CN104089937B - Marine bacteria based on laser-induced fluorescence (LIF) abundance and multifarious detection method in situ - Google Patents

Abstract

A laser-induced fluorescence-based in situ detection method for the abundance and diversity of marine bacteria with a wide range of applications, fast and easy analysis, and environmentally friendly. The technical solution is: it is characterized in that it includes the following steps: (1) transporting the tested water sample solution through a pump; (2) the water sample solution flows through the filter column; (3) the water sample flowing through the filter column enters the fluorescence pool; ( 4) Use a high-frequency pulse excitation light source-deep ultraviolet laser diode to pump a solid-state laser to generate a continuous-band light source, irradiate the fluorescent cell, use the spectral splitter-grating on the detection side of the fluorescent cell to perform spatial light splitting, and form λ ₁ after dispersion The spectral band of ‑λ ₂ ; (5) The CCD photodetector located at the detection window simultaneously collects the spectral data of λ ₁ ‑λ ₂ , and obtains the wavelength-light intensity two-dimensional spectrum through internal conversion and time series integration; (6) data The processing system calculates and analyzes the abundance and diversity of marine bacteria through the calculation of marine bacterial fluorescence recognition mode.

Description

In situ detection of abundance and diversity of marine bacteria based on laser-induced fluorescence

technical field

The invention belongs to the field of detection methods for in-situ abundance and diversity of marine bacteria, in particular to a detection method for in-situ abundance and diversity of marine bacteria based on laser-induced fluorescence, which has a wide range of applications, quick and easy analysis, and is environmentally friendly. This method uses the optical characteristics of the fluorescence spectrum produced by the laser-induced fluorescence process and the "characteristic spectrum" of various marine bacteria itself - that is, the fluorescence spectra produced by various marine bacteria in the laser-induced fluorescence process are compared in different bands, Combined with marine bacterial fluorescence recognition patterns, the abundance and diversity of marine bacteria can be analyzed.

Background technique

Marine bacteria are an important part of the marine ecosystem. The study of bacteria in the marine environment is not only one of the frontier fields of life science, but also an important part of the study of the structure of marine ecosystems. Marine bacteria participate in the process of degrading various marine pollutants and poisons, which help maintain the balance of the marine ecosystem and promote the self-purification ability of the ocean; marine bacteria are important producers of new antibiotics, amino acids, vitamins and other physiologically active substances; bacteria Participate in marine sedimentary diagenesis, such as participating in the formation of sulfur ore and deep-sea manganese nodules; bacteria play an important role in the process of marine oil and gas formation; seawater has a bactericidal effect due to the antagonism and lysis of marine bacteria , leading to the rapid death of terrestrial pathogenic bacteria; marine bacteria can be directly used as bait for marine economic animals; bacteria participate in the process of corrosion, denaturation, pollution and destruction of various marine substances; some marine bacteria are pathogenic bacteria of human or marine organisms ;Under certain conditions, the accumulation of marine bacterial metabolites will poison the aquaculture environment, such as the accumulation of ammonia and hydrogen sulfide, which endangers biological aquaculture; the metabolic activities of bacteria can also be used to improve the poisoned aquaculture environment, such as the oxidation of ammonia, etc., so The research on detection methods for the abundance and diversity of marine bacteria is of great significance for further understanding the ecological structure of marine bacteria and their role in the ocean carbon cycle.

In terms of research methods, at present, marine on-site sampling, laboratory isolation and culture, gene sequencing methods and fluorescence microscopy techniques are mainly used to observe and study the diversity of marine bacteria. Due to its high sensitivity, fluorescence detection technology has always been in a very important position in bacterial research. At present, the commonly used fluorescence detection technology in the application of bacteria mainly includes fluorescence microscopy, flow cytometry, rapid detection of ATP fluorescence, fluorescent protein labeling and so on. Because these techniques have defects such as the need to collect and prepare samples, strict requirements for sample injection, and the need to make fluorescent markers, it is difficult to apply them to the detection of bacteria in the marine field. At the same time, fluorescence microscope technology and plate culture methods have proved that in the ocean environment, only a small amount of marine bacteria can form colonies through traditional fixed plate technology, and some bacteria are in a living non-culturable state, which cannot be fully reflected by conventional isolation and culture methods. It is difficult to fully understand the abundance and diversity of bacteria, their ecological significance and their role in the ocean carbon cycle.

