Method for screening halogenated carbazole active degrading bacteria based on single cell Raman spectrum technology

Abstract

The invention relates to the technical field of biochemical detection and analysis, in particular to a method for screening halogenated carbazole active degrading bacteria based on a single-cell Raman spectrum technology, which utilizes the characteristic of more active substance exchange between high-activity microorganisms and the outside, adopts a stable isotope probe to anchor active strains, and performs targeted screening to obtain the halogenated carbazole active degrading bacteria. Therefore, the degrading bacteria are firstly screened and then cultured. Compared with other markers (13C and 15N), the deuterium (2D) marker is lower in price, and an obvious C-D tensile vibration band can be observed in a Raman silence region. According to the method, the time for screening the functional strain is shortened to several days from several months in the prior art, the performance of the active functional strain is improved by multiple times compared with that of the strain obtained through a traditional method, and the technical bottleneck that the single-active strain is difficult to effectively obtain through the traditional method is broken through.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical detection and analysis, and in particular to a method for screening halogenated carbazole active degrading bacteria based on single-cell Raman spectroscopy technology. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Polyhalogenated carbazoles (PHCZs) are a new type of pollutant with a structure similar to polychlorinated dibenzofurans in dioxins. They are a new class of "dioxin-like compounds" with potential persistence, bioaccumulation and toxicity. Studies have shown that PHCZs have been detected in a variety of environmental media. Their wide distribution and dioxin-like toxicity have attracted increasing attention in recent years.

[0004] However, the current research on the degradation and remediation of PHCZs is still very limited, mainly including technologies such as photodegradation, advanced oxidation and microbial degradation. Compared with physical and chemical technologies, microbial remediation technology has the characteristics of low cost, small environmental impact and no secondary pollution. However, traditional strain isolation technology is limited by unculturability, environmental adaptability deviation, difficulty in detecting low-abundance strains and lack of functional phenotypes, resulting in limited efficiency and breadth of active bacteria screening.

[0005] Single-cell Raman spectroscopy is an effective method for studying uncultivable microorganisms. More importantly, when it is used in combination with stable isotope probing (SIP), it has been proven that carbon dioxide fixation bacteria and active bacteria in the intestinal flora that metabolize sugars can be identified at the single-cell level by utilizing the shift in Raman peaks of proteins, lipids, pigments, etc. caused by the assimilation of stable isotopes by microorganisms. There are few types of microorganisms in the environment that can degrade pollutants and their activity is low. It is extremely difficult to isolate functional strains with high degradation activity using traditional screening methods. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides a method for screening halogenated carbazole active degrading bacteria based on single cell Raman spectroscopy technology.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] A method for screening halogenated carbazole active degrading bacteria based on single cell Raman spectroscopy technology, comprising:

[0009] (1) adding aerobic sludge to an enrichment medium containing halogenated carbazole, culturing for one cycle, transferring to a new medium containing halogenated carbazole, transferring several times, and obtaining an enriched bacterial solution;

[0010] (2) After the enriched bacterial solution is centrifuged, the precipitate is collected, and the precipitate is transferred to a medium containing halogenated carbazole labeled with isotope D2O for incubation;

[0011] (3) After the incubation is completed, the bacterial suspension is centrifuged and washed multiple times to collect the precipitate, and the precipitate is redissolved in a buffer solution for single-cell Raman detection;

[0012] (4) Screening at 2040-2300cm -1 The microbial cells with CD Raman peaks at the displacement and CD Raman peak ratios greater than or equal to the threshold value are halogenated carbazole active degrading bacteria.

[0013] In one or more embodiments, in step (1), the enrichment medium containing halogenated carbazole includes the following components and contents: NH4Cl 0.3 g / L, KH2PO4 0.2 g / L, Na2HPO4 0.13 g / L, NaCl 1 g / L, MgCl2·6H2O 0.5 g / L, CaCl2·2H2O 0.015 g / L, 50 μM 1,3,6,8-tetrabromocarbazole; the solvent is water, and the pH is 7.0.

[0014] In one or more embodiments, in step (1), the culture conditions during the culture cycle are: 20-37° C., 140-210 r / min, for 7-10 days.

[0015] In one or more implementations, in step (1), the number of transfers is 3 to 5 times.

[0016] In one or more embodiments, in step (2), the centrifugation condition is 4000-8000g for 3-5 min.

[0017] In one or more embodiments, in step (2), the volume fraction of D2O in the culture medium containing halogenated carbazole labeled with isotope D2O is 30 to 100%, preferably 50%.

