CN115369060B

CN115369060B - Triazolone degrading bacteria and microbial inoculum

Info

Publication number: CN115369060B
Application number: CN202211036684.1A
Authority: CN
Inventors: 施艳红; 操海群; 胡芃; 高全; 肖金京; 王莹
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2022-08-29
Filing date: 2022-08-29
Publication date: 2023-08-15
Anticipated expiration: 2042-08-29
Also published as: CN115369060A

Abstract

The invention belongs to the field of environmental microbiology engineering and technology, in particular to a degradation bacterium stenotrophomonas maltophilia SM3 for efficiently degrading triazolone, which is obtained through screening, and an immobilized microbial inoculum containing the degradation bacterium. The degrading bacteria of the invention are preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No:24515, date of preservation: 2022, 3 and 11. The invention also utilizes trehalose-polyvinyl alcohol-activated carbon to fix the degradation bacteria, can effectively maintain the activity of triazolone degradation bacteria, provides a new thought for the degradation of triazolone, and has better application prospect.

Description

Triazolone degrading bacteria and microbial inoculum

Technical Field

The invention belongs to the field of environmental microbiology engineering and technology, and in particular relates to a degradation bacterium stenotrophomonas maltophilia SM3 for efficiently degrading triazolone and an immobilized microbial inoculum containing the degradation bacterium.

Background

Triazolone (Triadimaefon, CAS: 43121-43-3), 1-4-chlorophenoxy) -3, 3-dimethyl-1, 2, 4-triazol-1-yl) -2-Ding Tong has the formula C ₁₄ Ｈ ₁₆ ClN ₃ O ₂ The bactericide which is widely used for preventing and treating plant diseases and is developed by German Bayer company has the characteristics of high efficiency, low toxicity, low residue, long lasting period, strong systemic property and the like. However, the triazole fungicide is lost to the environment in a large amount, which is also harmful to human society. Triazolone residues in surface waters of south soybean plants in the united states are detected at levels of about 0.291-1.150 μg/L, which can be a potential threat to aquatic animal and human health, albeit at trace levels. Related reports indicate that triazolone can generate toxicity such as genes, nerves and the like after gastrointestinal absorption and systemic circulation, and has reproductive development toxicity, endocrine disturbance, carcinogenesis and teratogenicity and the like on non-target organisms.

The degradation of triazolone includes photochemical degradation, and many students study to degrade triazolone in different media by using sunlight, ultraviolet light, mercury lamps and the like, and also report to degrade triazolone by using activated sodium persulfate and degrade triazolone by ozone oxidation, but the method has the problems of long time, high cost, influence on degradation efficiency due to pH, temperature and the like.

With the development of technology and the increase of screening and understanding of microorganisms, the degradation of pesticide residues by microorganisms has begun to be widely utilized. The microbial degradation pesticide residue has the advantages of low cost, easy operation and no secondary pollution caused by using chemical reagents. CN 201910125508.7 discloses that the separation of triazolone-degrading pale bacillus from fresh Daqu of Fenjiu factory can effectively degrade triazolone residue several tens times exceeding standard; CN 201911118613.4 discloses that alcaligenes faecalis WZ-2 obtained by screening can be used for degrading triazole bactericides; screening to obtain more triazolone degrading bacteria with better effect, and providing urgent requirements for degrading triazolone in water and soil.

Disclosure of Invention

In order to further popularize and biodegrade pesticide residues, particularly to degrade triazolone pollution in water, the invention obtains a novel strain capable of degrading triazolone through wide screening, and provides a novel thought for degrading triazolone. Specific:

the invention provides triazolone degrading bacteria, which are preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center, wherein the preservation number is CGMCC No:24515, date of preservation: 2022, 3 and 11.

In one aspect, the invention provides an application of stenotrophomonas maltophilia (Stenotrophomonas maltophilia) SM3 in degradation of triazolone.