In addition, the method adopts the mode of on-site sampling and laboratory analysis, that is, the method of on-site and real-time measurement cannot be realized, and other interfering substances are easily introduced during the sample transportation and processing process, which affects the accuracy of the analysis. The detection of marine bacterial abundance and diversity involves certain environmental conditions and complex processes, so it is impossible to guarantee that there will be no possibility of secondary pollution in this process. The incompatibility of the environment and the large difference between the laboratory environment and the marine environment lead to a large difference between the measurement results and the actual situation, and the accuracy and reliability of the results are questioned.

The above methods have the following defects to varying degrees: 1. It must be completed in a laboratory, and the application cannot be performed in real time on site, and the scope is limited. 2. The analysis takes a long time, at least several days. 3. The analysis process is complicated, the conditions are harsh, the energy consumption is high, and the technical level of the experimenters is high.

Due to the particularity of the marine environment and the unculturability of marine bacteria, on-site detection can more truly reflect the status of marine bacteria. Fluorescence measurement has the advantages of fast, convenient, continuous measurement, and no need for cultivation, which can better measure bacteria on site.

In recent years, as a new type of high-sensitivity detection method, laser-induced fluorescence (LIF) detection method has been rapidly developed and widely used in recent years. It is the most sensitive optical detection method so far. Laser-induced fluorescence detection technology The sensitivity is 1-3 orders of magnitude higher than that of ordinary fluorescence, and its detection limit for fluorescent substances can reach the annual nmol level, and even single-molecule detection can be realized under appropriate conditions. It does not require advantages such as cultivation. Therefore, the abundance and diversity of marine bacteria can be better detected by using in-situ laser-induced fluorescence technology, and it can be used as an important method for in-situ detection of marine bacterial abundance and diversity.

Contents of the invention

The invention provides a method for detecting the in-situ abundance and diversity of marine bacteria based on laser-induced fluorescence, which has wide application range, quick and easy analysis, and is environmentally friendly. This method organically combines laser-induced fluorescence technology with the different types, contents and ratios of fluorescent substances in bacteria, and establishes a detection mode for marine bacterial abundance and diversity based on laser-induced fluorescence, which provides on-site and rapid monitoring of marine bacterial abundance and diversity. Diversity provides the means.

In order to achieve the purpose of solving the above technical problems, the present invention adopts the following technical solutions:

A method for detecting the in-situ abundance and diversity of marine bacteria based on laser-induced fluorescence, characterized in that the method steps of the present invention are as follows:

(1) Transport the tested water sample solution through the pump;

(2) The water sample solution flows through the filter column under the action of the pump, and the inside of the filter column is filled with magnesium oxide-loaded cobalt-iron metal magnetic nanomaterials, and has a temperature control device. Since the water body contains phytoplankton with a large particle size, it passes The filter column can filter out phytoplankton with large particle size.

(3) The water sample flowing through the filter column enters the fluorescence pool. The window material of the fluorescence pool is made of nano-diamond material near the side of the excitation light source, which mainly eliminates Raman scattering of the water body. The side of the window near the detection window is made of graphene The silica glass of the film material mainly eliminates the fluorescence scattering of the water body.

