CN113481127B - Mutant strain of denitrifying alicyclic acidophile and application thereof - Google Patents

Mutant strain of denitrifying alicyclic acidophile and application thereof Download PDF

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CN113481127B
CN113481127B CN202110859319.XA CN202110859319A CN113481127B CN 113481127 B CN113481127 B CN 113481127B CN 202110859319 A CN202110859319 A CN 202110859319A CN 113481127 B CN113481127 B CN 113481127B
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李宁
左群
吕育财
龚大春
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract

By adopting the normal pressure room temperature plasma (ARTP) mutagenesis technology, the method is suitable for the treatment of the tumorAlicycliphilus denitrificansThe YLb10 bacterial strain is induced and screened to obtainAlicycliphilus denitrificans L1-3, the strain has higher Cr (VI) reduction capability. At the Cr (VI) concentration of 300mg/L, the mutant strain L1-3 can reduce 19.3 percent of Cr (VI) in 72 hours; under the condition of the Cr (VI) concentration of 400mg/L, the reduction rate of the mutant strain L1-3 to the Cr (VI) reaches 17.72 percent.

Description

Mutant strain of denitrifying alicyclic acidophile and application thereof
Technical Field
The invention relates to a biological preparation, in particular toAlicycliphilusdenitrificansMutagenic strain and its application.
Background
Chromium (Cr) is an important industrial heavy metal, and is present in the environment mainly in the form of Cr (III) and Cr (VI). Cr (VI) has toxicity, is a main pollution heavy metal in the environment, has the toxicity more than 100 times that of Cr (III), and is an essential nutrient element of organisms, so that the reduction of Cr (VI) to Cr (III) is a main repair path of Cr (VI) pollution in the environment.
The method has the advantages of good economic effect, simple process and the like and has wide development space for repairing Cr (VI) pollution in the environment by microorganisms. Obtaining high-quality Cr (VI) repairing microorganisms is the key for improving the microbial bioremediation of Cr (VI) pollution.
Many microorganisms capable of remediating Cr (VI) contamination are currently isolated. Comprises thatPseudomonas mandelii、Bacillus pumilus、Cellulosimicrobiumcellilans、sulfate-reducing bacteria、Desulfovibriodesulfuricans、MorganellamorganiiAnd the like. The repairing mode is mainly that the microorganism reduces Cr (VI) into Cr (III), and a bacterial enzyme system with NADH as a reducing agent converts the Cr (VI) into soluble NAD + -Cr (III) complex, chromate reduced to soluble chromium (III) complex. However, cr (VI) has a toxic effect on microorganisms, and many microorganisms cannot effectively reduce chromium or grow in a high-concentration Cr (VI) environment, so that the separation of Cr (VI) reducing strains with high performance from the environment or the acquisition of Cr (VI) reducing strains with high performance by genetic engineering means is still an important issue in the field of heavy metal bioremediation.
The invention relates to a Cr (VI) reducing strain existing in the laboratoryAlicycliphilusdenitrificansYLb10 is used as an initial strain, and is mutagenized by an ARTP method to further improve the Cr (VI) reducing capability of the strain.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defect of insufficient Cr (VI) reducing microorganism resources in the existing heavy metal pollution bioremediation technology and provides a method for restoring heavy metal pollution by using Cr (VI) reducing microorganism resourcesAlicycliphilusdenitrificansA mutagenized strain capable of reducing Cr (VI).
The invention aims to provide a mutagenic strainAlicycliphilusdenitrificansL1-3, preserved in China center for type culture Collection at 31 months 5 and 2021, the preservation number of the strain is CCTCC NO: m2021650. The mutant strain is rod-shaped and gram-negative bacteria.
The technical scheme of the invention is to use bacterial strainsAlicycliphilusdenitrificansYLb10 (from the light Industrial function Yeast stress laboratory in China, university of the three gorges, the strain is deposited inChina center for type culture Collection (Hubei, wuhan university), the preservation number of strains is CCTCC NO: m2020415) as an initial strain, performing ARTP mutagenesis, and screening to obtain a mutagenized strainAlicycliphilusdenitrificans L1-3。
