CN111982875A - Method for screening bacteria producing polyhydroxyalkanoate based on three-dimensional fluorescence spectrum analysis - Google Patents

Abstract

The invention belongs to the technical field of rapid screening of polyhydroxyalkanoate-producing bacteria, and provides a screening method for polyhydroxyalkanoate-producing bacteria based on three-dimensional fluorescence spectrum analysis. The screening method of the present invention comprises the following steps: (1) taking the bacterial liquid to be tested and separating the culture liquid to obtain a concentrated bacterial liquid, adjusting the concentration of the concentrated bacterial liquid and then freeze-drying to obtain a freeze-dried bacterial powder; (2) in step (1) ) adding n-hexane to the prepared freeze-dried bacterial powder, carrying out ultrasonic extraction, and then adding Nile red staining solution for dyeing to obtain a sample to be tested; (3) taking the sample to be tested in step (2) and carrying out three-dimensional fluorescence spectrum analysis . Compared with the traditional gas chromatography analysis method, the screening method provided by the invention saves labor and effort, greatly improves the screening efficiency of bacterial strains, and is a brand-new screening method for polyhydroxyalkanoate-producing bacteria.

Description

Screening of polyhydroxyalkanoate-producing bacteria based on three-dimensional fluorescence spectroscopy method

technical field

The invention relates to the technical field of rapid screening of polyhydroxyalkanoate-producing bacteria, in particular to a screening method for polyhydroxyalkanoate-producing bacteria based on three-dimensional fluorescence spectrum analysis.

Background technique

Polyhydroxyalkanoates (PHA) are a class of functional polyesters with similar structures in biological cells, which widely exist in microbial cells as energy storage substances. Since the mechanical properties and thermoplastic properties of PHA are similar to polyethylene and polypropylene, it can be developed and utilized as a polymer material. In addition, PHA is also biodegradable and biocompatible, so it is considered as an environmentally friendly bio-based polymer material, which has important applications in the packaging industry, organic agriculture, medical materials and advanced sensor research and development. application prospects.

As a kind of polymer, the different structural monomers, monomer arrangement and polymerization degree of PHA make the materials show different mechanical properties and have different application scenarios. At present, industrial-grade PHA production is highly dependent on efficient PHA-producing microbial strains. Studies have shown that there may be differences in the types of PHA produced by different species of microorganisms. Therefore, large-scale microbial strain screening is an important way to modify PHA materials in the future, accelerate industrial production capacity, and reduce production costs.

With the development of molecular biology technology, PHA-producing strains can be preliminarily screened by identifying the expression level of PHA synthetic genes. However, genotype is not equal to phenotype, and the ability of bacteria to produce PHA needs to be determined quantitatively or semi-quantitatively after culture. At present, the most used method for quantification of PHA is gas chromatography. The advantage of gas chromatography is that it is quantitatively accurate and can provide complex material composition information, but the disadvantage is that sample extraction is time-consuming and labor-intensive, chromatographic analysis requires a long sample pretreatment process, and the operator is also highly skilled. Not suitable for large-scale screening. On the other hand, Nile Red, a lipophilic fluorescent dye, can be combined with PHA and emit fluorescence in organic solvents to qualitatively determine whether bacteria produce PHA. However, the sensitivity of this method is low, especially when the current culture conditions are not prone to bacterial accumulation of PHA, and screening may occur. Three-dimensional fluorescence spectrometry is an emerging method for the detection of organic matter in the field of environmental chemistry in recent years. It is widely used in the study of natural organic matter and microbial analytes. Different from traditional fluorescence detection methods, three-dimensional fluorescence spectroscopy can provide continuous emission fluorescence characteristics of samples on continuous excitation light, with the advantages of high resolution and high resolution, and has a wide range of applications in materials science and environmental science. However, the current research shows that the autofluorescence characteristics of PHA are not obvious, and it is not suitable for fluorescence analysis.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides a screening method for polyhydroxyalkanoate-producing bacteria based on three-dimensional fluorescence spectrum analysis.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a screening method for polyhydroxyalkanoate-producing bacteria based on three-dimensional fluorescence spectrum analysis, comprising the following steps:

(1) get the bacterial liquid to be tested to separate the culture liquid, obtain the concentrated bacterial liquid, carry out freeze-drying after adjusting the concentration of the concentrated bacterial liquid, and obtain the freeze-dried bacterial powder;

(2) adding n-hexane to the freeze-dried bacterial powder obtained in step (1), carrying out ultrasonic extraction, and then adding Nile red staining solution for dyeing to obtain a sample to be tested;

(3) The sample to be tested in step (2) is taken for three-dimensional fluorescence spectrum analysis.

