Separation and detection method of algal chlorophyll a in bacteria-algal symbiotic system under complex environment

Abstract

This invention discloses a method for separating and detecting algal chlorophyll a in algal symbiotic systems under complex environments, belonging to the field of environmental monitoring and bioanalysis technology. The separation method is based on the differences in the growth morphology of algae and bacteria in the algal symbiotic system, classifying them into two types: suspended and attached algal symbiotic systems. Suspended algal systems undergo EDTA chemical dissociation, while attached algal systems are first rinsed with a mild alkaline solution, then centrifuged at low speed, and subsequently subjected to EDTA chemical dissociation. The separated algal-bacterial mixture is filtered through a fiber membrane. The fiber membrane containing the algal-bacterial mixture is then subjected to ultrasonic cavitation treatment, followed by high-speed centrifugation, and the supernatant is collected. The detection method involves measuring the absorbance of the supernatant at different wavelengths and calculating the chlorophyll a content using a formula. This invention can effectively separate algal chlorophyll a in different types of algal symbiotic systems, and the extracted chlorophyll is found in small-sized microalgae, making it suitable for separating and extracting chlorophyll a in complex environments.

Description

Technical Field

[0001] This invention belongs to the field of environmental monitoring and bioanalysis technology, specifically relating to a method for separating and detecting algal chlorophyll a in a bacterial-algal symbiotic system under complex environments. Background Technology

[0002] Chlorophyll content is a key parameter directly reflecting the growth of algae and the operational status of algal-microbe symbiotic systems. However, due to differences in the reactor structure and operating conditions of algal-microbe symbiotic systems, the biological communities and structures are complex. A large number of algae are encapsulated by extracellular polymers secreted by microorganisms or attached to various packing materials, making complete and rapid separation of algae difficult. This leads to lower detection values ​​for chlorophyll content in algal-microbe symbiotic systems and reduced data validity. Existing methods for separating and extracting chlorophyll from algae are mostly focused on extracting chlorophyll from single algal species with high algal content. For example, Chinese patent CN107253953A discloses a method for extracting chlorophyll from *Ulva prolifera*, which involves soaking the raw *Ulva prolifera* in water, filtering, adding cellulase, and enzymatically hydrolyzing at 40℃-45℃ to obtain a chlorophyll extract. However, this method is only applicable to single *Ulva prolifera* species, which have a large biomass and are easily separated from other impurities in the water. Furthermore, the extraction process requires the use of enzymatic hydrolysis of organic solvents, resulting in high operating costs. Chinese patent CN110156797A discloses a process for extracting chlorophyll from spirulina. After freeze-drying and dehydrating the spirulina with liquid nitrogen, a microwave-assisted enzymatic hydrolysis process is employed. The hydrolysate is then filtered under dark conditions and with xenon or ammonia protection to obtain chlorophyll. This method is applicable only to a single spirulina species and involves multiple agents such as nitrogen and biological enzymes during extraction. The process is complex, has a long testing cycle, and is costly, making it unsuitable for the rapid and low-cost chlorophyll detection requirements of algae-microbe symbiotic systems. Current technological developments largely focus on the research and development of novel extraction agents such as enzyme preparations or more powerful physical separation technologies.

