Chemical energy autotrophic bacterium detection method based on single cell Raman spectrum and stable isotope labeling

Abstract

The invention discloses a chemoautotrophic bacteria detection method based on a single cell Raman spectrum and 13C stable isotope labeling. 13C stable isotope is added into a bacterial culture solution or an environmental sample for enrichment culture, centrifugation is performed, cells are collected and used for Raman spectrum detection, and according to the deviation of two characteristic spectrum peaks, namely, the cytochrome c peak deviates from 747 cm <-1 >, 1112 cm <-1 >, 131 cm <-1 > and 1584 cm <-1 > to 725 cm <-1 >, 111 cm <-1 >, 1300 cm <-1 > and 1536 cm <-1 > under exciting light with the wavelength of 532 nm; the peak of phenylalanine shifts from 1002cm <-1 > to 965cm <-1 > under exciting light with the wavelength of 532nm, and chemoautotrophic bacteria can be identified. According to the detection method, the limitation of pure culture is overcome, the bottleneck that the metagenome cannot characterize the carbon sequestration activity is solved, and the single-cell and non-destructive detection of the chemoautotrophic bacteria in the natural environment is realized.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a chemoautotrophic bacteria detection method based on single-cell Raman spectroscopy and stable isotope labeling. Background Art

[0002] Carbon-fixing microorganisms are widely found in habitats such as terrestrial soil and oceans. These microorganisms include photoautotrophic and chemoautotrophic microorganisms, etc. They can use light energy or chemical energy to fix carbon dioxide and convert it into organic matter.

[0003] Traditional microbiology can study chemoautotrophic microorganisms by obtaining pure cultures of them, but most microorganisms are difficult to culture in the laboratory, and the culture conditions are difficult to determine. Metagenomic methods based on molecular biology can study chemoautotrophic bacterial groups in different environments under non-culture conditions. Currently, six major carbon fixation pathways for autotrophic microorganisms are known, namely the Calvin cycle (CBB), the reduced tricarboxylic acid cycle (rTCA), the reduced acetyl-CoA pathway (WL pathway), the 3-hydroxypropionic acid double cycle (3-HP), the 3-hydroxypropionic acid / 4-hydroxybutyric acid cycle (3H / 4HB), and the dicarboxylic acid / 4-hydroxybutyric acid cycle (DC / 4HB). Among them, the Calvin cycle is one of the most common and key pathways for fixing CO2. However, based on existing databases, metagenomic analysis may not be able to identify unknown or low-abundance chemoautotrophic bacterial groups, limiting the comprehensiveness of the analysis, and cells with related genes may not actually express gene functions, and their phenotypic carbon fixation activity cannot be determined. Metatranscriptomic technology can be used to target active genes and metabolic pathways in microbial communities and their changes under different environmental conditions, as well as to understand the metabolic dynamics of microorganisms. However, transcriptomic data are affected by spatiotemporal changes, and low-abundance transcripts may be ignored, resulting in inaccurate assessments of community functions.

[0004] Carbon fixation is the process of biosynthesis of organic matter, which requires a lot of energy to drive the reaction. Cytochrome c is widely present in cells and participates in the production of ATP. At the same time, as a heme protein, cytochrome c can show maximum electronic absorption around 550nm, which is compatible with 532nm laser and produces resonance Raman effect, which can enhance the Raman signal of cytochrome c relative to other cell components, making it an effective Raman biomarker. At the same time, at 1002cm -1 The ring breathing vibration of nearby phenylalanine is one of the most prominent Raman peaks in the phenylalanine spectrum. The conjugated structure of the porphyrin ring in cytochrome c and the benzene ring in phenylalanine may make their vibration frequencies susceptible to carbon isotope substitution. Therefore, in this study, cytochrome c and phenylalanine became the affected 13 C marks the main affected molecule.

[0005] Applying Raman microspectroscopy to the analysis of single microbial cells requires addressing two major challenges: weak Raman signals and interpretation of Raman spectral data (Li et al., Analytical Chemistry, 2013, 85: 1642-1649). At present, research has developed evidence of carbon fixation in photosynthetic algae based on the Raman peak shift of carotenoids; at the same time, based on the Raman spectral characteristics of cytochrome c, previous researchers have used labeling to identify the carbon fixation of photosynthetic algae. 15 N2 develops its CN bond shift to 1114 cm -1 However, since chemoautotrophic bacteria usually cannot synthesize carotenoids, the technology developed by predecessors cannot be applied. Therefore, there is still a lack of Raman spectral characteristic shift peaks of chemoautotrophic bacteria. There is currently no literature report on the characteristic shift peaks that can identify this group. Summary of the invention

[0006] The purpose of the present invention is to provide a chemoautotrophic bacteria detection method based on single-cell Raman spectroscopy and stable isotope labeling, which realizes single-cell level detection, does not rely on pure culture technology, and is particularly suitable for the detection method of chemoautotrophic bacteria in the environment.