The original strain Alicyclophila denitrificans: (Alicycliphilusdenitrificans) YLb10 is a gram-negative bacterium from Yangxi bottom mud, university of Sanxia, functional Yeast stress laboratory in light industry, china, university of Sanxia.
The invention makes the denitrified alicyclobacillus mutator strainAlicycliphilusdenitrificansApplication of L1-3 in Cr (VI) reduction.
The invention relates toAlicycliphilusdenitrificansApplication of L1-3 mutant strain in preparing Cr (VI) reduction biological agent.
A Cr (VI) reduced biological agent, the biological agent comprises the denitrified alicyclobacillus mutagenic strainAlicycliphilusdenitrificansL1-3。
In the biological preparationAlicycliphilusdenitrificansL1-3 effective viable count is 10 5 -10 8 CFU/mL or 10 5 -10 8 CFU/g.6. The alicyclobacillus denitrificans mutant strain according to claim 1AlicycliphilusdenitrificansUse of L1-3 for Cr (VI) tolerance.
The mutation strain of the alicyclic-philic denitrificansAlicycliphilusdenitrificansApplication of L1-3 in preparing Cr (VI) tolerant biological agents.
A Cr (VI) -tolerant biological agent, said biological agent comprising saidAlicycliphilusdenitrificansAnd (3) mutagenizing the strain.
In the biological preparationAlicycliphilusdenitrificansThe concentration of the mutant strain vegetative cell suspoemulsion is 10 5 -10 8 CFU/mL or 10 5 -10 8 CFU/g。
To be provided withAlicycliphilusdenitrificansYLb10 is the original strain, the cultured bacterial colony is used for preparing bacterial suspension by utilizing normal saline, and the bacterial concentration is adjusted to be 10 6 ~10 8 In the meantime. Will be provided with60-100uL of 60% glycerol is added into 0.9-1.3mL of bacterial suspension and mixed well. Taking 8-15uL of mixed bacterial suspension, putting the mixed bacterial suspension into an ARTP mutagenesis chamber, setting the power to be 100-150W, the processing time to be 100-150s and the argon flow to be 10.0SLM, and starting mutagenesis. The mutagenized bacterial suspension was transferred to fresh 10mL sterile LB, incubated at 28 ℃ for 2h for resuscitation. The recovered bacterial suspension was inoculated into a 96-well plate containing LB medium at a concentration of 450mg/L of Cr (VI), and cultured at 28 ℃. And observing the growth condition of the strain, and obtaining a culture strain with higher growth OD600 as a positive strain. After purification, cr (VI) reducing ability was measured as a target strain. Finally obtaining the strain through detection and screeningAlicycliphilusdenitrificans L1-3。
AlicycliphilusdenitrificansThe culture of L1-3 was carried out using LB medium containing Cr (VI). The main components are as follows: tryptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L, pH 7.2-7.6, cr (VI) 100 mg/L (K) 2 Cr 2 O 7 The concentration is adjusted as necessary). The strain is cultured at 28 deg.C in solid medium, and 1.5% agar powder is added.
Said strainAlicycliphilusdenitrificansL1-3, deposited in China center for type culture Collection at 31.5.2021 at the location of university in Wuhan, wuhan. The preservation number of the strain is CCTCC NO: m2021650.
The strain L1-3 is cultured for 72 hours under the condition that the environmental Cr (VI) concentration is 300mg/L, and the reduction rate of Cr (VI) is improved to 19.3 percent. When the Cr (VI) concentration in the culture environment was 400mg/L, the Cr (VI) reduction rate was 17.7% at 60 hours. It can be seen that the obtained strain L1-3 has remarkable ability of reducing Cr (VI) under the condition of high concentration Cr (VI) after ARTP mutagenesis;
the strain L1-3 has better tolerance to high-concentration Cr (VI). When the concentration of Cr (VI) in the environment is 400mg/L, the biomass growth is obvious after 60 hours of culture, and the OD600 can reach about 1.0. The strain L1-3 has better performance in resisting the inhibition of high-concentration Cr (VI) and effectively growing.
Another object of the present invention is to use said mutagenized strainAlicycliphilusdenitrificansThe L1-3 is applied to the microbial remediation of Cr (VI) polluted water and soil.
Drawings
FIG. 1 shows the ability of the L1, L2 cultures to reduce Cr (VI) in example 1.