[0003] Current methods for determining chlorophyll in water bodies mostly refer to the industry standard HJ897-2017 "Determination of Chlorophyll a in Water - Spectrophotometric Method". This method uses a glass fiber membrane to filter algae in the water. The algae trapped on the surface of the glass fiber membrane are mechanically ground and broken up using a grinding device such as a glass mortar. Chlorophyll is extracted from the ground and broken algal cells using acetone as the extraction solvent. After soaking in the acetone-added soaking solution for 2-24 hours, the solution is centrifuged at 3000-4000 r / min. The chlorophyll concentration of the supernatant after centrifugation is detected by a spectrophotometer. This method mainly relies on manual grinding to achieve the purpose of breaking algal cells and is mainly suitable for breaking up small amounts of algae dispersed in surface water. Because algae particles in algal symbiotic systems are small, they are easily carried out of the treatment system by the water. To improve the algae retention effect of algal symbiotic systems, two main types of cultivation and operation methods have gradually emerged in the industry: The first is the suspended algal symbiotic system, where algae and activated sludge form relatively dense, large-particle flocculent colloids or hard, granular algal complexes under the combined action of hydraulic shear and microbial activity, with bacteria and algae encapsulated by layers of extracellular polymers; the second is the attached algal symbiotic system, where algae grow primarily on the surface and inside of the packing material through extracellular polymers (EPS) or by direct attachment. The protein content is higher than that of suspended bacterial-algae symbiotic systems, with a denser polysaccharide-protein coating and stronger adhesion. Compared with ordinary microbial flocs or particles mainly composed of activated sludge, the bacterial-algae symbiotic system forms a stable symbiotic structure between bacteria and algae through electrostatic attraction of EPS and protein bridging, and its binding strength is significantly higher than that of pure bacterial systems. In the bacterial-algae symbiotic system, the EPS secreted by bacteria adheres to and encapsulates a large number of algae, making it difficult for traditional reagents and mechanical separation methods to effectively contact or interact with algae, thus failing to effectively separate algae from bacterial-algae complexes, or causing chlorophyll denaturation due to excessive physicochemical separation intensity, affecting the detection results. Meanwhile, acetone is classified as a hazardous chemical (listed as No. 137 in the "List of Hazardous Chemicals (2025 Edition)"), and its purchase, use, storage, and waste disposal are subject to strict legal regulations. Furthermore, acetone is highly toxic to humans, and long-term repeated exposure can damage the liver, kidneys, and nervous system. The SL88 method for acetone extraction has a long extraction time (generally over 20 hours), which increases the time personnel are exposed to acetone and the risk of toxicity. At the same time, the samples are easily affected by environmental factors and contamination, affecting the accuracy of the test results. In addition, the long detection time seriously affects the real-time judgment and operation adjustment of algal activity in the bacterial symbiotic system, affecting the stable operation of the biochemical treatment system.

[0004] Most existing methods use rotational speed as the main control index for chlorophyll separation. However, in actual operation, there are significant differences in centrifugal force between centrifuges of different specifications at the same rotational speed. This leads to significant differences in chlorophyll separation effect when using centrifuges of different specifications at the same rotational speed. Therefore, in actual operation, relative centrifugal force rather than rotational speed should be used as the control index.

[0005] In summary, since algae and microorganisms in the bacterial-algae symbiotic system often form bacterial flocs by encapsulating and adsorbing onto EPS or adsorbing onto the surface of fillers and physical membrane materials, they are easily affected by factors such as physical encapsulation, chemical bonding, and steric hindrance. Existing technologies for separating and extracting chlorophyll in bacterial-algae symbiotic systems often suffer from defects such as low extraction rate, high reagent toxicity, large data deviation, inability to repeat verification, and long and expensive processing procedures. As a result, the test results cannot truly reflect the chlorophyll content in the bacterial-algae symbiotic system. Summary of the Invention

[0006] To address the problem in algae-microbe symbiotic systems where algae attach to filler materials and granular sludge surfaces via EPS (expanded polystyrene) or encapsulate with bacterial flocs, traditional chemical and mechanical separation methods struggle to effectively contact or interact with algae, and existing technologies cannot guarantee the separation and extraction efficiency of chlorophyll in algae-microbe symbiotic systems. This invention, based on the attachment and growth characteristics of algae in algae-microbe symbiotic systems and the adsorption and bridging effect of calcium and magnesium ions in EPS, provides a method for the separation and detection of chlorophyll a from algae in algae-microbe symbiotic systems under complex environments. Through gentle dissociation with a small amount of chemical reagents, short-duration and efficient ultrasonic vibration, and centrifugation, a chlorophyll separation, extraction, and detection method suitable for algae-microbe symbiotic systems is constructed, employing a "staged dissociation-synergistic cell disruption-spectrophotometric detection" approach. This method achieves gentle and rapid separation of algae from microorganisms and filler materials under tight encapsulation and attachment conditions, ensuring the accuracy of detection data while shortening the detection time and improving the timeliness of the results.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a method for separating algal chlorophyll a in a bacterial-algal symbiotic system under complex conditions, comprising the following steps:

[0009] Classification of microbial-algae symbiotic systems: Based on the differences in the growth morphology of microorganisms and algae in microbial-algae symbiotic systems, they are divided into two types: suspended and attached. Among them, the suspended microbial-algae symbiotic system consists of microbial communities and algae suspended in water in the form of flocs or granular sludge, while the attached microbial-algae symbiotic system consists of microbial communities and algae attached to the surface of packing materials and physical membrane components.