[0007] Raman spectroscopy is a powerful method for studying biomacromolecules. Since the Raman spectrum of water is weak and the spectrum is simple, Raman spectroscopy can be used to study the structure and changes of biomacromolecules in a state close to nature and activity. Raman spectroscopy is based on the vibration of chemical bonds of biomacromolecules inside cells. The large amount of water components in cells has little interference with Raman signals. Secondly, single-cell Raman spectroscopy has low requirements on the type and physiological state of sample cells, causes less damage to cells, is simple to operate, and can achieve real-time detection of environmental microorganisms.

[0008] Raman spectroscopy can not only characterize the information of cellular macromolecules, but also reflect components of different physiological states and metabolic activities, and can provide a fingerprint of microbial composition at the single-cell level. The inventors found that for chemical components synthesized within bacterial cells, such as phenylalanine and cytochrome c, by selecting appropriate excitation light and matching the excitation light with the energy level of the pigment electronic transition, characteristic spectral signal peaks can be generated. On this basis, stable isotope labeling is added. When bacteria absorb and assimilate isotopes, the lighter atoms in the original cell are replaced by heavy isotopes, which can produce a shift in the characteristic spectral signal peak. In addition, the Raman spectroscopy method is a non-destructive detection, and the identified microbial cells can continue to be sorted and sequenced, which greatly promotes the functional mining of environmental microorganisms. Therefore, single-cell Raman spectroscopy technology has great application potential in the detection of chemoautotrophic bacteria.

[0009] However, applying Raman microscopy to the analysis of single microbial cells requires addressing two major challenges: weak Raman signals and interpretation of Raman spectral data. Due to the high sensitivity of Raman spectroscopy, factors such as bacterial species, growth environment, growth state, and specimen preparation process can affect the morphology of the spectrum. The excitation light wavelength, sample pretreatment, and spectral data processing of Raman spectroscopy are not uniform, which further affects the accuracy of subsequent measurement results (Neugebauer et al., International Journal of Antimicrobial Agents, 2015, 461: S35-S39).

[0010] The inventors of this application have found through a large amount of data measurement that when chemoautotrophic bacteria absorb inorganic carbon sources, the Raman peaks of cytochrome c and phenylalanine will produce obvious Raman peak position shifts. This is because the porphyrin ring in cytochrome c and the benzene ring of phenylalanine both have specific conjugated structures, making their vibration frequencies more sensitive to carbon isotope substitution, while other carbon-containing molecules may be insensitive to isotope substitution or the changes are not obvious due to the selectivity of metabolic pathways or the bias in the carbon assimilation process. Under the action of 532nm wavelength excitation light, phenylalanine at 1002cm -1 The Raman shift near the -1 ; cytochrome c at 747, 1125, 1312 and 1584 cm -1 The Raman shifts nearby are shifted to about 725, 1115, 1300, and 1536 cm -1 . By using the appearance of characteristic shifted spectral peaks, it is possible to determine whether the observed cells are chemoautotrophic bacteria, and the cells are less damaged during the Raman detection process and can be used for subsequent sorting, sequencing and cultivation. Based on this discovery, the applicant established a single-cell Raman spectroscopy detection method for chemoautotrophic bacteria, which enables the study of chemoautotrophic bacteria groups in complex environments at the single-cell level, and promotes the study of cells in this group.

[0011] In one aspect, the present invention provides a method for detecting chemoautotrophic bacteria based on single-cell Raman spectroscopy and stable isotope labeling, the method comprising the following steps:

[0012] (1) Test samples 13 C stable isotope labeling, and then the bacteria in the sample are collected and prepared into single-cell morphology;

[0013] (2) Single-cell bacteria were detected using Raman spectroscopy, with a laser wavelength of 400–550 nm;

[0014] (3) After preprocessing the collected Raman spectra, subsequent data analysis was performed. According to the cytochrome c peaks at 747, 1125, 1312, and 1584 cm-1 Shift to 725, 1115, 1300, 1536cm -1 and / or phenylalanine peak from 1002cm -1 Offset to 965cm -1 The characteristic shift spectral peak can be used to determine whether chemoautotrophic bacteria exist in the sample to be tested.