(4) Use a high-frequency pulse excitation light source-deep ultraviolet laser diode to pump a solid-state laser to generate a continuous wave band, irradiate the fluorescent cell, and use the spectral spectroscopic device-grating on the detection side of the fluorescent cell to perform spatial splitting, and form λ ₁ - after dispersion The spectral band of λ ₂ ;

(5) The CCD photodetector located at the detection window simultaneously collects the spectral data of λ ₁ -λ ₂ , and obtains the wavelength-light intensity two-dimensional spectrum through internal transformation and time series integration;

(6) First, aiming at the possible detection errors caused by pico-algae in the water body, a high-frequency pulsed excitation light source is used, the excitation adopts 400nm, the emission adopts 685nm, and the CCD photodetector collects spectral data as the water algae influence factor;

(7) Secondly, in view of the impact of CDOM in water, according to CDOM has its own characteristic fluorescence (excitation 340/emission 430), the same high-frequency pulse excitation light source is used, the excitation adopts 340nm, the emission adopts 430nm, and the CCD photodetector collects Spectral data, as water body CDOM impact factors;

(8) Finally, use a high-frequency pulsed excitation light source-deep ultraviolet laser diode to pump a solid-state laser to generate a continuous-wave band light source of 275-450nm, irradiate the fluorescent cell, and use the spectral spectroscopic device-grating on the detection side of the fluorescent cell to perform spatial spectroscopy After dispersion, the spectral band of 280-535nm is collected, and the CCD photodetector located at the detection window collects the spectral data of 280-535nm at the same time, and the wavelength-light intensity two-dimensional spectrum is obtained through internal transformation and time series integration;

(9) In the data processing system, the established marine bacterial diversity fluorescence recognition mode is adopted, and the abundance and diversity of marine bacteria are calculated and analyzed through the marine bacteria fluorescence recognition mode.

In the present invention, it also has the following technical features: the flow rate of the water sample is 5.0-10.0ml/min.

In the present invention, it also has the following technical features: the interior of the filter column is filled with magnesium oxide-loaded cobalt-iron metal magnetic nanomaterials, and the temperature range of the temperature control device is 10-15°C.

In the present invention, it also has the following technical features: using a high-frequency pulsed excitation light source-deep ultraviolet laser diode pumped solid-state laser, which has the characteristics of small size, no need for water cooling, low fluctuation noise, simple power supply, etc., and the high frequency range is 100-150KHz . In addition, the continuous excitation pulse excitation light source is used to improve the measurement accuracy to a certain extent. At the same time, the typical fluorescent substances in bacteria can be excited by deep ultraviolet excitation, without the need for dyeing and other steps.

In the present invention, it also has the following technical features: the established marine bacteria diversity fluorescence recognition mode is based on the fact that the bacteria contain some components that can be excited to produce fluorescence. "Characteristic spectrum", the different types, contents or proportions of these components lead to large differences in fluorescence peaks. Through the collection and analysis of fluorescence signals, the method of principal component fluorescence matrix is used to identify bacterial diversity. At the same time Considering that environmental factors such as algae, CDOM, and inorganic particles may affect the fluorescence, the principle component fluorescence spectrum analysis method was used to establish the fluorescence recognition mode of marine bacterial diversity.

In the present invention, it also has the following technical features: the pump is a peristaltic pump, and the pipeline is made of polytetrafluoroethylene.

In the present invention, it also has the following technical features: the CCD detection element adopts the American Ocean Optics back-illuminated two-dimensional area array CCD spectrometer.

In the present invention, it also has the following technical features: the microcomputer data analysis and processing system adopts the marine bacteria diversity fluorescence recognition mode, which has a good correction effect on algae and CDOM and other influencing factors, and can eliminate the impact of these influencing factors on the calculation Analyzing the impact of marine bacterial abundance and diversity.

Effect of the present invention is:

The present invention uses the laser-induced fluorescence method to detect the abundance and diversity of marine bacteria, which is an important part of the current marine ecological environment monitoring system. The method utilizes the phenomenon that the fluorescent substances in the marine bacteria will emit "characteristic spectra", and uses laser-induced fluorescence technology to detect the abundance and diversity of marine bacteria. Organically combined with the different types, contents and ratios of fluorescent substances in bacteria, the plane dispersion diagram with wavelength as the abscissa and spectral sequence as the ordinate is obtained through the spectroscopic system, and then imported into the CCD detection element, and the optical signal is processed and converted by photoelectric detection For electrical signal output, the output electrical signal is converted by a weak signal amplification circuit, amplified to a certain voltage range and sent to the A/D conversion channel of the data processing part for quantification, and the full spectrum is obtained after time series integration processing.