FIG. 2 shows the growth of the L1 and L2 cultures in Cr (VI) -containing culture broth in example 1.
FIG. 3 is a graph showing the reduction ability of Cr (VI) by the pure culture strain isolated in example 1.
FIG. 4 is a graph showing the growth ability of the pure cultured strain isolated in example 1 in a Cr (VI) environment.
FIG. 5 shows the growth pattern of the mutagenized strain L1-3 in example 2 on LB medium containing Cr (VI).
FIG. 6 is the Cr (VI) reduction and growth capacity of strain L1-3 of example 3 at a concentration of 400 mg/LCr (VI).
Detailed Description
Example 1
ARTP mutagenesis and Strain L1-3 acquisition
(1) Original strainAlicycliphilus denitrifians Plate activation of YLb10
The original strain to be preservedAlicycliphilusdenitrifiansYLb10 is activated (preserved in China center for type culture Collection in 2020, 8 months and 10 days, with the preservation number of CCTCC NO: M2020415, classified and named as:AlicycliphilusdenitrifiansYLb10, deposited at China, wuhan university), inoculated onto LB medium plate, and cultured in 28 ℃ incubator for 3 days. LB culture medium: the LB culture medium comprises the following main components: 10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of sodium chloride and pH =7.2-7.6.
Scraping a small amount of YLb10 bacterial colonies on the plate, inoculating the bacterial colonies into LB liquid culture medium, measuring the OD600 value of the YLb10 bacteria at regular time, and drawing a growth curve.
Preparing a YLb10 bacterial suspension: centrifugally collecting the bacteria liquid cultured to logarithmic phase, washing with normal saline for 2 times, and diluting with normal saline to obtain the product with OD600 value of 0.6-0.8 or bacteria concentration of 10 6 ~10 8 The bacterial suspension in between. Bacterial strainsAlicycliphilus denitrifians Ylb10。
(2) The ARTP mutagenesis was performed as follows
1) Absorbing 84 mu L of glycerol with the mass concentration of 60% into the prepared YLb10 bacterial suspension, and oscillating on a vortex mixer for 2-3 min to fully and uniformly mix the glycerol with the suspension;
2) Placing a metal slide on an alcohol lamp outer flame on an ultra-clean bench for burning for 30 s, cooling, placing the metal slide on a disposable flat plate or a sterilized glass flat plate, and uniformly coating 10 mu L of bacterial liquid on the slide;
3) Moving the flat plate with the sample slide to an ARTP-M normal temperature and pressure plasma mutation breeding instrument, placing the slide to a corresponding hole position by using sterile forceps, adjusting a knob below a carrier table to enable the slide to be positioned at a position 2 mm away from an airflow port, and closing a bin gate;
4) Set instrument power 120W, process time 120 s, air flow parameter 10.00 SLM. Clicking a' start processing button to start processing a sample;
5) After the sample is processed, placing the slide glass into a centrifuge tube filled with 10mL of LB culture medium by using sterile forceps;
6) Vibrating the centrifugal tube on an oscillator for 1 min, and eluting microorganisms attached to the bacterial slide into liquid to form new bacterial suspension;
7) And (3) placing the centrifugal tube containing the bacterial suspension into a constant-temperature incubator at 28 ℃ for 2 hours, so that the mutagenized bacterial strain can be revived, and the screening of the bacterial strain at the later stage is facilitated.
(3) ARTP mutagenesis screening
The bacterial suspension after mutagenesis is titrated into a 96-well plate containing LB liquid culture medium of 450mg/L Cr (VI) by using a drop titration device in a laboratory, and the plate is placed in an incubator at 28 ℃ to screen strains capable of growing. Measuring the change of OD600 in the pore plate once by using a microplate reader for 12 hours, counting the change value, screening the pores with obvious growth, transferring all the pores into a test tube, expanding the pores by using LB liquid culture medium containing 300mg/L Cr (VI) with the pH of =7.2-7.6, and placing the pores in an incubator at 28 ℃ for standing culture.
(4) Double sieve