[0015] In another embodiment of the present invention, in step (1) of the method, the process of treating the sample to be tested includes: 13 The bacteria were collected by density gradient centrifugation after enrichment with C isotope labeling.

[0016] In another embodiment of the present invention, in step (1) of the method, the treatment process of the sample to be tested includes adding NaH 13 The culture was enriched with CO3 and then the bacteria were collected by density gradient centrifugation.

[0017] In another embodiment of the present invention, in step (1) of the method, the process of treating the sample to be tested includes treating the sample solution 13 The bacteria were collected by C isotope labeling enrichment and density gradient centrifugation, and then an appropriate amount of solvent was added to disperse the bacteria into a single cell form. The solvent was sterile water, a buffer solution of pH 6-8, or any aqueous solution suitable for bacterial survival and not affecting Raman spectroscopy detection. The bacterial concentration was adjusted to 10 3 -10 6 / ml, used for Raman spectroscopy detection.

[0018] In another embodiment of the present invention, in step (2) of the method, bacteria in single-cell morphology are detected using Raman spectroscopy, and the laser wavelength is selected to be 500-550 nm, more preferably 532 nm.

[0019] In another embodiment of the present invention, in step (2) of the method, the conditions for detecting by Raman spectroscopy are: objective lens, spectral range (500-3200cm -1 ), exposure time 1-5s, number of cycles 1-5 times, maximum acquisition power, laser wavelength selection 400-550nm.

[0020] In another embodiment of the present invention, in step (2) of the method, the conditions for detecting by Raman spectroscopy are: objective lens (100×), spectral range (500-3200cm -1 ), exposure time 1-5s, number of cycles 1-5 times, maximum acquisition power, laser wavelength 532nm.

[0021] In an embodiment of the present invention, the preprocessing of the collected Raman spectrum in step (3) of the method includes background removal and normalization. In another preferred embodiment, the conditions for subsequent data analysis after the preprocessing of the collected Raman spectrum are: using software to process the cell Raman spectrum, preprocessing the spectrum by baseline subtraction, rubberband subtraction, etc., obtaining the sample average Raman spectrum by math merging, etc., and using software for nonlinear fitting and drawing of some spectra.

[0022] Chemoautotrophic bacteria absorb 13 When C isotope-labeled inorganic carbon sources are used, they prefer to assimilate intracellular phenylalanine and cytochrome c, so that under the action of excitation light in the wavelength range of 400–550 nm (most preferably 532 nm), cytochrome c is excited at 747, 1125, 1312, and 1584 cm -1 The Raman shifts nearby are shifted to about 725, 1115, 1300, and 1536 cm -1 ; Phenylalanine at 1002cm -1 The Raman shift near the -1 Therefore, the conditions for determining whether chemoautotrophic bacteria exist in the sample to be tested in step (3) of the method are:

[0023] (1) Microbial cells are excited by 532nm wavelength light and originally have wavelengths of 747, 1125, 1312 and 1584cm -1 The characteristic peaks of cytochrome c appeared near the surface and shifted to about 725, 1115, 1300, and 1536 cm -1 , then it is regarded as a chemoautotrophic bacteria group;

[0024] (2) Originally at 1002cm -1 The characteristic peak of phenylalanine shifted to about 965 cm -1 , they are regarded as chemoautotrophic bacteria.

[0025] The two conditions for determining whether chemoautotrophic bacteria exist in the sample to be tested can be determined independently. If both conditions are met at the same time, the determination result will be more accurate.

[0026] To further confirm the population of chemoautotrophic bacteria, single cells with characteristic shifted spectral peaks can be selected based on step (3), and the target population cells can be sorted using a single cell ejection sorter. After sorting, the cells can be amplified using a single cell whole genome amplification kit and subjected to gene sequence analysis. The judgment is based on:

[0027] Genomic information identification criteria: The single-cell genome obtained by multiple displacement amplification is identified as a chemoautotrophic bacteria group by using genome phylogenetic analysis or functional gene annotation to determine its group affiliation or the presence of carbon fixation functional genes.

[0028] The inventors of the present application have particularly noticed that when chemoautotrophic bacteria absorb and fix isotope-labeled inorganic carbon, they can assimilate the lighter carbon atoms in their own intracellular compounds into heavier carbon atoms. 13 C isotopes, with cytochrome c peaks at approximately 747, 1125, 1312, and 1584 cm -1 The Raman peaks shifted to about 725, 1115, 1300, and 1536 cm -1 ; Phenylalanine peak at about 1002cm -1 There is a shift to about 965cm -1 . The appearance of characteristic shifted spectral peaks can be used to determine whether the observed cells are chemoautotrophic bacteria. Based on this discovery, the applicant established a single-cell Raman spectroscopy detection method for chemoautotrophic bacteria, which enables the study of chemoautotrophic bacteria groups in complex environments at the single-cell level and promotes the study of cells in this group.