The full spectrum obtained after time series integration processing, according to the "characteristic spectrum" of fluorescent substances in various bacteria, is analyzed by the marine bacterial diversity fluorescence recognition model software to obtain the abundance and diversity of marine bacteria.

The invention uses a laser-induced fluorescence method to detect the abundance and diversity of marine bacteria. The method can accurately, continuously and quickly analyze and test the abundance and diversity of marine bacteria, and can work reliably in a harsh environment for a long time.

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Description of drawings

Fig. 1 is a flow chart of the working principle of the method of the present invention;

Fig. 2 is the detection device structure schematic diagram that the inventive method adopts;

Figure 3 is a pattern diagram of laser-induced fluorescence recognition of marine bacterial diversity.

Explanation of symbols in the figure: 1. Water sample; 2. Water sample pump; 3. Pulse excitation light source; 4. Nanodiamond; 5. Fluorescence cell; 6. Silicon glass with graphene attached; 7. CCD photodetector; .Control part; 9. Data processing part; 10. Waste liquid collection; 11. Filter column; 12. Spectroscopic device.

detailed description

See Figure 1 and Figure 2,

Method steps of the present invention are as follows:

(1) Transport the tested water sample solution through the pump 2;

(2) The water sample solution flows through the filter column 11 under the action of the pump 2, and the inside of the filter column 11 is filled with magnesium oxide-loaded cobalt-iron metal magnetic nanomaterials, and has a temperature control device. , so the phytoplankton with large particle size can be filtered out through the filter column.

(3) The water sample flowing through the filter column enters the fluorescent pool 5, and the window material of the fluorescent pool adopts nano-diamond 4 material near the side of the excitation light source to mainly eliminate Raman scattering of the water body, and the window material near the detection window side adopts a The silicon glass 6 of the graphene thin film material mainly eliminates the fluorescence scattering of the water body.

(4) Utilize the 100KHz pulsed excitation light source 3-deep ultraviolet laser diode to pump the solid-state laser to produce a continuous wave band, irradiate the fluorescent cell 5, and use the spectral spectroscopic device-grating on the detection side of the fluorescent cell to perform spatial light splitting, and form λ1- after dispersion The spectral band of λ2;

(5) The CCD photodetector 7 located at the detection window collects the spectral data of λ1-λ2 at the same time, and obtains the wavelength-light intensity two-dimensional spectrum through internal conversion and time series integration;

(6) First of all, for the possible detection error caused by pico-algae in the water body, a pulsed excitation light source 3 of 100KHz is used, 400nm is used for excitation, 685nm is used for emission, and spectral data is collected by the CCD photodetector 7 as an influence factor of algae in the water body;

(7) Secondly, in view of the impact of CDOM in water, according to CDOM has its own characteristic fluorescence (excitation 340/emission 430), also use 100KHz pulse excitation light source 3, excitation adopts 340nm, emission adopts 430nm, CCD photodetector 7 Collect spectral data as the water body CDOM impact factor;

(8) Finally, utilize the 100KHz pulsed excitation light source 3-deep ultraviolet laser diode to pump the solid-state laser to produce a continuous-wave band light source 275-450nm, irradiate the fluorescent cell, and utilize the spectral spectroscopic device 12-grating on the fluorescent cell 5 to detect one side. Spatial spectroscopic, collecting the spectral band of 280-535nm after dispersion, the CCD photodetector 7 located at the detection window simultaneously collects the spectral data of 280-535nm, and obtains the wavelength-light intensity two-dimensional spectrum through internal transformation and time series integration;

(9) In the data processing system, the established marine bacterial diversity fluorescence recognition mode is adopted, and the abundance and diversity of marine bacteria are calculated and analyzed through the marine bacteria fluorescence recognition mode.