In one aspect, the invention provides a microbial inoculum, which is a liquid microbial inoculum, a freeze-dried powder microbial inoculum and an immobilized microbial inoculum, wherein the microbial inoculum comprises stenotrophomonas maltophilia (Stenotrophomonas maltophilia) SM3. The liquid microbial inoculum is a fermentation product of stenotrophomonas maltophilia (Stenotrophomonas maltophilia) SM3 and also comprises necessary stabilizers, nutritional agents, dispersing agents and the like; in order to maintain the activity and stability of the microorganism and facilitate long-term storage and use, the preparation of the fermentation product of the microorganism into freeze-dried powder is also a feasible method, and the preparation of the freeze-dried powder can freeze-dry the microorganism together with freeze-drying protective agents such as trehalose, glycerol and the like and stabilizers. The immobilized microbial agent can be immobilized by adopting various carriers, in particular various forms of porous carriers, so that the immobilized microbial agent has various pore diameters and is convenient for immobilization and stabilization of microorganisms. The porous carrier can be montmorillonite, porous silica, porous carbon, polymer high molecular porous microsphere, porous membrane microcapsule carrier, etc.

In one aspect of the invention, the invention further provides an immobilized microbial agent. According to the invention, the screened triazolone degrading bacteria Stenotrophomonas maltophilia (named SM 3) is used as an immobilization object, sodium alginate-polyvinyl alcohol-activated carbon (PSC) is used as a composite carrier, the performance of the prepared immobilized bacteria ball (PSC-SM 3) is characterized, and the triazolone in the water body can be efficiently degraded by the preparation of the bacteria ball by the immobilization method. Preferably, the immobilized microbial inoculum comprises 1-4 parts of sodium alginate, 4-16 parts of polyvinyl alcohol (PVA) and 2-7 parts of activated carbon.

The invention provides a preparation method of an immobilized bacterial agent for degrading triazolone, which comprises the steps of uniformly mixing sodium alginate, polyvinyl alcohol, active carbon and triazolone degrading bacterial suspension, dripping the mixture into a cross-linking agent, taking out the cross-linked mixture after cross-linking, washing the cross-linked mixture with a buffer solution, absorbing water, and preserving the cross-linked mixture for later use; the triazolone degrading bacteria are stenotrophomonas maltophilia (Stenotrophomonas maltophilia) SM3.

Advantageous effects

According to the invention, a large number of screening is carried out to obtain the degradation bacterium stenotrophomonas maltophilia (Stenotrophomonas maltophilia) SM3 capable of efficiently degrading triazolone, so that a new thought is provided for researching the degradation of triazolone and knowing the characteristics of stenotrophomonas maltophilia. Meanwhile, the trehalose-polyvinyl alcohol-activated carbon (PSC) compound prepared by the method can effectively fix the degradation bacteria stenotrophomonas maltophilia (Stenotrophomonas maltophilia) SM3 and keep high activity of the degradation bacteria stenotrophomonas maltophilia (Stenotrophomonas maltophilia) SM3, and provides a solution for the fixation of microorganisms. In a word, the technical scheme of the invention provides theoretical value and practical application significance for reasonably evaluating the immobilized cell technology to repair triazolone pollution in water.

Drawings

Fig. 1: trehalose-polyvinyl alcohol-activated carbon (PSC) embedded SM3 pellet morphology.

Fig. 2: scanning electron microscope micrograph of PSC.

Fig. 3: scanning electron microscope micrograph of PSC-SM 3.

Fig. 4: effect of PSC embedded strain SM3 (PSC-SM 3) to degrade triazolone in domestic sewage.

Fig. 5: effect of PSC embedded strain SM3 (PSC-SM 3) to degrade triazolone in industrial wastewater.

Fig. 6: effect of PSC embedded strain SM3 (PSC-SM 3) on degradation of triazolone in farm irrigation water.

Detailed Description

The invention is further described below in connection with specific embodiments. The technical scheme of the invention is a conventional mode in the field unless specifically stated, and the reagent or the material is a conventional reagent and is derived from commercial channels unless specifically stated.