[0029] Compared with other detection methods, the chemoautotrophic bacteria detection method based on single-cell Raman spectroscopy provided by the present invention has the following characteristics and advantages:

[0030] (1) Sample preparation is simple, and pure culture is not required. Chemoautotrophic bacteria can be detected at the single-cell level by simply enriching with isotope labeling and adjusting the cell density of environmental samples.

[0031] (2) In the prior art, metagenomics can annotate carbon fixation functional genes, but it can only indicate that the cells of this group have carbon fixation potential, and cannot characterize whether the carbon fixation genes of this group are expressed. The study of carbon fixation groups by single-cell Raman spectroscopy is limited to photosynthetic carbon fixation groups, while chemoautotrophic bacteria are widely distributed. The present invention can effectively identify chemoautotrophic bacteria at the single-cell level through carbon fixation phenotypic information, and the detection will not destroy the cells, which can be used for subsequent sorting, sequencing and cultivation.

[0032] (3) Based on Raman spectroscopy detection, cells can be further sorted for single-cell whole genome amplification, and chemoautotrophic groups can be identified through genomic information, making the detection results more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The representative chemoautotrophic strain Thiobacimonas profunda (deep-sea sulfur-oxidizing monas) CGMCC1.12377 was subjected to 532nm laser wavelength. 13 C culture and 12 Average Raman spectra measured after C incubation.

[0034] Figure 2 Environmental sediments and enriched pure strains were subjected to 532nm laser wavelength. 13 C culture and 12 C The average Raman spectra measured after cultivation. Figure a is environmental sediment, and Figure b is the enriched pure culture strain.

[0035] Figure 3 Genome sequencing and functional annotation of environmental sediments and sorted chemoautotrophic bacteria reconstructed carbon fixation and sulfur oxidation metabolic pathways.

[0036] Figure 4 Assembling a genomic phylogenetic tree for the metagenomes of enriched cultures. DETAILED DESCRIPTION

[0037] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those not indicating specific techniques or conditions in the embodiments are carried out according to the technical conditions described in the literature in this area or according to the product specification. Those not indicating the manufacturer of reagents used or instruments are all conventional products that can be obtained commercially.

[0038] The representative chemoautotrophic strain was Thiobacimonas profunda CGMCC 1.12377, which was purchased from China General Microbiological Culture Collection Center.

[0039] The method of the present invention comprises:

[0040] a) Isotope labeling and Raman analysis sample preparation for pure culture chemoautotrophic bacteria

[0041] According to the published literature, MB 2216 culture medium purchased from BD-Difco, USA, was selected and activated and revived at 30°C.

[0042] In a 40 mL serum bottle, 20 mL of artificial seawater medium was injected, and then 400 μL of activated Thiobacimonas profunda CGMCC 1.12377 was inoculated. After sealing, a mixed N2 and O2 gas (v / v, 4 / 1) was used to replace the headspace gas. The serum bottles that completed the above steps were set up into three parallel sample groups and three biological replicates. Group 1: Add 13C-labeled NaHCO3 (99atom%, purity 99%) was used as the carbon source substrate and placed in a constant temperature shaking incubator (150rpm) away from light. The final concentration of the carbon source was 2mM. 12 C-labeled NaHCO3 was used as the carbon source substrate, and the cells were placed in a constant temperature shaking incubator (150 rpm) away from light, with a final carbon source concentration of 2 mM; Group 3: No additional carbon source was added, and the cells were placed in a constant temperature shaking incubator (150 rpm) away from light. Bacteria were cultured in different treatments and recovered after 3 days for subsequent analysis. The formula of artificial seawater culture medium is shown in Table 1:

[0043] Table 1 Artificial seawater culture medium formula

[0044]

[0045] Use sterile water to make up to 1000 mL, pH 7.8-8.0.