The flow rate of the water sample in the method is 5.0-10.0ml/min.

The filter column in the method is filled with magnesium oxide-loaded cobalt-iron metal magnetic nanomaterials, and has a temperature control device with a temperature range of 10-15°C.

The method uses a 100KHz pulsed excitation light source-deep ultraviolet laser diode pumped solid-state laser, which has the characteristics of small size, no need for water cooling, low fluctuation noise, and simple power supply. In addition, a continuous excitation type 100KHz pulsed excitation light source is used to improve In order to improve the measurement accuracy, at the same time, the typical fluorescent substances in bacteria can be excited by using deep ultraviolet excitation, without steps such as staining.

The window material of the fluorescence pool in the method uses nano-diamond materials on the side close to the excitation light source, mainly to eliminate Raman scattering of the water body, and uses silicon glass with a thin film material-graphene on the side close to the detection window, mainly to eliminate fluorescence scattering of the water body .

The established marine bacteria diversity fluorescence recognition mode is based on the fact that bacteria contain some components that can be excited to produce fluorescence. During the process of detecting marine bacteria by laser-induced fluorescence method, they will emit "characteristic spectra". The different types of these components, The difference in content or ratio leads to a large difference in fluorescence peaks. Through the collection and analysis of fluorescence signals, the method of principal component fluorescence matrix is used to identify the diversity of bacteria, taking into account environmental factors such as algae, CDOM, and inorganic Particles may have an impact on fluorescence, and the principal component fluorescence spectrum analysis method was used to establish a fluorescence recognition model for marine bacterial diversity.

The method pump is a peristaltic pump, and the pipeline is made of polytetrafluoroethylene.

The CCD detection element of the method adopts the American Ocean Optics back-illuminated two-dimensional area array CCD spectrometer.

The microcomputer data analysis and processing system of the method adopts the marine bacterial diversity fluorescence recognition model, which has a good correction effect on algae and CDOM and other influencing factors, and can eliminate the impact of these influencing factors on the calculation and analysis of marine bacterial abundance and diversity. Impact.

The working principle of the present invention is:

The laser-induced fluorescence method is used to identify the bacterial category, mainly because the bacteria contain some components that can be excited to produce fluorescence, such as tryptophan, tyrosine, phenylalanine, DNA, RNA, NADH, NADPH, FAD and extracellular Enzymes, etc., the different types, contents or ratios of these components lead to large differences in fluorescence peaks. The excitation wavelengths of these substances range from 250-450nm, and the maximum peaks of the emission wavelengths that produce fluorescence appear between 280-540nm.

The maximum excitation wavelength and emission wavelength of different fluorescent targets in and out of marine bacteria are shown in Table 1:

Table 1: Optical Characteristics of Fluorescent Targets

By using ultraviolet laser-induced fluorescence technology to detect the characteristic fluorescent signals produced by marine bacteria under excitation of different wavelengths, through the collection and analysis of fluorescent signals, find out the fluorescence differences between different bacteria, and realize the diversity detection of marine bacteria. Detection requires the establishment of a marine bacterial diversity fluorescence recognition model, which mainly uses the principal component fluorescence matrix method to identify bacterial diversity.

The following calculation methods are mainly used:

I _f ＝φ·I ₀ ·(1-10 ^{- ε· l · c} )

where If is the fluorescence intensity, φ is the quantum yield, _I0 is the incident light intensity, ε is the molar absorbance, _l is the optical path of the sample, and c is the molar concentration of the fluorescent substance.

At the same time, considering that environmental factors such as algae, CDOM, and inorganic particles may have an impact on fluorescence, the principal component fluorescence spectrum analysis method is used to analyze and process the collected fluorescence signals to find the best identification index for the fluorescence classification of marine bacteria. Laser-induced fluorescence recognition patterns of marine bacterial diversity (Fig. 3).

