CN113462611B - Nicotine-degrading Arthrobacter ureafaciens and application thereof - Google Patents

Abstract

The invention discloses urea-producing arthrobacterium for degrading nicotine and application thereof, wherein the urea-producing arthrobacterium is urea-producing arthrobacterium CZY1 with a preservation number of CCTCC NO: M2021466, and the obtained strain CZY1 can be directly used for degrading nicotine in waste tobacco liquid extract in a reconstituted tobacco production process, and comprises activation, transfer culture and inoculation degradation, and the strain CZY1 is activated in an LB liquid culture medium; transfer culture: transferring the activated strain into a new LB liquid culture medium, culturing to a logarithmic phase, centrifugally stirring, washing and then re-suspending; transferring the heavy suspension bacteria liquid into BSM liquid culture medium containing a certain amount of nicotine, culturing under the conditions of a certain temperature, a certain pH and an inoculation amount, sampling and determining nicotine concentration and OD_600。The method is simple to operate, and the obtained urea-producing arthrobacter CZY1 has high tolerance and high degradability to nicotine.

Description

Nicotine-degrading Arthrobacter ureafaciens and application thereof

Technical Field

The invention belongs to the technical field of microbial engineering, and particularly relates to urea-producing arthrobacterium capable of degrading nicotine and application thereof.

Background

As a special agricultural product, the tobacco has high domestic yield and generates a great amount of waste. The waste tobacco liquid extracting solution (TWE) in the reconstituted tobacco production process is liquid homogenate obtained by crushing tobacco wastes (tobacco stems, tobacco powder and partial low-grade tobacco leaves), performing solid-liquid separation after water extraction and performing film concentration, wherein the TWE is complex in physicochemical components and contains high-concentration nicotine, and degradation treatment of harmful substances such as nicotine is required before discharge, so that the ecological environment is prevented from being damaged.

Nicotine (Nicotine), also known as Nicotine, is an alkaloid present in plants of the solanaceae family (solanum genus) and is also one of the important constituents of tobacco. Nicotine, as an alkaloid, accounts for 95% of the total tobacco alkaloid and is a major factor responsible for cigarette addiction. As a highly toxic N-heterocyclic compound, nicotine can cross biological membranes and the blood brain barrier, interact with acetylcholine receptors, and cause various adverse reactions and even death. The United States Environmental Protection Agency (USEPA) has specified tobacco production waste streams with nicotine contents in excess of 500 mg/kg as "toxic release list" (TRI) chemicals (Dorgan, c.a. Statistical accepted of the environment, 3rd ed), and the european union has also established regulations that when nicotine contents are above 0.05% (w/w), they are classified as hazardous waste and must not be discharged directly to the environment without treatment, and the nicotine waste streams need to be treated separately and not allowed to be treated with other municipal waste or wastewater.

At present, physical, chemical and biological methods are mainly used in industry to treat nicotine in various wastes. Wherein the physical method mainly comprises an adsorption method and an extraction method, but the physical method does not substantially change the structure of the nicotine; the chemical methods have many kinds, but the basic principles are the same, namely, a strong oxidant is utilized to destroy the properties of functional groups or chemical bonds of nicotine so as to degrade the nicotine, however, the properties of the nicotine are stable, the boiling point is 274 ℃ and the melting point is-79 ℃, so that the chemical methods have the defects of high energy consumption, low efficiency, a large number of byproducts, secondary pollution easily caused by improper operation and the like, and further, the chemical methods are accompanied by high temperature and high heat, even open fire, and certain potential safety hazards exist in the production process. Under the condition, a green, environment-friendly and sustainable biological method is applied. The biological method utilizes some microorganisms which can take nicotine as a carbon and nitrogen source to degrade the nicotine, and has the advantages of low cost, easy culture, safety, environmental protection and the like.

At present, domestic reports on microorganisms with high nicotine tolerance are few, and related researches are not deeply developed.

Disclosure of Invention

The invention aims to provide urea arthrobacterium for degrading nicotine and application thereof, and urea arthrobacterium (A), (B) and (C)Paenarthrobacter ureafaciens) CZY1 shows better nicotine tolerance with some nicotine degradation ability.