Example 1

1.1 Obtaining and identifying triazolone degrading bacteria stenotrophomonas maltophilia (Stenotrophomonas maltophilia) SM3

Inoculating the stable cultured intestinal bacteria bacterial liquid into enrichment culture medium containing triazolone (50 mg/L) with an inoculum size of 5%, filling nitrogen to ensure anaerobic environment, culturing in a constant temperature oscillator (180 r/min) at 37 ℃ for 3-5 days in a dark place, re-inoculating the stable cultured intestinal bacteria bacterial liquid into enrichment culture medium containing triazolone, repeatedly culturing for three times, inoculating the stable cultured bacterial liquid into inorganic salt culture medium with triazolone as a unique carbon source with an inoculum size of 2%, culturing for 3 days, and transferring to a new inorganic salt culture medium, and culturing for 2-3 weeks.

The bacterial liquid with better growth condition is selected to be diluted by sterile water (10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 ) Then, the mixture was spread on LB (Luria-Bertani) solid medium, and cultured at 37℃for 48 h. Single colonies were transferred to inorganic salt solid medium (containing 100 mg/L triazolone) and single bacteria were picked up in liquid inorganic salt medium containing 50 mg/L triazolone. 37. Culturing in shaking table at r/min for 48 hr, detecting triazolone content in the culture solution by HPLC, and selecting strain with good growth, stable passage and good degradation capability for storage. Wherein a gram-negative bacterium is isolated, which is a yellowish, round, smooth, moist raised microcolonie with a pungent ammonia taste named SM3 on LB medium.

And (3) amplifying and sequencing the strain SM3 by 16S rRNA, comparing the obtained result at NCBI, and carrying out homology analysis on sequences with high similarity to construct a phylogenetic tree. Further analysis in combination with the handbook of identification of common bacterial systems, SM3 was identified asStenotrophomonas maltophiliaThe most closely related to it isStenotrophomonas- maltophiliaKV24, similarity up to 99.4%, and yellow single fungusThe genus Cytophytes is widely present in water, soil, human and animal body surfaces and the gastrointestinal tract.

1.2 Preparation of stenotrophomonas maltophilia SM3 bacterial liquid:

preparation of stenotrophomonas maltophilia SM3 medium: LB (Luria-Bertani) medium (g/L): yeast extract 5 g/L, tryptone 10 g/L, sodium chloride 10 g/L, pH 7.2+ -0.1; if a solid culture medium is prepared, 2% of agar powder is added. All vessels and media were autoclaved at 120℃for 20 min.

The stenotrophomonas maltophilia SM3 was inoculated into LB medium, cultured in a constant temperature shaker at 30℃and 150 r/min for 24 h (OD 600 = 1), centrifuged at 6000 r/min, washed with 10 XPBS for 5min each, and resuspended in 10mL of 10 XPBS buffer at 4℃for further centrifugation.

Example 2

Preparation of stenotrophomonas maltophilia SM3 immobilized bacteria ball

Inoculating SM3 strain stored in-80deg.C refrigerator into LB culture medium, and culturing to OD ₆₀₀ When the bacterial suspension is 1, the bacterial suspension is placed in a high-speed centrifuge 6000 r.min ^-1 Under the condition, the mixture was centrifuged 2 times with 10 XPBS as solvent for 5min each time, and the mixture was washed and resuspended in 10 XPBS buffer of 10mL at 4℃for further use. An embedding method is adopted to prepare the immobilized bacteria ball. Uniformly mixing 1.5% Sodium Alginate (SA), 6% polyvinyl alcohol (PVA), 2.5% Active Carbon (AC) and 0.02g/mL bacterial suspension, injecting the mixed solution into a 5mL syringe, and dripping the mixed solution into 1.5% cross-linking agent CaCl ₂ In the process, after 8. 8 h is taken out and washed in 10 XPBS buffer solution, the surface moisture is sucked by filter paper, and the surface moisture is stored in a refrigerator at 4 ℃ for standby, and SEM observation of the prepared immobilized pellets is shown in figure 3, the surface of PSC can be obviously observed to have rod-shaped bacteria attached, and the area is large, which means that bacteria can be embedded in the PSC, and immobilized triazolone degrading bacteria are prepared. In summary, a method for preparing immobilized microorganisms for bioremediation of triazolone-contaminated water using PSC as a carrier is possible. Wherein 10 XPBS (10 XPBS): weighing 14.4 g KH ₂ PO ₄ 、2 g Na ₂ HPO ₄ 80g NaCl, 2.7g KCl in solutionAnd (3) in 800mL of distilled water, regulating the solution to 7.4 by using HCl, and finally adding distilled water to a volume of 1L to obtain the 0.1M PBS buffer solution.