[0046] The recovered bacterial suspension was centrifuged at 5000-6000g for 3 minutes and washed with sterile water 2-3 times. An appropriate amount of sterile water was added to adjust the concentration of the bacterial suspension. 2 μl of the bacterial suspension was dropped on the aluminum foil sample area and naturally air-dried at room temperature. Single-cell Raman spectroscopy was collected using the LabRAM Aramis (HORIBA Jobin-Yvon) laser confocal microscopy Raman system for cell observation and spectral signal collection. -1 The silicon peak at 520.6±0.5cm was used as the calibration peak. The 100x objective lens focused on the silicon wafer, the grating was set to 300g / mm, the laser wavelength was 532nm, the exposure time was 5s, the number of cycles was 1, and the silicon peak was calibrated to 520.6±0.5cm -1 After the calibration is completed, the Raman spectrum of the sample is collected. The collection parameters are: objective lens (100×), spectrum range (500-3200cm -1 ), exposure time 5s, cycle number 1, maximum acquisition power, laser wavelength selected 532nm; 20 Raman spectra / sample; LabSpec5 software (HORIBA Jobin-Yvon) was used to process the cell Raman spectrum data, and the spectrum was preprocessed by baseline subtraction, rubberband subtraction, etc., and the average Raman spectrum of the sample was obtained by math merging, and some spectra were nonlinearly fitted and drawn using Origin software.

[0047] b) Sample preparation for bacterial Raman detection in environmental sediments

[0048] 20 mL of artificial seawater medium was injected into a 40 mL serum bottle, and 2 g of sediment was added to conduct the sediment microcosm labeling experiment. After the serum bottle was sealed, argon (Ar) was used to continuously aerate for about 10 min to replace the headspace gas in the sealed serum bottle, and the headspace gas was mixed with N2 and O2 at a volume ratio of 4:1. Three parallel sample groups and three biological replicates were set for the sediment. Group 1: In addition 13 C-labeled NaHCO3 was used as the carbon source substrate and placed in the dark for 5 days under natural conditions for labeling. The final concentration of the carbon source was 2 mM. Group 2: 12 C-labeled NaHCO3 was used as the carbon source substrate and was placed in the dark under natural conditions for 5 days for labeling, and the final concentration of the carbon source was 2 mM; Group 3: No additional carbon source was added and it was placed in the dark under natural conditions for 5 days for labeling.

[0049] The microorganisms contained in the sediment after labeling and culture are extracted by iodine hexol density gradient centrifugation. The steps are as follows: about 0.5g of sediment is mixed with 5mL of PBS, wherein the PBS formula is (l-1): 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate, and 0.24g of potassium dihydrogen phosphate. Then, 0.5% (v / v) Tween 20 (Aladdin) surfactant is added, and the above mixture is placed on a vortex instrument and vigorously shaken for 30 minutes to separate the cells attached to the sediment particles to obtain a sediment suspension. A 1.42g / mL (80% w / v) iodine hexol (Nycodenz, ≥98%, Aladdin) solution is prepared, and the above sediment suspension is slowly added to a centrifuge tube containing 5mL of iodine hexol solution. Subsequently, centrifugation is carried out at a speed of 14000g and a temperature of 4°C for about 30 minutes. After centrifugation, the white cell layer containing microorganisms in the middle was aspirated, and 5 mL of PBS was added and centrifuged at 6000 rpm for 10 min. Finally, it was washed three times with sterile water, and then an appropriate amount of sterile water was added to adjust the cell concentration for subsequent Raman spectroscopy detection.

[0050] c) Raman analysis sample preparation of pure cultures isolated from environmental sediments

[0051] Weigh about 1g of sediment sample and add it to a centrifuge tube containing 9mL of sterile water. Vortex for 20min to mix it thoroughly to make a sediment suspension. After diluting it 100 times, take 200μL of the dilution and inject it into a sterile culture dish containing solid culture medium, spread it evenly, and invert it to culture in an environment of 28℃.

[0052] Inject 20 mL of artificial seawater culture medium into a 40 mL serum bottle, pick a single colony and add it to the culture medium for cultivation. After sealing the serum bottle, use argon (Ar) to continuously aerate for about 10 minutes to replace the headspace gas in the serum bottle, and mix the headspace gas with N2 and O2 at a volume ratio of 4:1. A total of three parallel sample groups and three biological replicates were set up. Group A samples: add 13 C-labeled NaHCO3 was used as the carbon source substrate and placed in a constant temperature shaking incubator (150 rpm) away from light. The final concentration of the carbon source was 2 mM. Group B samples: 12 C-labeled NaHCO3 was used as the carbon source substrate and placed in a constant temperature shaking incubator (150 rpm) away from light. The final concentration of the carbon source was 2 mM. Group C samples: no additional carbon source was added and placed in a constant temperature shaking incubator (150 rpm) away from light. Bacteria were cultured in different treatments and recovered after 3 days for subsequent analysis.