Arthrobacter ureafaciens for degrading nicotinePaenarthrobacter ureafaciens) CZY1, depository: china center for type culture Collection; address: the institute of Life sciences of Wuhan university in Lojia mountain in Wuchang district, Wuhan, Hubei province; preservation day: 2021, 4 months and 27 days; the preservation number is CCTCC NO: M2021466.

Application of urea-producing arthrobacter CZY1 in nicotine degradation by waste tobacco liquid water extraction in a reconstituted tobacco production process.

Further, the applications include activation, transfer culture and inoculation degradation, and specifically include:

1) and (3) activation: activating the strain CZY1 in LB liquid culture medium;

2) transfer culture: transferring the activated strain into a new LB liquid culture medium, culturing to a logarithmic phase, centrifugally stirring, washing and then re-suspending;

3) transferring the re-suspended bacterial liquid into BSM liquid culture medium containing a certain amount of nicotine, culturing under the conditions of a certain temperature, a certain pH and an inoculation amount, sampling and measuring nicotine concentration and OD_600。

Further, the temperature is 30-40 ℃, the PH is 5-8, the initial nicotine concentration is 2-14 g/L, and the inoculation amount is 1-6% (vol/vol).

The invention has the beneficial effects that: the screened Arthrobacter ureafaciens CZY1 has high tolerance and can effectively degrade nicotine.

Drawings

FIG. 1 is a perspective electron micrograph of strain CZY 1;

FIG. 2 is a phylogenetic evolutionary tree of CZY 1;

FIG. 3 shows the growth of the strain under nicotine conditions at different temperature conditions;

FIG. 4 shows the growth of strains under nicotine conditions at different pH values;

FIG. 5 shows the growth of the strains under nicotine conditions at different initial nicotine concentrations;

FIG. 6 shows the growth of strains under different inoculum size conditions in a nicotine environment;

FIG. 7 is a growth curve and a degradation curve of the strain in a medium.

Detailed Description

The invention is further illustrated by the following examples and figures of the specification.

Sample source: arthrobacter ureafaciens (Paenarthrobacter ureafaciens) CZY1 was isolated from TWE samples from hangzhou li group environmental protection paper industries ltd.

Example 1

Screening to obtain urea-producing arthrobacterium (II)Paenarthrobacter ureafaciens）CZY1：

1) Collecting a waste smoke liquid water extract sample:

a sample of the aqueous extract of waste tobacco smoke containing high concentration of nicotine was collected from Hangzhou Liqun environmental protection paper industries, Ltd, sealed with a sterilized culture bottle, and stored at 4 ℃.

2) Screening of high-tolerance nicotine-degrading strains:

for the primary screening of the high-tolerance nicotine-degrading bacterial strain, 25 mL of waste smoke liquid water extract is diluted into 100 mL by using sterile water, centrifuged at 3000 rpm for 3 min at normal temperature, supernatant is centrifuged at 5000 rpm for 2 min at normal temperature, supernatant is discarded, 5 mL of sterile water is used for resuspension and precipitation, centrifuged at 5000 rpm for 2 min at normal temperature, and the steps are repeated twice to wash out residual impurities. The precipitate was resuspended in 5 mL of sterile water to obtain crude cells. The crude cells were inoculated into 100 mL of Basic inorganic salt (BSM) liquid medium containing 8 g/L nicotine, and cultured at 30 ℃ and 180 rpm for 4 days. After the culture medium is turbid, a small amount of bacterial liquid dipped by an inoculating loop is streaked and separated on a BSM solid plate with the same concentration, and the culture is carried out in an inverted mode at the temperature of 30 ℃. After the single colony grows out, the single colony is streaked on a BSM solid plate with the same concentration again, inverted culture is carried out at 30 ℃ until a purified single colony is obtained, the pure single colony is inoculated into 100 mL of BSM liquid culture medium containing 8 g/L nicotine, and culture is carried out at 30 ℃ and 180 rpm for 4 days for re-screening verification.