Example 3

Efficiency test of degradation of triazolone by stenotrophomonas maltophilia SM3 immobilized bacteria ball (PSC-SM 3)

Waste water containing triazolone is produced in the production and use processes of the triazolone, and the discharge standard of the total discharge port of the waste water treatment facilities of the triazolone technical production enterprises is 5 mg/L specified in the discharge standard of the water pollutants of the heterocyclic pesticide industry (GB 21523-2008) in China. Therefore, the invention sets four concentration values to simulate triazolone wastewater, and triazolone solution is added into domestic sewage, industrial wastewater and farm irrigation water to make the final concentration reach 1 mg/L, 2 mg/L, 5 mg/L and 10 mg/L respectively, and the wastewater is placed in an outdoor natural environment. After 1 d, 3 d, 5 d, 7 d and 14 d are respectively extracted and purified from the same amount of wastewater, the triazolone residual quantity is detected, and the degradation rate of PSC-SM3 on the triazolone is calculated corresponding to the initial substrate quantity, so that the degradation effect of the immobilized pellets added into the water body on the triazolone is evaluated.

The PSC-SM3 has the effects of degrading 1 mg/L, 2 mg/L, 5 mg/L and 10 mg/L triazolone in domestic sewage as shown in figure 4. The line graph shows the effect of PSC-SM3 on triazolone removal, with initial triazolone addition as a control. The bar graph shows the effect of removing triazolone after the actual correction, against unfixed SM3. As can be seen from the line graph, the removal effect of PSC-SM3 on triazolone is gradually increased with time at the addition concentration of triazolone of 1 mg/L, 2 mg/L and 5 mg/L, and reaches about 70% at the time of 7 d. Whereas the degradation rate of PSC-SM3 to triazolone was significantly slowed in the range of 1-7 d at a triazolone addition concentration of 10 mg/L, and 53.60% at 7. 7 d. And the degradation rate of PSC-SM3 to triazolone is also reduced at 1 d along with the increase of the initial substrate amount, the degradation rate of 1 d is 47.45 percent and 53.58 percent under the actions of 1 mg/L and 2 mg/L of triazolone, and the degradation rate of 1 d is 32.18 percent and 31.26 percent under the actions of 5 mg/L and 10 mg/L of triazolone. The final degradation rates of PSC-SM3 pair 1 mg/L, 2 mg/L, 5 mg/L, 10 mg/L triazolone at 14 d were 80.21%, 80.03%, 79.72%, 76.64%, respectively. Meanwhile, compared with SM3 alone, the degradation rate of triazolone of PSC-SM3 is obviously improved at each concentration, and P is less than 0.05.

The effect of PSC-SM3 on degrading 1 mg/L, 2 mg/L, 5 mg/L, 10 mg/L triazolone in industrial wastewater is shown in FIG. 5. The line graph shows the effect of PSC-SM3 on triazolone removal, with initial triazolone addition as a control. The bar graph shows the effect of removing triazolone after the actual correction, against unfixed SM3. As can be seen from the line graph, in 7 d, the degradation efficiency of PSC-SM3 on four kinds of triazolone is slowed down when the triazolone is added at the concentration of 10 mg/L, and the degradation rates under the actions of 1 mg/L, 2 mg/L and 5 mg/L are all about 60%. As the initial substrate amount increases, PSC-SM3 also reduces triazolone degradation at 1 d, the degradation rate of 1 d is 43.24% under the action of 1 mg/L and 2 mg/L triazolone, the degradation rate of 1 d is 21.87% under the action of 5 mg/L and 10 mg/L triazolone, and the degradation rate of 1 d is 27.99%. The final degradation rates of PSC-SM3 pair 1 mg/L, 2 mg/L, 5 mg/L and 10 mg/L triazolone at 14 d are all higher than 80%, respectively 89.50%, 86.86%, 81.06% and 80.72%. Meanwhile, compared with SM3 alone, the degradation rate of triazolone of PSC-SM3 is obviously improved at each concentration, and P is less than 0.05.