[0053] d) Single cell resonance Raman detection and genome amplification

[0054] Take 2 μl of bacterial suspension and drop it on the aluminum foil Raman sheet. After air drying at room temperature, it can be used for Raman spectroscopy detection. This experiment uses a Raman spectrometer (LabRAM Aramis, HORIBA Jobin-Yvon Ltd) for cell Raman spectroscopy detection. The experimental parameters are: objective lens (100×), spectral range (500-3200cm -1 ), exposure time 5s, cycle number 1, maximum acquisition power, excitation wavelength selected as 532nm, 300g / mm grating, take 2μl sample spot on aluminum foil sorting sheet, and air dry naturally. Single cell ejection sorter (HOOKE, PRECI SCS-1) was used for target population cell sorting, 10 cells were sorted per group, 3 parallels each; sorting parameter was 45, after sorting, cells were collected into 3μl PBS solution, and single cell whole genome amplification was performed using REPLI-g single cell whole genome amplification kit (QIAGEN).

[0055] e) Identification of chemoautotrophic bacteria

[0056] Raman peak discrimination criteria: When chemoautotrophic bacteria absorb isotopic carbon sources, they replace the lighter carbon atoms in their intracellular compounds with heavier ones. 13 C isotope, resulting in a red shift in the Raman spectrum. Under 532nm wavelength excitation light, cytochrome c has wavelengths of about 747, 1125, 1312 and 1584cm -1 The following deviations occur to 725, 1115, 1300, 1536cm -1 ; while phenylalanine is at about 1002cm -1 Appears to be shifted to 965cm -1 ( Figure 1 ).

[0057] Genomic information identification criteria: The single-cell genome obtained by multiple displacement amplification is identified as a chemoautotrophic group by using genome phylogenetic analysis or functional gene annotation to determine its group affiliation or the presence of carbon fixation functional genes.

[0058] Example 1 Detection of single-cell genomics of chemoautotrophic bacteria in coastal sediments

[0059] Stable isotope labeling of sediment samples. Coastal sediment samples were collected from the Zhoushan area of ​​the East China Sea. 20 mL of artificial seawater culture medium was injected into a 40 mL serum bottle, and 2 g of sediment was added for the sediment microcosm labeling experiment. After sealing the serum bottle, argon (Ar) was used to continuously aerate for about 10 min to replace the headspace gas in the sealed serum bottle, and the headspace gas was mixed with N2 and O2 in a volume ratio of 4:1. Three parallel sample groups and three biological replicates were set up for the sediment. Group 1: In addition 13 C-labeled NaHCO3 was used as the carbon source substrate and placed in the dark for 5 days under natural conditions for labeling. The final concentration of the carbon source was 2 mM. Group 2: 12 C-labeled NaHCO3 was used as the carbon source substrate and was placed in the dark under natural conditions for 5 days for labeling, and the final concentration of the carbon source was 2 mM; Group 3: No additional carbon source was added and it was placed in the dark under natural conditions for 5 days for labeling.

[0060] Sediment sample bacteria acquisition. The microorganisms contained in the labeled cultured sediment were extracted using the iodine hexol density gradient centrifugation method. The steps are as follows: about 0.5 g of sediment was mixed with 5 mL of PBS, wherein the PBS formula was (l-1): 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate. Then, 0.5% (v / v) Tween 20 (Aladdin) surfactant was added, and the mixture was placed on a vortex instrument and vigorously shaken for 30 minutes to separate the cells attached to the sediment particles to obtain a sediment suspension. A iodine hexol (Nycodenz, ≥98%, Aladdin) solution with a density of 1.42 g / mL (80% w / v) was prepared, and the sediment suspension was slowly added to a centrifuge tube containing 5 mL of the iodine hexol solution. Subsequently, the mixture was centrifuged at a speed of 14000 g and a temperature of 4°C for about 30 minutes. After centrifugation, the white cell layer containing microorganisms in the middle was aspirated, and 5 mL of PBS was added and centrifuged at 6000 rpm for 10 min. Finally, it was washed three times with sterile water, and then an appropriate amount of sterile water was added to adjust the cell concentration for subsequent Raman spectroscopy detection.

[0061] Single cell Raman spectroscopy detection. Raman spectroscopy detection was performed with 532nm wavelength excitation light, and the detection conditions were set as objective lens (100×), spectral range (500-3200cm-1 ), exposure time 5s, cycle number 1, maximum acquisition power, 300g / mm grating, 2μl sample spot was placed on the aluminum foil sorting sheet, and the sample was placed at about 1002cm -1 The Raman peak of phenylalanine shifted to about 965 cm -1 and at about 747, 1125, 1310, 1584cm -1 The cytochrome c Raman peaks that appeared shifted to approximately 725, 1115, 1300, and 1531 cm -1 The cells are considered as chemoautotrophic cells ( Figure 2 a).