[0018] In one or more embodiments, in step (2), the incubation time is 18 to 36 hours, preferably 24 hours.

[0019] In one or more embodiments, in step (3), the centrifugation condition is 4000-8000g for 3-5 min.

[0020] In one or more embodiments, in step (3), the buffer solution is PBS buffer.

[0021] In one or more embodiments, in step (3), the single-cell Raman detection conditions are: the excitation light is 532 nm, 50-200 mW; the acquisition time is 0.5-1 s; the objective lens is 50x-100x, preferably 50x.

[0022] In one or more embodiments, in step (3), the formula for the CD Raman peak ratio is:

[0023] CD Raman peak ratio = 2040-2300 cm -1 CD Raman peak intensity at displacement / 2800~3100cm -1 The CH Raman peak intensity at the displacement is ×100%.

[0024] In one or more embodiments, in step (3), the threshold value is the average value of the CD Raman peak ratio obtained by single-cell Raman detection according to the method in step (3) after the precipitate is transferred to the medium containing halogenated carbazole for incubation in step (2) + 3 times the standard deviation;

[0025] Wherein, the isotope D2O label is not added to the culture medium of the halogenated carbazole.

[0026] The average Raman CD ratio in the threshold is determined according to the formula of the CD Raman peak ratio. The threshold is calculated by calculating the average CD Raman peak ratio of multiple bacteria (≥50) collected, and the average CD Raman peak ratio of multiple bacteria (≥50) + 3 times the standard deviation is determined as the threshold.

[0027] The beneficial effects of the present invention are:

[0028] The present invention utilizes the characteristics of highly active microorganisms exchanging substances with the outside world more actively, uses stable isotope probes to anchor active bacteria, and performs targeted screening, thereby achieving the "screening before breeding" of degrading bacteria. Compared with other markers ( 13 C and 15 N), deuterium ( 2 D) The labeling price is lower and obvious CD stretching vibration bands can be observed in the Raman silent area. The present invention shortens the time for screening functional strains from the traditional several months to several days, and the performance of active functional strains is improved many times compared with the traditional method of obtaining strains, breaking through the technical bottleneck of the traditional method that is difficult to effectively obtain a single active strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0030] Figure 1 A flow chart of a method for screening halogenated carbazole active degrading bacteria based on single-cell Raman spectroscopy;

[0031] Figure 2 This is the Raman spectrum of some bacteria in Example 1;

[0032] Figure 3 It is the CD Raman peak ratio of some single cells at different incubation times in Example 1. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0036] In the following examples, the components and contents of the medium containing halogenated carbazole and the enriched medium containing halogenated carbazole are the same, and the components and contents of the two are: NH4Cl 0.3g / L, KH2PO4 0.2g / L, Na2HPO4 0.13g / L, NaCl 1g / L, MgCl2·6H2O 0.5g / L, CaCl2·2H2O 0.015g / L, 50μM 1,3,6,8-tetrabromocarbazole; the solvent is water, and the pH is 7.0.

[0037] The LB solid culture medium includes components and contents: yeast powder 10 g / L, peptone 5.0 g / L, sodium chloride 10.0 g / L, agar 15.0 g / L, and the rest is water, with a pH of 6.0-8.0.

[0038] Figure 1This is a flow chart of the method for screening halogenated carbazole active degrading bacteria based on single-cell Raman spectroscopy technology, refer to Figure 1 Completed the screening of active halogenated carbazole-degrading bacteria.

[0039] Example 1

[0040] Determination of isotope D2O incubation time:

[0041] (1) Collect the activated sludge mixed liquid from the aerobic pool of a sewage plant in Qingdao and add it to the culture medium containing halogenated carbazole. The culture conditions are 30°C, 180r / min, and shaker culture for 7 days. Take the final enriched bacterial liquid of the above culture and transfer it to a new culture medium containing halogenated carbazole at an inoculation rate of 5wt%. Repeat this process 4 times to obtain the enriched bacterial liquid.

[0042] (2) The enriched bacterial suspension was centrifuged at 6000g for 5 min, the supernatant was discarded, the precipitate was washed three times with 95% physiological saline, and resuspended in a medium containing halogenated carbazole. In order to determine a suitable incubation time, the active microbial markers in the bacterial suspension were sufficiently D. 0.5 mL of the above bacterial suspension and 0.5 mL of heavy water D2O were added to a 2 mL centrifuge tube, incubated at 30°C, and Raman spectroscopy was performed after 0 hour, 12 hours, 18 hours, 24 hours, and 36 hours of incubation.