After primary screening, bacterial liquid cultured for 3-5 days is centrifuged at 8000 r/min for 5 min for collecting thalli, and the thalli are inoculated in a fresh LB-Cr culture medium (the concentration of Cr (VI) is 450 mg/L), the initial OD600 is 0.2, the temperature is 28 ℃, each group is provided with three repetitions, and OD600 is detected at 0 d, 3d and 6 d, and the content of Cr (VI) is changed. Finally obtaining 2 cultures L1 and L2 with higher Cr (VI) reduction efficiency, wherein the reduction rates of the cultures L1 and L2 to Cr (VI) are respectively as follows: 21.02% and 24.89% (results are shown in FIG. 1).
Cultures of both L1 and L2 mutagenized strains were cultured in LB-Cr (Cr (VI) =450 mg/L) medium with an initial OD600 of 0.2, an OD600 of 3d, L1 of 1.6, and an OD600 of L2 of 1.7. It was shown that the microorganisms in the culture in 2 were able to tolerate the inhibition of high concentrations of Cr (VI) while growing efficiently, and the results are shown in FIG. 2.
In order to obtain a pure culture strain capable of efficiently reducing Cr (VI). Performing plate line drawing and purification on the L1 and L2 strain cultures, and finally separating to obtain 6 pure culture strains: respectively, 3 strains derived from the L1 culture: l1-3, L1-5, L1-15; and 3 strains derived from L2 culture: l2-7, L2-9 and L2-15.
L1-3, L1-5, L1-15, L2-7, L2-9, L2-15 were cultured in LB-Cr (Cr (VI) =300 mg/L) broth, and the Cr (VI) concentration and strain biomass (OD 600) were measured at 0 hours and 72 hours, respectively. The 6 strains were examined for their ability to reduce Cr (VI) and for their ability to grow in a Cr (VI) environment.
The results show that the reduction rates of the L1-3, L1-5, L1-15, L2-7, L2-9 and L2-15 strains to 300mg/L Cr (VI) at 72 hours of the 6 pure culture strains are respectively as follows: 19.3%, 12.3%, 8.2%, 17.3%, 6.1%, 8.3%. The strain L1-3 has the strongest Cr (VI) reduction capability, and the result is shown in figure 3.
The growth of each pure culture strain is shown in FIG. 4. After the initial culture OD600 of each strain is adjusted to about 0.2, and the initial culture is carried out for 72 hours, the biomass (OD 600) of L1-3, L1-5, L1-15, L2-7, L2-9 and L2-15 is respectively increased as follows: 0.263, 0.067, 0.141, 0.13, 0.178, 0.233. The growth of biomass of the strain L1-3 is highest in 72 h. Therefore, the mutagenized strain L1-3 has better Cr (VI) resistant growth capacity.
The purified strain L1-3 has the highest Cr (VI) reduction efficiency and growth capacity in a culture solution of 300mg/L Cr (VI). Therefore, L1-3 was selected as a final Cr (VI) reducing strain obtained by ARTP mutagenesis and directed selection of YLb 10.
The obtained mutagenized strain L1-3 was classified and namedAlicycliphilus denitrificans L1-3, preserved in China Center for Type Culture Collection (CCTCC) in 2021, 5 months and 31 days, and the preservation number of the strains is CCTCC NO: m2021650, deposit address: wuhan, wuhan university.
Example 2
Morphology of Strain L1-3:
the colony morphology of the mutagenized strain L1-3 is shown in FIG. 5. L1-3 can grow on LB solid medium containing 300mg/L Cr (VI), and the colony morphology is round, white and regular on the edge.
Example 3
Cr (VI) reducing ability and growing ability of mutagenic strain L1-3 under 400mg/L Cr (VI)
Mutagenizing the strain L1-3 (10) 6 CFU/mL) were cultured in LB-Cr broth at a Cr (VI) concentration of 400mg/L, and the ability of the strain to reduce Cr (VI) and grow was examined at higher Cr (VI) concentrations. The results are shown in FIG. 6.
The mutant strain L1-3 can reduce Cr (VI) under the condition of Cr (VI) concentration of 400mg/L. The Cr (VI) concentration in the culture solution gradually decreased with the increase of the culture time, and after the culture for 60 hours, the Cr (VI) concentration in the culture solution was decreased from 402.65mg/L to 331.32 mg/L. The reduction efficiency of Cr (VI) was 17.7%. At a Cr (VI) concentration of 400mg/L, the biomass of L1-3 increased with increasing cultivation time. The OD600 increased from 0.22 at the time of initial culture to 1.01 at 60 h. The above structure shows that the L1-3 strain can tolerate the inhibition of Cr (VI) of 400mg/L, grow and effectively reduce Cr (VI).
The mutagenized strain L-3 can reduce Cr (VI) and grow under the condition of high concentration of Cr (VI), which is beneficial to the application of the strain in the microbiology of Cr (VI) pollution and the preparation of the microbial agent for repairing the Cr (VI) pollution.