Example 2

Separating strain morphology, physiological and biochemical identification:

the characteristics of the bacterial strains were further characterized using microbiological experiments (second edition, zhao bin, forest meeting, joshao kingdom) with the colony shape being circular, yellow, convex, smooth in surface, opaque in edges, purple in gram staining results, indicating positive, the result photographed by electron microscopy is shown in fig. 1, CZY1 being rod-shaped, having no spores, and having no flagella. The genus of the strain is determined according to 16S rRNA, the strain is determined according to Bergey' S handbook of bacteria, the strain is inoculated into LB liquid culture medium for overnight activation, and Ezup column type bacterial genome DNA extraction kit is used for extracting (biological engineering, Shanghai, China) total genome DNA according to the instruction. 16S rRNA was amplified using the universal primer, forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'-GGTTACCTTGTTACGACTT-3'), and bacterial 16S rRNA sequences were amplified using the high fidelity polymerase 2 × Phanta Master Mix (Takara) in the following PCR reaction: 1 μ L of genomic DNA; 2 × Phanta Master Mix 25 μ L; primer F (10 μm) 2 μ L; primer R (10 μm) 2 μ L; ddH₂O was supplemented to 50. mu.L. The PCR reaction procedure was as follows: initial temperature 95 ℃ for 3 minutes, 95 ℃ denaturation for 15 seconds, 60 ℃ annealing for 15 seconds, 72 ℃ extension for 15 seconds, cycle number 30, 72 ℃ final extension for 5 minutes, final hold at 16 ℃, PCR reaction fluid exchange by beijing optimak for sequencing, sequence alignment by NCBI with BLAST database, MEGA-X construction phylogenetic tree, ClusterW and adjacency method.

The results of the biochemical tests are shown in table 1:

TABLE 1 physiological and biochemical experimental results of strain CZY1

Detecting items	CZY1
		Color of colony	Light yellow
Gram stain	+
		Optimum pH	7.0
Optimum temperature	30
		Catalase Activity	+
Methyl Red reaction	-
		Urease activity	+
Amylase Activity	-
		Indole formation	-
Acid production by glucose fermentation	-
		Fructose fermentation acid production	-
Nitrate reduction reaction	+
		Hydrolysis reaction of gelatin	-

16s rRNA sequencing results: 16S rRNA was amplified by using the universal primer, forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'-GGTTACCTTGTTACGACTT-3'). The sequencing result of the PCR reaction solution is shown as SEQ ID NO. 1.

The specific result of the evolutionary tree is shown in FIG. 2, and the result shows that the strain CZY1 belongs to Arthrobacter ureafaciens.

Example 3

Optimum growth temperature experiment of isolated strains in nicotine environment:

activating the strain of example 1 in LB liquid culture medium overnight, transferring into new LB liquid culture medium, culturing at 30 deg.C and 180 rpm to logarithmic phase, centrifuging part of the strain at 5000 rpm for 2 min, washing with sterile water for 2 times, resuspending with appropriate amount of sterile water, transferring the resuspended strain into BSM liquid culture medium containing 2g/L nicotine under conditions of initial pH7.0, initial nicotine concentration 2g/L and inoculum size 2% (vol/vol), culturing at 180 rpm at different temperatures (30 deg.C, 35 deg.C, 37 deg.C and 40 deg.C) for 96 h, sampling, and determining OD₆₀₀The optimum temperature was determined by setting 3 replicates for each group, and the result is shown in FIG. 3, where the optimum temperature was 30 ℃.

Example 4

Optimum growth PH conditions of the isolated strain in nicotine environment:

using the resuspended suspension of example 3, the suspension was incubated at 30 ℃ with an initial nicotine concentration of 2g/L and an inoculum size of 2% (vol/vol) at 180 rpm at different pH's (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) for 96 h, and the OD was determined by sampling₆₀₀The optimum pH was determined by setting 3 replicates for each group and was 7.0 as shown in FIG. 4.

Example 5

Optimum initial nicotine concentration conditions of the isolated strain in nicotine environment:

the resuspended suspension of example 3 was used at a temperature of 30 ℃ and pH7.0 with an inoculum size of 2% (vol/vol) at different initial nicotine concentrations (2, 4, 6, 8,10. 12, 14, 16 g/L) at 180 rpm for 96 h, sampling and determining the OD₆₀₀Each group was set to 3 replicates and the optimum initial nicotine concentration was determined, as can be seen from figure 5, the optimum initial nicotine concentration was 2 g/L.