[0043] Before Raman spectroscopy detection, in order to remove heavy water and culture medium in the bacterial suspension, the bacterial suspension was centrifuged at 6000g for 5 min, the supernatant was discarded, and the suspension was washed twice with sterile water and reconstituted with PBS buffer.

[0044] Drop 2 μL of sample on a quartz slide, air-dry at room temperature, and collect Raman spectra under a 50x objective lens, 100 mW laser, and 1 s spectrum collection time of the RACS-Seq single-cell Raman optical tweezers sorter (Qingdao Single-cell Biotechnology). In addition, to eliminate background interference, background removal is required before collecting the spectrum, that is, 3 to 6 spectra are collected in the blank area near the target cell, and the background spectrum is obtained and the background-reduced Raman spectrum is obtained after background removal.

[0045] The collected Raman spectra are Figure 2 As shown, from Figure 2 It can be seen that some bacteria are between 2040 and 2300 cm -1 The CD bands were shown at the shifted positions, indicating that they actively incorporate D2O-derived D into cellular components such as lipids and proteins. Figure 3The CD Raman peak ratios of 50 single cells collected from each sample are shown. The CD Raman peak ratio in the sample increases with the increase of incubation time until the incubation time reaches 18h, and the CD Raman peak ratio reaches stability. The proportion of active microorganisms in the sample did not show changes when the incubation time was 18h, 24h and 36h. Therefore, 24h incubation is selected as a reasonable incubation time, under which the active microorganisms in the sample can be marked with sufficient D.

[0046] In addition, the threshold value was determined to be 2.6% based on the average value of the CD Raman peak ratio of 50 single cells incubated with unweighted water (0 h) + 3 times the standard deviation. When the CD Raman peak ratio in the sample is greater than this threshold, it indicates that the cells are in an active state. The formula for the CD Raman peak ratio is: CD Raman peak ratio = 2040-2300 cm -1 CD Raman peak intensity at displacement / 2800~3100cm -1 The CH Raman peak intensity at the displacement is ×100%.

[0047] Example 2

[0048] (1) Collect the activated sludge mixed liquid from the aerobic pool of a sewage plant in Qingdao and add it to the culture medium containing halogenated carbazole. The culture conditions are 30°C, 180r / min, and shaker culture for 7 days. Take the final enriched bacterial liquid of the above culture and transfer it to a new culture medium containing halogenated carbazole at an inoculation rate of 5wt%. Repeat this process 4 times to obtain the enriched bacterial liquid.

[0049] (2) The enriched bacterial suspension was centrifuged at 6000 g for 5 min, the supernatant was discarded, the precipitate was washed three times with 95% physiological saline, and resuspended in a medium containing halogenated carbazole. According to the isotope D2O incubation time determined in Example 1, 0.5 mL of the above bacterial suspension and 0.5 mL of heavy water D2O were added to a 2 mL centrifuge tube and incubated at 30° C. for 24 hours.

[0050] Take 1 mL of the bacterial solution after incubation with heavy water and add it to a 1.5 mL sterile centrifuge tube. Wash the bacterial solution three times with sterile water. Use PBS buffer to adjust the sample concentration to 5.0×10 6 ~5.0×10 8 cells / mL.

[0051] Take 20μL of sterile water and drop it on the liquid inlet of the microfluidic chip (Qingdao Single-cell Biotechnology), take 10-20μL of sample and drop it on the sample outlet, then put the chip into the vacuum injection device, and stop when the vacuum reaches -95kPa to complete the injection. Use a syringe to absorb 1mL of buffer, and connect the hose after the bubbles inside the syringe are discharged. Fix the syringe on the instrument injection pump and insert the other end of the hose into the liquid inlet of the chip. Keep the injection pump flow rate at 9μL / min, run for 10min, and then insert the capillary into the sample outlet of the chip. Observe the sample under a 50x objective lens, and set the injection pump flow rate to 9μL / min. Use the same Raman acquisition conditions as when determining the heavy water incubation time above. When cells with CD peaks appear at 2040-2300cm, use optical tweezers (1064nm laser 2000mA) to capture, and the XY movement speed is set to 0.3mm / s.

[0052] Screening is at 2040~2300cm -1 There is a CD Raman peak at the displacement, and the CD Raman peak ratio at the displacement is greater than or equal to the threshold value of the microbial cell. According to the activity threshold determined in Example 1, the single cell with a CD Raman peak ratio greater than 2.6% is cultured, and the cultured microorganisms are subjected to high-throughput sequencing. From the sequencing results, it can be seen that the genera screened are mainly Bacillus sp (Bacillus), Alcaligenes sp (Alcaligenes) and Brevundimonas sp (Brevundimonas).