[0062] Single cell sorting and genome amplification. Take 2 μl of sample and spot it on the aluminum foil sorting sheet and air dry it naturally; use a single cell ejection sorter (HOOKE, PRECI SCS-1) to sort the target population cells, and sort 10 cells per group; the sorting parameter is 45, and the sorted cells are collected into 3 μl PBS solution for single cell whole genome amplification; the amplification kit is REPLI-g single cell whole genome amplification kit (QIAGEN).

[0063] Multiple displacement amplification. The REPLI-g single-cell whole genome amplification kit (QIAGEN) was used for whole genome amplification of cells. The specific method is as follows: add 1.5μl Buffer D2 to the cell suspension obtained in the above step, blow it evenly; 65℃ metal bath for 10min; add 1.5μl stop solution, and let the sample stand on ice; add 20μl Master Mix, flick to mix, centrifuge and reflux, and add 3μl vegetable oil for liquid sealing; incubate at 30℃ for 12h; 65℃ metal bath for 15min, and store the amplified product at -80℃.

[0064] Phylogenetic analysis of the genome. The multiple displacement amplification products were sequenced by the second-generation sequencing platform Illumina HiSeq4000. The sequencing results were assembled using the assembly, binning and bin_refinement modes in the MetaWRAP software package, and functional gene annotation was performed using Prokka software. In order to reveal the carbon fixation metabolic potential of the sorted cell genomes and the enrichments and original sediments, different genome sequences were mapped to the KEGG metabolic pathways, and the complete Calvin cycle metabolic pathway was reconstructed for these functional genes ( Figure 3 ). Through the reconstruction of the genomic metabolic pathway, the presence of functional genes related to the carbon fixation pathway was successfully identified, confirming that single-cell Raman sorting can effectively separate chemoautotrophic carbon-fixing bacteria in coastal sediments.

[0065] Example 2 Detection of pure cultures of chemoautotrophic bacteria in coastal sediments

[0066] Isolation and cultivation of pure culture. Weigh about 1g of sediment sample and add it to a centrifuge tube containing 9mL of sterile water. Vortex for 20min to mix it thoroughly to make a sediment suspension. After diluting 100 times, pipette 200μL of the dilution into a sterile culture dish containing solid culture medium, spread it evenly, and invert it to culture in an environment of 28°C. Inject 20mL of artificial seawater culture medium into a 40mL serum bottle, pick a single colony and add it to the culture medium for cultivation. After sealing the serum bottle, use argon (Ar) to continuously aerate for about 10min to replace the headspace gas in the serum bottle, and mix the headspace gas with N2 and O2 in a volume ratio of 4:1. A total of three parallel sample groups and three biological replicates were set up. Group A samples: add 13 C-labeled NaHCO3 was used as the carbon source substrate and placed in a constant temperature shaking incubator (150 rpm) away from light. The final concentration of the carbon source was 2 mM. Group B samples: 12 C-labeled NaHCO3 was used as the carbon source substrate and placed in a constant temperature shaking incubator (150 rpm) away from light. The final concentration of the carbon source was 2 mM. Group C samples: no additional carbon source was added and placed in a constant temperature shaking incubator (150 rpm) away from light. Bacteria were cultured in different treatments and recovered after 3 days for subsequent analysis.

[0067] Single cell Raman spectroscopy detection. Take 2μl of bacterial suspension and drop it on the aluminum foil Raman sheet. After air drying at room temperature, it can be used for Raman spectroscopy detection. This experiment uses a Raman spectrometer (LabRAM Aramis, HORIBA Jobin-Yvon Ltd) for cell Raman spectroscopy detection. The experimental parameters are: objective lens (100×), spectral range (500-3200cm -1 ), exposure time 5s, cycle number 1, maximum acquisition power, laser (532nm, 785nm), 300g / mm grating. It was found that at about 749, 1125, 1312, 1584cm -1 The cytochrome c Raman peaks that appeared shifted to approximately 725, 1115, 1300, and 1534 cm -1 and at about 1003cm -1 The Raman peak of phenylalanine shifted to about 985 cm -1 The cells are considered as chemoautotrophic cells ( Figure 2 b).