Preferably, the method for adjusting the concentration of the concentrated bacterial solution is to add sterile water to the concentrated bacterial solution to adjust its OD value at 600 nm to 1.5-1.8.

Preferably, the temperature of the freeze-drying is -50～-70°C, and the time is 20～28h.

Preferably, the volume ratio of the n-hexane to the bacterial solution to be tested is 1:0.8-1.5.

Preferably, the power of the ultrasonic extraction is 300-500 W, and the ultrasonic extraction time is 3-8 min.

Preferably, the added amount of the Nile red staining solution is 0.01-0.05% of the volume of the bacterial solution to be tested.

Preferably, the concentration of the Nile red staining solution is 0.005-0.015 mg/mL.

Preferably, the conditions for the three-dimensional fluorescence spectrum analysis are: detection excitation light wavelength range 200-600nm, step 2nm, detection emission light wavelength 246.977-824.286nm, step 4nm, detection integration time 0.1s.

The screening method for PHA-producing bacteria based on three-dimensional fluorescence spectrum analysis provided by the present invention relates to the screening and detection of microbial resources of environment-friendly bio-based polymer materials (PHA). It emits strong fluorescence in a specific wavelength region, and the potential yield of PHA by microorganisms is evaluated based on the intensity of the fluorescence. Compared with traditional gas chromatography analysis, the screening method provided by the invention saves labor and labor, greatly improves the screening efficiency of bacteria, and is a brand-new screening method for PHA-producing bacteria.

Description of drawings

The electrophoresis diagram of the N.aquimarinus TN28-1 bacteria product composition in the embodiment 1 of Fig. 1;

The three-dimensional fluorescence characteristic peaks of pure PHB in Fig. 2 embodiment 1 and the three-dimensional fluorescence characteristic peaks of P.putida KCTC1751 ^T and N.aquimarinus TN28-1 bacterial product components;

Figure 3 is the peak time of pure PHB in Comparative Example 1 and the peak time of P.putida KCTC1751 ^T and N.aquimarinusTN28-1 bacterial product components;

Figure 4 shows the similarity of the gas chromatographic peak areas and three-dimensional fluorescence integration volumes of pure PHB in Comparative Example 1 and PHB produced by P.putida KCTC1751 ^T and N.aquimarinus TN28-1 bacteria.

Figure 5 shows the cell morphology of P.putida KCTC1751 ^T and N.aquimarinus TN28-1 bacteria in Comparative Example 2 under a fluorescence microscope (pure PHB has no cell morphology, so there is no comparison).

Detailed ways

The invention provides a screening method for polyhydroxyalkanoate-producing bacteria based on three-dimensional fluorescence spectrum analysis, comprising the following steps:

(1) get the bacterial liquid to be tested to separate the culture liquid, obtain the concentrated bacterial liquid, carry out freeze-drying after adjusting the concentration of the concentrated bacterial liquid, and obtain the freeze-dried bacterial powder;

(2) adding n-hexane to the freeze-dried bacterial powder obtained in step (1), carrying out ultrasonic extraction, and then adding Nile red staining solution for dyeing to obtain a sample to be tested;

(3) The sample to be tested in step (2) is taken for three-dimensional fluorescence spectrum analysis.

In the present invention, the bacterial liquid to be tested is separated from the culture liquid to obtain the concentrated bacterial liquid, and the concentration of the concentrated bacterial liquid is adjusted and then freeze-dried to obtain the freeze-dried bacterial powder.

In the present invention, the method for separating the culture liquid is preferably centrifugation, and the centrifugation conditions are preferably 10,000-15,000×g, more preferably 11,000-14,000×g, and still more preferably 13,000×g; The time is preferably 1min.

In the present invention, the method for adjusting the concentration of the concentrated bacterial liquid is preferably as follows: adding sterile water to the concentrated bacterial liquid, the concentration preferably has an OD value of 1.5-1.8 at 600 nm, more preferably 1.6-1.7, and even more preferably is 1.6.