[0053] Example 3

[0054] The strain selected in Example 2 was inoculated into a conical flask containing 50 mL of LB solid medium and incubated at 30°C and 150 rpm for 24 h. After centrifugation at 4°C and 8000 g for 5 min, the amplified bacteria were collected and sterile saline (0.9% NaCl) was added to adjust the OD of the bacterial suspension to 600 When the pH reaches about 0.2, this is used as the inoculum. 1 mL of the bacterial suspension is inoculated into 100 mL of an enrichment medium containing halogenated carbazole and incubated on a rotary shaker at 150 rpm and 30°C.

[0055] The residual amount of 1,3,6,8-tetrabromocarbazole in the inorganic salt culture medium was detected by high performance liquid chromatography (HPLC). The detection conditions of HPLC were as follows: the mobile phase was acetonitrile: water (80:20, V / V); the analytical column was Grace Alltima Cl8 Column (4.6×250 mm, 5 μm); the wavelength was 262 nm; the flow rate was 1 mL / min; the injection volume was 10 μL; and the column temperature was 25°C.

[0056] According to the results of high performance liquid chromatography, the degradation rates of 1,3,6,8-tetrabromocarbazole by Bacillus, Brevimonas and Alcaligenes were 79.79%, 65.12% and 42.67% respectively within 72 hours.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for screening halogenated carbazole active degrading bacteria based on single cell Raman spectroscopy, characterized in that: include: (1) adding aerobic sludge to an enrichment medium containing halogenated carbazole, culturing for one cycle, transferring to a new medium containing halogenated carbazole, transferring several times, and obtaining an enriched bacterial solution; (2) After centrifuging the enriched bacterial solution, collecting the precipitate, transferring the precipitate to a medium containing halogenated carbazole labeled with isotope D2O, and incubating the medium; (3) After the incubation is completed, the bacterial suspension is centrifuged and washed multiple times to collect the precipitate, and the precipitate is redissolved in a buffer solution for single-cell Raman detection; (4) Screening at 2040-2300cm -1 The microbial cells with CD Raman peaks at the displacement and CD Raman peak ratios greater than or equal to the threshold value are halogenated carbazole active degrading bacteria.

2. The method according to claim 1, characterized in that In step (1), the enrichment medium containing halogenated carbazole includes the following components and contents: 0.3 g / L NH4Cl, 0.2 g / L KH2PO4, 0.13 g / L Na2HPO4, 1 g / L NaCl, 0.5 g / L MgCl2·6H2O, 0.015 g / L CaCl2·2H2O, and 50 μM 1,3,6,8-tetrabromocarbazole; the solvent is water, and the pH is 7.

0.

3. The method according to claim 1, characterized in that In step (1), the culture conditions during the culture period are: 20-37° C., 140-210 r / min for 7-10 days; Or, in step (1), the number of transfers is 3 to 5 times.

4. The method according to claim 1, characterized in that In step (2), the centrifugation condition is 4000-8000g for 3-5 minutes; Alternatively, in step (2), in the culture medium containing halogenated carbazole labeled with isotope D2O, the volume fraction of D2O is 30 to 100%, preferably 50%.

5. The method according to claim 1, characterized in that In step (2), the incubation time is 18 to 36 hours, preferably 24 hours.

6. The method according to claim 1, characterized in that In step (3), the single cell Raman detection conditions are: excitation light is 532nm, 50-200mW; acquisition time is 0.5-1s; The objective lens is 50x to 100x, preferably 50x.

7. The method according to claim 1, characterized in that In step (3), the formula for the CD Raman peak ratio is: CD Raman peak ratio = 2040-2300 cm -1 CD Raman peak intensity at displacement / 2800~3100cm -1 The CH Raman peak intensity at the displacement is ×100%.

8. The method according to claim 1, characterized in that In step (3), the threshold value is the average value of the CD Raman peak ratio obtained by single-cell Raman detection according to the method in step (3) after the precipitate is transferred to the medium containing halogenated carbazole for incubation in step (2) + 3 times the standard deviation; Wherein, the isotope D2O label is not added to the culture medium of the halogenated carbazole.

9. The method according to claim 1, characterized in that In step (2), the centrifugation condition is 4000-8000g for 3-5 minutes; Alternatively, in step (3), the centrifugation condition is 4000-8000 g for 3-5 min.

10. The method according to claim 1, characterized in that In step (3), the buffer solution is PBS buffer.

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