[0068] Genome phylogenetic analysis. The pure culture isolated from the sediment was sequenced by whole genome sequencing. The sequencing results were annotated with functional genes using Prokka software, and a complete Calvin cycle carbon fixation metabolic pathway ( Figure 3 ), indicating that it has carbon fixation potential. Combined with the genome data of the enriched strains, the core genome phylogenetic tree was constructed using IQ-Tree software. Figure 4As shown, four high-quality metagenomic assemblies with a completeness greater than 50% and a contamination less than 10% were obtained from the sediment enrichment, among which bin1 belonged to the genus Guyparkeria, which was the same taxonomic group as the pure culture isolated ( Figure 4 ), has the ability of chemoautotrophic carbon fixation.

[0069] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, substitute and vary the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A method for detecting chemoautotrophic bacteria based on single-cell Raman spectroscopy and stable isotope labeling, the method comprising the following steps: (1) Test samples 13 C stable isotope labeling, and then the bacteria in the sample are collected and prepared into single-cell morphology; (2) Raman spectroscopy is used to detect bacteria in single-cell form, with a laser wavelength of 400–550 nm; (3) After preprocessing the collected Raman spectra, subsequent data analysis was performed. According to the cytochrome c peaks at 747, 1125, 1312, and 1584 cm -1 Shift to 725, 1115, 1300, 1536cm -1 and / or phenylalanine peak from 1002cm -1 Offset to 965cm -1 The characteristic shift spectral peak can be used to determine whether chemoautotrophic bacteria exist in the sample to be tested.

2. The chemoautotrophic bacteria detection method according to claim 1, characterized in that: In step (1) of the method, the process of treating the sample to be tested includes: 13 The bacteria were collected by density gradient centrifugation after enrichment with C isotope labeling.

3. The chemoautotrophic bacteria detection method according to claim 2, characterized in that: In step (1) of the method, the process of treating the sample to be tested includes adding NaH 13 The culture was enriched with CO3 and then the bacteria were collected by density gradient centrifugation.

4. The chemoautotrophic bacteria detection method according to claim 2, characterized in that: In step (1) of the method, the process of treating the sample to be tested includes: 13 The bacteria were collected by C isotope labeling enrichment and density gradient centrifugation, and then an appropriate amount of solvent was added to disperse the bacteria into a single cell form. The solvent was sterile water, a buffer solution of pH 6-8, or any aqueous solution suitable for bacterial survival and not affecting Raman spectroscopy detection. The bacterial concentration was adjusted to 10 3 -10 6 / ml, used for Raman spectroscopy detection.

5. The chemoautotrophic bacteria detection method according to claim 4, characterized in that: In step (2) of the method, the laser wavelength is selected to be 532 nm.

6. The chemoautotrophic bacteria detection method according to claim 1, characterized in that: In step (2) of the method, the conditions for detecting by Raman spectroscopy are: objective lens, spectral range (500-3200cm -1 ), exposure time 1-5s, number of cycles 1-5 times, maximum acquisition power, laser wavelength selection 400-550nm.

7. The chemoautotrophic bacteria detection method according to claim 1, characterized in that: The preprocessing of the collected Raman spectrum in step (3) of the method includes background removal and normalization processing.

8. The chemoautotrophic bacteria detection method according to claim 7, characterized in that: In step (3) of the method, the conditions for subsequent data analysis after preprocessing the collected Raman spectra are as follows: using software to process the cell Raman spectrum, preprocessing the spectrum by baseline subtraction, rubberband subtraction, etc., obtaining the sample average Raman spectrum by math merging, etc., and using software for nonlinear fitting and drawing of some spectra.

9. The chemoautotrophic bacteria detection method according to claim 1, characterized in that: In step (3) of the method, the conditions for determining whether chemoautotrophic bacteria exist in the sample to be tested are: (1) Microbial cells are excited by 532nm wavelength light and originally have wavelengths of 747, 1125, 1312 and 1584cm -1 The characteristic peaks of cytochrome c appeared near the surface and shifted to about 725, 1115, 1300, and 1536 cm -1 , they are considered as chemoautotrophic bacteria; and / or (2) Originally at 1002cm -1 The characteristic peak of phenylalanine shifted to about 965 cm -1 , they are regarded as chemoautotrophic bacteria.

10. The chemoautotrophic bacteria detection method according to claim 1, characterized in that: In step (3) of the method, in order to further confirm the population of chemoautotrophic bacteria, single cells with characteristic shifted spectral peaks can be selected based on step (3), and a single cell ejection sorter can be used to sort the target population cells. After sorting, the cells are amplified using a single cell whole genome amplification kit, and gene sequence analysis is performed. The judgment basis is: Genomic information identification criteria: The single-cell genome obtained by multiple displacement amplification is identified as a chemoautotrophic bacteria group by using genome phylogenetic analysis or functional gene annotation to determine its group affiliation or the presence of carbon fixation functional genes.

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