cycloaliphatic (Alicyclobacillus aeris) A-YW into the domestic sewage containing the anionic surfactant, and the experimental result shows that the bacillus cycloaliphatic (Alicyclobacillus aeris) A-YW has the degradation effect on the anionic surfactant. The strain is an environmental treatment strain which has potential significance in treating the domestic sewage containing the anionic surfactant.

The invention obtains the alicyclic acid bacillus (Alicyclobacillus aeris) A-YW by separating sludge collected from a sewage outlet of a detergent manufacturer, and degradation tests of an anionic surfactant show that the strain has good degradation effect on the anionic surfactant, is an environment-friendly strain with potential significance in sewage treatment, has the characteristics of high degradation efficiency, high degradation rate and the like, and provides help for environmental protection in China.

Drawings

FIG. 1 is a scanning electron micrograph of a strain of Alicyclobacillus which degrades anionic surfactants according to the present invention.

Detailed Description

The following is a detailed description of the embodiments of the present invention, which is implemented on the premise of the technical solution of the present invention, and the detailed implementation and specific operation procedures are given, but the protection scope of the present invention is not limited to the following embodiments. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Screening and identification of strains

The method for screening the bacterial strain capable of efficiently degrading the anionic surfactant pollutants in the domestic sewage comprises the following steps:

1. isolation of the Strain

(1) Weighing 1g of sludge sample, and oscillating in a triangular flask containing 9ml of sterile water to obtain suspension;

(2) respectively inoculating 10ml of suspension, and shake culturing at 30 ℃ and 200rpm in a shaking table at 90ml of enrichment culture solution in a 250ml triangular flask. The enrichment medium contains 0.05g yeast extract/liter, 0.3g K₂HPO₄·2H₂O，0.15g KH₂PO₄，0.025g MgSO₄，0.1g(NH₄)₂SO₄，0.001g FeSO₄·7H₂O, 100ppm surfactant, pH 6.0, autoclaving at 121 deg.C for 20 min;

(3) taking 1ml of the original culture from the 5 days as an acclimatization period, transferring the original culture into a fresh culture medium under the same conditions at the end of each acclimatization period, and carrying out 3 acclimatization periods in total;

(4) coating, separating and inverting the culture after domestication on a primary screening flat plate, and culturing for 2 days at 30 ℃;

(5) selecting single colonies according to the growth condition of the colonies on the plate, and streaking and purifying the single colonies on a new plate;

(6) picking a single colony from the flat plate and inoculating the single colony to a slant culture medium;

(7) and identifying the strain.

2. Strain morphology identification

The isolated and purified strain was streaked on a 90mm diameter plate TYS (tryptone 0.5g/L, yeast extract 0.25g/L, NaCL 0.25g/L medium adjusted to 6.0 with dilute sulfuric acid, followed by autoclaving at 121 ℃ for 20 min) medium and cultured in a 30 ℃ incubator to obtain a single colony. The formed colonies are all milky white, have tidy edges, are wet, have slightly raised centers, are glossy, have concentric circles, are semitransparent, are not easy to lift and are circular. Positive by gram stain. The scanning electron microscope photo shows that the bacteria are rod-shaped, most of the bacteria generate spores after being cultured in a culture medium for 3 days, the spores are generated terminally or secondarily, the cells of the bacteria are expanded, the width of the bacteria is 0.3-0.8 um, and the length of the bacteria is 2.0-6.0 um, and the figure 1 shows.

3. 16S rRNA gene amplification, cloning and sequencing

Removing a small amount of strains from the stored inclined plane by using an inoculating loop, inoculating the strains into a test tube filled with 5ml of TYS liquid culture medium, culturing for 24 hours at the temperature of 25 ℃ by using a shaking table at 200rpm, collecting bacterial liquid, transferring the bacterial liquid into a 10ml EP tube, and centrifuging for 10 minutes at 5000rpm in a centrifuge. Discarding the supernatant, adding 480 mu L of TE buffer solution, shaking to dissolve the thalli, adding 5 mu L of lysozyme, and preserving the heat at 37 ℃ for 3-5 h; adding 20% SDS, shaking, adding 5 μ L proteinase K, and water-bathing at 55-65 deg.C for 5-10 min until the mixture is clarified; adding 100 μ L of NaCl with concentration of 5mol/L and 80 μ L of CTAB/NaCl, and water-bathing at 65 deg.C for 10 min; adding equal volume of chloroform: isoamyl alcohol (24: 1), centrifuging at 12000r/min for 10 min; adding equal volume of isopropanol into the supernatant, centrifuging at 12000r/min for 5min to remove the supernatant, washing the precipitate with 70% ethanol twice, and air drying; adding 200 mu LTE to dissolve DNA, adding 5 mu LRNA enzyme, and preserving heat for 2-5 h at 37 ℃; an equal volume (24: 1) of chloroform was added: re-extracting isoamyl alcohol, precipitating DNA by using isopropanol, washing twice by using 70% ethanol, airing, and adding 30-40 mu LTE for later use. 16S rRNA gene amplification was performed on a gradient PCR apparatus (Biometra) using 27F (5'-AGAGTTTGATCCTGGCTCAG-3')/1492R (5'-GGTTACCTTGTTACGACTT-3') primer set using the extracted genomic DNA as a template. The reaction system is as follows: mu.L 10 XTaq Buffer, 2.5. mu.L MgCl 21. mu.L 10 XdNTPs, 1. mu.L primer 27f, 1. mu.L primer 1492R, 1. mu.L LTaq DNA polymerase, 1. mu.L template DNA, and sterile water was added to the 25. mu.L system. In the control group, the template DNA was replaced with an equal volume of sterile water. Amplification conditions: pretreatment at 94 ℃ for 5min, denaturation at 94 ℃ for 40s, annealing at 52 ℃ for 45s and extension at 72 ℃ for 90s for 30 cycles, and finally heat preservation at 72 ℃ for 10 min. And (3) purifying the amplification product by using a recovery kit (OMEGA) after agarose gel electrophoresis, connecting the amplification product with pGEM-Tvecter by using a connection kit (Promega) and transforming the amplification product into E.coil DH5 alpha, coating a plate, screening positive clones, detecting PCR (polymerase chain reaction) clones, and determining that the clones which are successfully transformed are sent to Shanghai Bioengineering Co., Ltd for sequencing. And constructing a phylogenetic tree by MEGA3.1 software according to the 16S rRNA gene sequences of the similar species, and carrying out phylogenetic analysis.