Example 6

Optimum inoculum size of the isolated strain in nicotine environment:

using the resuspended suspension of example 3, the suspension was incubated at 30 ℃ and pH7.0 for 96 h at an initial nicotine concentration of 2g/L at 180 rpm with different inoculum sizes (2%, 3%, 4%, 5%, 6%, vol/vol), and the OD was determined by sampling₆₀₀In this case, 3 replicates were set for each group, and the optimum inoculum size was determined, as shown in FIG. 6, which was 2% (vol/vol).

From the experiments of examples 3 to 6, the optimum growth conditions for CZY1 are: the temperature is 30 ℃, the pH is 7.0, the inoculation amount is 2 percent, the initial nicotine concentration is 2g/L, and the experimental result of the initial nicotine concentration shows that the maximum nicotine tolerance of CZY1 is 14 g/L.

Example 7

Strains (OD) which have been activated in LB liquid medium₆₀₀=0.6 ± 0.05) into BSM liquid medium containing 2g/L nicotine, culturing at 180 rpm under optimum conditions, sampling every 8h, detecting nicotine residue concentration by High Performance Liquid Chromatography (HPLC) and drawing degradation curve, and sampling every 8h to detect OD₆₀₀And a growth curve is plotted. The specific operation is as follows:

(1) drawing of degradation curves

Preparation of nicotine standard solution and preparation of standard curve: measuring a certain amount of nicotine stock solution, diluting with Up water to obtain standard solutions of 0.25 g/L, 0.50 g/L, 0.75 g/L and 1.00 g/L, respectively, measuring peak areas of the nicotine standard solutions by HPLC, wherein each concentration is parallel to three, the nicotine concentration is used as abscissa, and the corresponding peak area is used as ordinate to draw a standard curve. The HPLC method is as follows: the sample size was 10. mu.L, and the mobile phase was 0.01M KH, as determined by Agilent 1260 HPLC₂PO₄(prepared with Up water, pH adjusted to 3.0): methanol = 90: 10,the flow rate was 1.0 mL/min, the column was an Agilent SB-C18 column (4.6 mm. times.150 mm), the column temperature was room temperature, and the detection wavelength was 254 nm using a Diode Array (DAD) detector.

Detecting the concentration of the residual nicotine in the sample: 2mL of the bacterial solution is taken every 8 hours (including 0 hour), centrifuged at 12000 rpm for 10 min at normal temperature, and a proper amount of supernatant is sucked by a disposable sampler, filtered through a 0.22 mu m filter membrane and put into a liquid bottle. The peak area of the sample is obtained by the method in the step I, the concentration of the residual nicotine is calculated by a nicotine standard curve, and a nicotine degradation curve is drawn according to the concentration.

(2) Drawing of growth curves

2mL of the bacterial solution was taken every 8 hours (including 0 hour), centrifuged at 12000 rpm at room temperature for 10 min, and the supernatant was discarded. Resuspend the cells in the same volume (2 mL) of sterile water and measure OD at 600 nm by UV spectrophotometer₆₀₀And drawing a growth curve according to the growth curve.

As shown in FIG. 7, after 104h of culture, CZY1 could completely degrade 2g/L nicotine, while CZY1 could grow to OD₆₀₀≈3.0。

Sequence listing

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<120> urea-producing arthrobacterium capable of degrading nicotine and application thereof

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Claims (3)

1. Arthrobacter ureafaciens for degrading nicotinePaenarthrobacter ureafaciens) CZY1 with the preservation number of CCTCC NO: M2021466.

2. The use of Arthrobacter uregenes CZY1 as claimed in claim 1 in the degradation of nicotine by water extraction from waste tobacco liquid in reconstituted tobacco production process.

3. The use according to claim 2, wherein the use comprises activation, transfer culture and seed degradation, in particular comprising:

1) and (3) activation: activating the strain CZY1 in LB liquid culture medium;

2) transfer culture: transferring the activated strain into a new LB liquid culture medium, culturing to a logarithmic phase, centrifugally stirring, washing and then re-suspending;

3) transferring the resuspended bacterial liquid into a BSM liquid culture medium with the initial nicotine concentration of 2-14 g/L, culturing at the temperature of 30-40 ℃ and the pH of 5-8, and sampling to determine the nicotine concentration and OD₆₀₀。

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