In the present invention, the concentrated bacterial liquid after concentration adjustment is preferably placed in a sealable glass test tube and then freeze-dried.

In the present invention, the temperature of the freeze-drying is preferably -50～-70°C, more preferably -55～-65°C, still more preferably -60°C; the time is preferably 20～28h, more preferably 22～ 25h, more preferably 24h.

After the freeze-dried bacterial powder is obtained, n-hexane is added to the freeze-dried bacterial powder to perform ultrasonic extraction, and then Nile red staining solution is added for dyeing to obtain a sample to be tested.

In the present invention, the volume ratio of the n-hexane to the bacterial solution to be tested is preferably 1:0.8-1.5, more preferably 1:0.9-1.2, and still more preferably 1:1.

In the present invention, the power of the ultrasonic extraction is preferably 300-500W, more preferably 360-440W, and even more preferably 400W; the ultrasonic extraction time is preferably 3-8min, more preferably 4-7min, and then More preferably, it is 5 min.

In the present invention, the addition amount of the Nile red staining solution is preferably 0.01-0.05% of the volume of the bacterial solution to be tested, more preferably 0.02-0.03%, and even more preferably 0.02%.

In the present invention, the Nile Red staining solution is prepared by dissolving Nile Red in acetone.

In the present invention, the concentration of the Nile Red staining solution is preferably 50-150 ng/mL, more preferably 70-120 ng/mL, and still more preferably 100 ng/mL.

Three-dimensional fluorescence spectrum analysis can be performed after the sample to be tested is prepared.

In the present invention, the conditions for the three-dimensional fluorescence spectrum analysis are: the wavelength range of the detection excitation light is preferably 200-600 nm, the step is preferably 2 nm, the wavelength of the detection emission light is preferably 246.977-824.286 nm, the step is preferably 4 nm, and the detection The integration time is preferably 0.1s.

After measuring the fluorescence intensity of the sample, according to the fluorescence amount of the sample, compare it with the background fluorescence amount, and preliminarily evaluate the ability of the strain to produce PHA.

The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

Example 1

PHB (poly 3-hydroxyvalerate) is a polymer with the simplest structure, the most studied and the most thorough among all PHA substances, and its structural unit is trihydroxyvaleric acid. In this example, the detection ability of this method for substances with similar physicochemical properties such as PHA is characterized by directly detecting PHB.

In this example, three-dimensional fluorescence spectrum analysis was performed on the product components of pure PHB, commercial PHA-producing bacteria Pseudomonas putida KCTC1751 ^T and environmental screened PHA-producing strain Nitratireductor aquimarinus TN28-1.

In this example, pure PHB was purchased commercially; strain P.putida KCTC1751 ^T was purchased from the Korean Collection for Type Cultures, numbering KCTC1751, which has been proven to be a high-efficiency PHA-producing microorganism (its The main component of the PHA produced is PHB), which is widely used in industrial production; bacterial strain N.aquimarinusTN28-1 is screened from the bottom mud of Shenzhen Dapeng Bay sea area, and the deposit number is MCCC 1K04607 (MCCC, Marine Culture Collection of China Marine Microorganism Culture Collection and Management Center) ). The phaC synthase gene was amplified by polymerase chain reaction (PCR), the primers were: phaC1F1 (5'-TGGARCTGATCCAGTAC-3') and phaC1R1 (5'-CGGGTTGAGRATGCTCTG-3'), the conditions were: 94 ℃ for 5 min, then 1 min at 94 °C, 1 min at 54 °C, 1 min at 72 °C, a total of 30 cycles, and finally 10 min at 72 °C. The PCR products were separated by agarose gel electrophoresis, 1% agarose was used, and the voltage was 120V for 15min. The gel image (as shown in Figure 1) determined that there was a clear band at 500bp, which proved that the strain had the potential to produce PHA.

In this example, the preparation method of the pure PHB sample to be tested is as follows: first, add 3 mL of n-hexane solution to a stoppered quartz fluorescence cuvette, use a micropipette to draw 1 μL of pure PHB into the cuvette, cover and mix. Then 1 μL of Nile red staining solution (mass concentration of 100 ng/mL, dissolved in acetone) was added, and then the three-dimensional fluorescence spectrum analysis method was used for detection.