According to the above reaction principle, it can be seen that in the process of detecting marine bacteria by laser-induced fluorescence method, a "characteristic spectrum" will be emitted. The laser-induced fluorescence technology is organically combined with the different types, contents and ratios of fluorescent substances in the bacteria, and the plane dispersion diagram with the wavelength as the abscissa and the spectral sequence as the ordinate is obtained through the spectroscopic system, and then through the CCD detection element. The full spectrum can be obtained at one time by the direct reading method, and the abundance and diversity of marine bacteria can be detected by analyzing the full spectrum through the software.

In recent years, as a new type of high-sensitivity detection method, laser-induced fluorescence (LIF) detection method has been rapidly developed and widely used in recent years. It is the most sensitive optical detection method so far. Laser-induced fluorescence detection technology The sensitivity is 1-3 orders of magnitude higher than that of ordinary fluorescence, and its detection limit for fluorescent substances can reach the annual nmol level, and even single-molecule detection can be realized under appropriate conditions. It does not require advantages such as cultivation. Therefore, the abundance and diversity of marine bacteria can be better detected by using in-situ laser-induced fluorescence technology. It can be used as an important method for in-situ detection of marine bacterial abundance and diversity. A very ideal means, with the characteristics of on-site, fast, simple and sensitive.

CCD (Charge Coupled Devices) is a charge-coupled array detector. It is a device that expresses the light intensity by charge and transmits charge by coupling. It has self-scanning, wide spectral range, large dynamic range, small size, It has the advantages of low power consumption, long life, and high reliability. Put the CCD two-dimensional line array on the spectrum surface, and the entire spectrum can be obtained with one exposure. It has the following characteristics:

1. Wide spectral range, high quantum efficiency (up to 90%), small dark current, low noise, can realize multi-channel simultaneous data acquisition, and its structural characteristics can be used as a full-spectrum direct-reading detection element in a spectrum analyzer , and collect data at the same time to obtain a wavelength-intensity two-dimensional spectrum.

2. Very weak spectral signals can be detected by time series integration. Acquire time-resolved spectral signals, and then realize rapid analysis of spectra. The spectral signal can be collected by combining the CCD detection element with the laser-induced fluorescence optical system. After time series integration, the two-dimensional spectral map of the spectral intensity changing with the wavelength can be obtained, which can be analyzed through the processing software combined with the marine bacterial diversity fluorescence recognition mode. Marine bacterial abundance and diversity.

The advantage of the CCD detection element is that all the pixels (N) are exposed at the same time, and the entire spectrum can be obtained at the same time, which is N times faster than the general single-channel spectrum system to detect the same spectrum, and no spectrometer is needed in the process of capturing the entire spectrum. With mechanical scanning, there is no error of wavelength non-repetition caused by the mechanical system; the relative intensity error of spectral lines caused by unstable light source intensity is reduced; the dynamic process of spectral changes can be measured.

The whole system is a photoelectric detection system integrating light, machine, electricity and calculation. According to the working modules, it can be divided into six parts: the first part is laser-induced fluorescence, which is mainly aimed at some components that can excite and generate fluorescence in bacteria; the second part is the optical spectroscopic system part, which mainly separates the fluorescent bands in space through spectroscopic devices , follow the principle of light dispersion; the third part is the photoelectric conversion and amplification part, based on the principle of photoelectric effect, to convert the spatially separated optical signals into electrical signals, we mainly use CCD detection elements; the fourth part is data acquisition and recording Part, this part completes the acquisition, A/D conversion, transmission and storage of electrical signals: the fifth part is the control part, which is mainly responsible for the timing control in the process of photoelectric signal acquisition; the sixth part is the software processing part, which mainly combines the diverse marine bacteria Analyze and calculate the fluorescent recognition pattern.