The PSC-SM3 degradation of 1 mg/L, 2 mg/L, 5 mg/L, 10 mg/L triazolone in farm irrigation water is shown in FIG. 6. The line graph shows the effect of PSC-SM3 on triazolone removal, with initial triazolone addition as a control. The bar graph shows the effect of removing triazolone after the actual correction, against unfixed SM3. As can be seen from the line graph, with the increase of the initial substrate amount, PSC-SM3 also has a decrease in triazolone degradation at 1 d, and the degradation rate of 1 d is 44.64%, 40.34% under the action of 1 mg/L and 2 mg/L triazolone, and the degradation rate of 1 d is 28.30% and 26.31% under the action of 5 mg/L and 10 mg/L triazolone. 10 Under the action of mg/L triazolone, PSC-SM3 in 1-7 d can degrade triazolone more slowly than under the actions of 1 mg/L, 2 mg/L and 5 mg/L triazolone, 45.05% is adopted in 7 d, and 53.37% -73.99 is adopted under the actions of 1 mg/L, 2 mg/L and 5 mg/L triazolone. Under the action of 2 mg/L triazolone, PSC-SM3 has almost similar trend increase to the degradation rate of triazolone along with time, and under the actions of 1 mg/L, 2 mg/L and 5 mg/L triazolone, the degradation rate in 1-7 d is relatively gentle, and the degradation rate in 7-14 d is relatively fast. The final degradation rates of PSC-SM3 for 1 mg/L, 2 mg/L, 5 mg/L, 10 mg/L triazolone at 14 d were 84.50%, 84.39%, 76.76%, 74.47%, respectively. Meanwhile, compared with SM3 alone, the degradation rate of triazolone of PSC-SM3 is obviously improved at each concentration, and P is less than 0.05.

The foregoing is a further elaboration of the present invention in connection with the detailed description, and it is not intended that the invention be limited to the specific embodiments shown, but rather that a number of simple deductions or substitutions be made by one of ordinary skill in the art without departing from the spirit of the invention, should be considered as falling within the scope of the invention as defined in the appended claims.

Claims

1. A triazolone degrading bacterium is characterized in that the degrading bacterium is stenotrophomonas maltophiliaStenotrophomonas maltophilia) SM3, preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No:24515, date of preservation: 2022, 3 and 11.

2. The use of the degrading bacterium according to claim 1 for degrading triazolone.

3. A triazolone degrading bacterial agent, which is characterized by comprising the degrading bacterial agent in claim 1.

4. The microbial inoculum according to claim 3, wherein the microbial inoculum is a liquid microbial inoculum, a freeze-dried powder microbial inoculum or an immobilized microbial inoculum.

5. The microbial inoculant of claim 4, wherein the immobilized microbial inoculant further comprises trehalose, polyvinyl alcohol, and activated carbon.

6. The microbial inoculant of claim 5, wherein the immobilized microbial inoculant comprises 1-4 parts of sodium alginate, 4-16 parts of polyvinyl alcohol (PVA) and 2-7 parts of activated carbon.

7. The preparation method of the immobilized bacterial agent for degrading triazolone is characterized by comprising the steps of uniformly mixing sodium alginate, polyvinyl alcohol, active carbon and triazolone degrading bacterial suspension, dripping the mixture into a cross-linking agent, taking out the cross-linked mixture after cross-linking, washing the cross-linked mixture in a buffer solution, absorbing water, and preserving the cross-linked mixture for later use; the triazolone degrading bacterium is the degrading bacterium of claim 1.

8. The preparation method according to claim 7, comprising uniformly mixing 1.5% sodium alginate, 6% polyvinyl alcohol, 2.5% active carbon and 0.02g/mL bacterial suspension, injecting the mixture into a 5mL syringe, and dropwise adding the mixture to 1.5% cross-linking agent CaCl ₂ After 8. 8 h, the sample was taken out and washed in 10 XPBS buffer, and the surface water was sucked off with filter paper and stored in a refrigerator at 4℃for further use.