Claims (2)

1. Mutant strain of alicyclic-philic denitrificaion bacteriumAlicycliphilus denitrificans Application of L1-3 in Cr (VI) with tolerance concentration of 400mg/L, and denitrified alicyclobacillus mutant strainAlicycliphilus denitrificans L1-3, classified and namedAlicycliphilus denitrificans L1-3, preserved in China center for type culture Collection at 31 months 5 and 2021, the preservation number of the strain is CCTCC NO: m2021650, deposit address: wuhan university, wuhan, china.
2. A biological agent resistant to Cr (VI) at a concentration of 400mg/L, comprising the mutated strain of Alicyclobacillus denitrificans of claim 1Alicycliphilus denitrificans L1-3, in said biological preparationAlicycliphilus denitrificansL1-3 effective viable count is 10 5 -10 8 CFU/mL or 10 5 -10 8 CFU/g, and the concentration of Cr (VI) in the process of reducing Cr (VI) is 400mg/L.
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MX2018005790A (en) * 2018-05-09 2019-11-11 Univ Mexico Nac Autonoma Klebsiella pneumoniae strain for the biotransformation of chrome, iron, selenium and uranium metals.
CN111996138A (en) * 2020-08-10 2020-11-27 三峡大学 Reduced strain Ylb10 and application thereof in reduction of Cr (VI)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2018005790A (en) * 2018-05-09 2019-11-11 Univ Mexico Nac Autonoma Klebsiella pneumoniae strain for the biotransformation of chrome, iron, selenium and uranium metals.
CN111996138A (en) * 2020-08-10 2020-11-27 三峡大学 Reduced strain Ylb10 and application thereof in reduction of Cr (VI)

Non-Patent Citations (3)

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Title
Alicycliphilus denitrificans Ylb10 的 Cr( VI) 还原性质研究;左群等;《生物学杂志》;20210210;36-40 *
Enrichment and characterization of an effective hexavalent chromium-reducing microbial community YEM001;Yucai Lyu等;《Environmental Science and Pollution Research》;20210106;19866-19877 *
Potential of bacterial isolates from a stream in manaus-amazon to bioremediate chromium-contaminated environments;Ydrielly Veras Teles等;《Water Air Soil Pollut》;20180731;1-5 *

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