The method of real-time qualitative and quantitative analysis of some components that can be excited to generate fluorescence in bacteria by integrating laser-induced fluorescence, optical system, and CCD detection components is currently a very effective in-situ detection method for the abundance and diversity of marine bacteria. direction of development.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention to other forms. Any skilled person who is familiar with this profession may use the technical content disclosed above to change or remodel it into an equivalent change. Example. However, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims (7)

1. A detection method based on laser-induced fluorescence in situ abundance and diversity of marine bacteria, characterized in that it comprises the following steps: (1) Transport the tested water sample solution through the pump; (2) The water sample solution flows through the filter column under the action of the pump, and the inside of the filter column is filled with magnesium oxide-loaded cobalt-iron metal magnetic nanomaterials, and has a temperature control device. Since the water body contains phytoplankton with a large particle size, it passes The filter column can filter out phytoplankton with large particle size; (3) The water sample flowing through the filter column enters the fluorescence pool. The window material of the fluorescence pool is made of nano-diamond material near the side of the excitation light source, which mainly eliminates Raman scattering of the water body. The side of the window near the detection window is made of graphene Silica glass as a thin film material mainly eliminates the fluorescence scattering of water bodies; (4) Use a high-frequency pulse excitation light source-deep ultraviolet laser diode to pump a solid-state laser to generate a continuous wave band, irradiate the fluorescent cell, and use the spectral spectroscopic device-grating on the detection side of the fluorescent cell to perform spatial splitting, and form λ ₁ - after dispersion The spectral band of λ ₂ ; (5) The CCD photodetector located at the detection window simultaneously collects the spectral data of λ ₁ -λ ₂ , and obtains the wavelength-light intensity two-dimensional spectrum through internal transformation and time series integration; (6) In the data processing system, the established marine bacteria diversity fluorescence recognition mode is adopted, and the abundance and diversity of marine bacteria are calculated and analyzed through the marine bacteria fluorescence recognition mode. It is possible to have an impact. The principal component fluorescence spectrum analysis method is used to analyze and process the collected fluorescence signals, to find the best identification index for the fluorescence classification of marine bacteria, and to establish a laser-induced fluorescence identification mode for the diversity of marine bacteria. 2. the detection method based on the in situ abundance and diversity of marine bacteria based on laser-induced fluorescence according to claim 1, is characterized in that the pulsed excitation light source of described high frequency, excites and adopts 400nm, emits and adopts 685nm, CCD photoelectric detection The spectral data collected by the instrument is used as the influencing factor of water algae; The high-frequency pulse excitation light source adopts 340nm for excitation and 430nm for emission, and the CCD photodetector collects spectral data as the water body CDOM impact factor; The high-frequency pulsed excitation light source-deep ultraviolet laser diode-pumped solid-state laser generates a continuous wave band of 275-450nm, irradiates the fluorescent pool, uses the spectral spectroscopic device-grating on the detection side of the fluorescent pool to perform spatial splitting, and collects 280-450nm after dispersion. For the 535nm spectral band, the CCD photodetector located at the detection window simultaneously collects 280-535nm spectral data, and obtains the wavelength-light intensity two-dimensional spectrum through internal conversion and time series integration. 3. The method for detecting the in situ abundance and diversity of marine bacteria based on laser-induced fluorescence according to claim 1, characterized in that the flow rate of the water sample is 5.0-10.0ml/min. 4. the detection method based on the in situ abundance and diversity of marine bacteria based on laser-induced fluorescence according to claim 1, is characterized in that the interior of the filter column is filled with magnesium oxide-loaded cobalt-iron metal magnetic nanomaterials, and the temperature control device The temperature range is 10-15°C. 5. the detection method based on the in situ abundance and diversity of marine bacteria based on laser-induced fluorescence according to claim 1, the high-frequency range of the described pulsed excitation light source-deep ultraviolet laser diode pumped solid laser utilizing high frequency 100-150KHz. 6. The method for detecting in-situ abundance and diversity of marine bacteria based on laser-induced fluorescence according to claim 1, wherein the pump is a peristaltic pump, and the pipeline is made of polytetrafluoroethylene. 7. The method for detecting in-situ abundance and diversity of marine bacteria based on laser-induced fluorescence according to claim 1, wherein the technical feature is that the CCD detection element adopts the American Ocean Optics back-illuminated two-dimensional array CCD spectrometer.