At the same time, P.putida KCTC1751 ^T and N.aquimarinus TN28-1 bacteria were cultured on nutrient agar medium, respectively. The composition of the nutrient agar medium according to the concentration includes: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride, and the pH value is 7.3. The culture medium was used to obtain the test P.putida KCTC1751 ^T bacteria liquid and the test N.aquimarinus TN28-1 bacteria liquid respectively.

Take the P.putida KCTC1751 ^T bacterial solution to be tested and centrifuge at 13000 × g for 1 min, separate the medium to obtain a concentrated bacterial solution; then use sterile water to adjust the concentration of the concentrated bacterial solution so that the OD value at 600nm is 1.6. Take 5mL of the concentration-adjusted concentrated bacterial solution into a sealable glass test tube, freeze-dry it at -60°C for 24 hours to obtain freeze-dried bacterial powder; add 5 mL of freeze-dried bacterial powder into the sealable glass test tube containing the freeze-dried bacterial powder. n-hexane, and then the container was sealed and ultrasonically extracted at 400 W for 5 min. Afterwards, 1 μL of Nile red staining solution with a concentration of 100 ng/mL was added to obtain the test sample of P.putida KCTC1751 ^T bacteria. Similarly, the samples to be tested of N. aquimarinus TN28-1 were prepared according to the above method, and three-dimensional fluorescence spectrum analysis was performed.

The test samples of pure PHB, P.putida KCTC1751 ^T bacteria and N.aquimarinusTN28-1 bacteria were analyzed according to the following parameters. Detection related parameters are as follows: sample detection volume 3mL. The wavelength range of detection excitation light is 200-600nm, the step is 2nm, the wavelength of emission light is 246.977-824.286nm, the step is 4nm, and the detection integration time is 0.1s. During detection, firstly, 1 μL of Nile red staining solution (mass concentration 100 ng/mL, solvent is acetone) was added to blank n-hexane solvent (chromatographically pure, 95%) as a fluorescent blank control. After the sample was detected, the blank control was deducted as the amount of fluorescence produced by the reaction, and the PHA yield of the strain was evaluated by the amount of fluorescence. The resulting data were visualized and quantitatively analyzed using the drEEM tool. In this example, the three-dimensional fluorescence spectrum analysis used the Aqualog synchronous absorption-three-dimensional fluorescence detection scanning spectrometer produced by HORIBA Company.

The results are shown in Figure 2. Commercially pure PHB showed significant fluorescence characteristic peaks at Ex=500nm, Em=550nm, and the peak height was about ^700A.U .; Em=550nm, peak height 450A.U.; The fluorescence characteristic peak position of N.aquimarinus TN28-1 bacteria is similar to PHB, roughly at Ex=490nm, Em=530nm, peak height 120A.U. Through three-dimensional fluorescence spectrum analysis, comparing the peak heights of the two strains at the characteristic peak positions, P.putida KCTC1751 ^T has a higher PHB yield, while N. aquimarinus TN28-1 has a lower yield.

Comparative Example 1

In order to further verify the accuracy of the three-dimensional fluorescence spectroscopic analysis, a gas chromatography-mass spectrometer was used to analyze the commercially purchased pure PHB. In the gas chromatography analysis, the peak time of PHB was 3.65 min (as shown in Figure 3). And gas chromatography-mass spectrometry was performed on the components produced by the two strains to be tested. The detection method is as follows: take 5mL of the fermentation broth of P.putidaKCTC1751 bacteria and N.aquimarinus TN28-1 bacteria respectively, centrifuge at 5000×g for 20min, remove the supernatant, and then add 1mL of chloroform respectively, 1mL contains 15% (v/v) Methanol solution of concentrated sulfuric acid, fully mixed, and oil bath at 100 °C for 150 min to carry out methyl esterification reaction under acidic conditions. After the reaction was completed, it was cooled in an ice bath for 5 min, and 1 mL of deionized water was added respectively, fully mixed for 30 s, and allowed to stand for 1 min. 150 μL of the lower organic phase was taken and placed in a GC small tube for chromatographic analysis. The analysis results are shown in Figure 3. In the gas chromatography-mass spectrometry analysis, the peak time of the product of P.putida KCTC1751 was 3.587min, and the peak time of the product of N.aquimarinus TN28-1 was 3.650min. At the same time, the PHB characteristic peaks of gas chromatography and three-dimensional fluorescence spectrometry were integrated. The form of a scatterplot characterizes the consistency of the results of the two methods. It can be seen from Figure 4 that the PHB characteristic peak integration results of the three-dimensional fluorescence have strong consistency with the traditional gas chromatography analysis results. Therefore, it is demonstrated that three-dimensional fluorescence spectrometry can be used as a potential PHA yield detection method. In addition, the results of Example 1 and Comparative Example 1 show that it is feasible to use three-dimensional fluorescence spectroscopic analysis to detect the fluorescence characteristic peaks of lipophilic dyes combined with PHB in an organic solvent environment.