Sequence analysis results show that the genetic relationship between the A-YW and the Alicyclobacillus (Alicyclobacillus aeris) is the closest, and the 16S rDNA sequence similarity of the A-YW and the Alicyclobacillus (Alicyclobacillus aeris) reaches 99.2 percent.

Secondly, measuring the degradation capability of the strain to the anionic surfactant

1. Cultivation of the Strain

Selecting activated colonies, inoculating the colonies in a seed culture medium, and culturing A-YW bacteria at 30 ℃ and 200rpm for 3d to enable the A-YW bacteria to grow in a logarithmic phase to prepare a seed solution; the seed culture medium contains 0.5g peptone, 0.5g sucrose, 0.2g K per liter₂HPO₄、0.1g MgSO₄·7H₂O、0.1g(NH4)₂SO₄、0.001g FeSO4·7H₂O、0.02g CaCl₂Autoclaving at 121 deg.C for 20min at pH 6.0.

2. Comparison of the change in LAS content before and after treatment of the wastewater

Inoculating the A-YW strain seed solution cultured to logarithmic phase into 100mL of domestic sewage by using the inoculation amount of 1%, then performing purification treatment on a shaking table at the conditions of 30 ℃ and 110rpm for 7 days, and taking supernatant to determine the LAS content in the sewage; the change in the removal rate of the anionic surfactant was measured after the treatment. The results are shown in Table 1.

TABLE 1 variation of LAS content in Water

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.

Claims

1. A bacillus alicyclolyticus for the degradation of anionic surfactants, comprising: the Alicyclobacillus is Alicyclobacillus (Alicyclobacillus aeris) A-YW, which is preserved in China Center for Type Culture Collection (CCTCC) in 2021, 4 months and 25 days, and the preservation number of the strain is CCTCC NO: m2021451.

2. The use of the alicyclobacillus for degrading anionic surfactant according to claim 1 in efficiently degrading anionic surfactant in domestic sewage.

3. The use of the alicyclobacillus for degrading anionic surfactant according to claim 2 in efficiently degrading anionic surfactant in domestic sewage, wherein: inoculating the A-YW strain seed liquid cultured to logarithmic phase to domestic sewage for degradation treatment of anionic surfactant.

4. The use of the alicyclobacillus for degrading anionic surfactant according to claim 3 in efficiently degrading anionic surfactant in domestic sewage, wherein: activated colonies were picked and inoculated in a seed medium, and then A-YW was cultured at 30 ℃ for 3d at 200rpm to grow at a logarithmic phase to prepare a seed solution.

5. The use of the alicyclobacillus for degrading anionic surfactant according to claim 4 in efficiently degrading anionic surfactant in domestic sewage, wherein: the seed culture medium contains 0.5g of peptone, 0.5g of sucrose and 0.2g K per liter₂HPO₄、0.1g MgSO₄·7H₂O、0.1g (NH4)₂SO₄、0.001g FeSO₄·7H₂O、0.02g CaCl₂Autoclaving at 121 deg.C for 20min at pH 6.0.

6. The use of the alicyclobacillus for degrading anionic surfactant according to claim 4 in efficiently degrading anionic surfactant in domestic sewage, wherein: the inoculation amount of the A-YW strain seed liquid is 1-3%.

7. The use of the alicyclobacillus for degrading anionic surfactant according to claim 6, wherein the alicyclobacillus for degrading anionic surfactant is used for efficiently degrading anionic surfactant in domestic sewage, and the method comprises the following steps: the inoculation amount of the A-YW strain seed liquid is 1 percent.