Comparative Example 2

This comparative example further verifies the accuracy of the three-dimensional fluorescence spectrum analysis by the PHA fluorescence reaction observation method. The steps of PHA fluorescence reaction observation are as follows: take 1 mL of the fermentation broth of P.putida KCTC1751 and N.aquimarinus TN28-1 respectively, centrifuge at 13000×g for 1 min, and remove the supernatant. Add 1 μL of Nile red staining solution (10 μg/mL DMSO (dimethyl sulfoxide)) to the remaining cells, mix well, add 1.5 μL of Nile red staining solution dropwise to suspend on the washed glass slide, Dry for 5 seconds, carefully covering the surface with a coverslip. Using a fluorescence microscope, the cell morphology was photographed in bright field and fluorescence at 562 nm for excitation light and 594 nm for emission light, respectively.

By comparing the cell morphology of the two strains under a fluorescence microscope (as shown in Figure 5), similar results to the three-dimensional fluorescence analysis can also be obtained. And after Nile red staining, the ability of N. aquimarinus TN28-1 to produce PHA is difficult to determine under the fluorescence microscope, which shows that three-dimensional fluorescence spectroscopy is superior to ordinary staining and has higher precision.

To sum up, the present invention provides a screening method for PHA-producing bacteria based on three-dimensional fluorescence spectrum analysis, which can avoid the time-consuming and labor-intensive procedures of traditional gas chromatography analysis, and greatly improve the screening efficiency of bacteria. Response observation method, with higher accuracy.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (8)

1. A method for screening bacteria producing polyhydroxyalkanoate based on three-dimensional fluorescence spectrum analysis is characterized by comprising the following steps:

(1) taking a bacterium solution to be detected to separate a culture solution to obtain a concentrated bacterium solution, adjusting the concentration of the concentrated bacterium solution, and then carrying out freeze drying to obtain freeze-dried bacterium powder;

(2) adding normal hexane into the freeze-dried bacterial powder prepared in the step (1), carrying out ultrasonic extraction, and then adding a nile red staining solution for staining to obtain a sample to be detected;

(3) and (3) carrying out three-dimensional fluorescence spectrum analysis on the sample to be detected in the step (2).

2. The screening method according to claim 1, wherein the method for adjusting the concentration of the concentrated bacterial liquid comprises: and adding sterile water into the concentrated bacterial liquid, and adjusting the OD value of the concentrated bacterial liquid at 600nm to be 1.5-1.8.

3. The screening method according to claim 1 or 2, wherein the freeze-drying temperature is-50 to-70 ℃ and the time is 20 to 28 hours.

4. The screening method according to claim 3, wherein the volume ratio of the n-hexane to the bacterial liquid to be tested is 1: 0.8-1.5.

5. The screening method according to claim 1, wherein the power of the ultrasonic extraction is 300-500W, and the time of the ultrasonic extraction is 3-8 min.

6. The screening method according to claim 1, wherein the nile red staining solution is added in an amount of 0.01 to 0.05% by volume of the bacterial solution to be tested.

7. The screening method according to claim 1 or 6, wherein the concentration of the Nile Red staining solution is 50 to 150 ng/mL.

8. The screening method according to claim 1, wherein the conditions of the three-dimensional fluorescence spectrum analysis are: the wavelength range of the detection excitation light is 200-600 nm, the step is 2nm, the wavelength of the detection emission light is 246.977-824.286 nm, the step is 4nm, and the detection integration time is 0.1 s.

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