[0010] Pretreatment is carried out according to the classification of bacterial-algae symbiotic systems: the pretreatment of suspended bacterial-algae symbiotic systems involves extracting a uniformly mixed bacterial-algae mixture from the reactor during the system stirring or aeration stage, adding ethylenediaminetetraacetic acid (EDTA), and gently shaking to mix and react.

[0011] The pretreatment of the attached bacterial-algae symbiotic system involves draining the water from the packing material and membrane module, cutting them into small pieces, and rinsing them repeatedly in a dilute alkaline solution. Then, the mixture containing the inorganic packing material and bacterial-algae mixture is packaged and centrifuged. The separated bacterial-algae mixture is then diluted with pure water, and ethylenediaminetetraacetic acid is added and gently shaken to mix.

[0012] Filtration and separation of bacterial-algae mixture: After pretreatment according to the classification of bacterial-algae symbiotic system, a certain amount of bacterial-algae mixture is transferred and filtered through a fiber membrane. The fiber membrane that retains the bacterial-algae mixture after filtration is divided into samples of the target area.

[0013] Ultrasonic cavitation treatment: Place each sample in a centrifuge tube, add ethanol solution, control the ultrasonic frequency, and perform oscillation treatment;

[0014] High-speed centrifugation: After ultrasonic cavitation treatment, the centrifuge tubes are placed in a centrifuge, and the RCF is controlled to perform centrifugation separation. The supernatant of all centrifuge tubes is taken out, mixed evenly, and then used for later use.

[0015] Furthermore, in the pretreatment of suspended algal symbiotic systems, which are pretreated according to the classification of algal symbiotic systems, ethylenediaminetetraacetic acid (EDTA) is C. 10 H 14 N2O8Na2·2H2O, the concentration of ethylenediaminetetraacetic acid salt added is 1mM-10mM.

[0016] Furthermore, in the pretreatment of the suspended bacterial-algae symbiotic system according to the classification of bacterial-algae symbiotic systems, the reaction time is controlled at 30-60 min, the temperature at 15℃-25℃, and the pH value at 6-9.

[0017] Furthermore, in the pretreatment of attached algal symbiotic systems, which are pretreated according to the classification of algal symbiotic systems, the volume of the uniformly cut small pieces is 0.5 cm. 3 -1cm 3 Control the temperature of the dilute alkaline solution to 20℃-25℃, the pH value to 8-9, and rinse repeatedly 5-8 times; control the relative centrifugal force to 300xg-1000xg, and the centrifugation time to 5min-15min;

[0018] EDTA-1-acetate is C 10 H 14 N2O8Na2·2H2O, the concentration of ethylenediaminetetraacetic acid salt added is 1mM-5mM.

[0019] Furthermore, in the pretreatment of the attached algal symbiotic system according to the classification of algal symbiotic systems, the reaction time is controlled at 15 min-45 min, the temperature at 15℃-25℃, and the pH value at 6-9.

[0020] Furthermore, in the filtration and separation of the bacterial-algae mixture, the volume of the bacterial-algae mixture drawn by the pipette is 10mL-50mL; the pore size of the fiber membrane is controlled at 0.45μm with a diameter of 5cm; the filtration pressure is controlled at 0.05MPa-0.2MPa; and the target area of ​​the fiber filter membrane that retains the bacterial-algae mixture after filtration is 3cm². 2 -5cm 2 .

[0021] Furthermore, in the ultrasonic cavitation treatment, the effective volume of the centrifuge tube is 5 mL, the mass fraction of the ethanol solution is 95%, and the amount of ethanol solution added is 4 mL.

[0022] The ultrasonic frequency is controlled at 30kHz-60kHz, the oscillation time is 0.5min-3min, and the reaction temperature is ≤4℃.

[0023] Furthermore, during high-speed centrifugation, the centrifugation process is repeated 1 to 3 times depending on the centrifugation effect; the relative centrifugal force is controlled at 4500xg-20000xg, the centrifugation time is 10min-20min, and the reaction temperature is ≤4℃.

[0024] Furthermore, in the pretreatment and high-speed centrifugation of the attached algae-bacteria symbiotic system, the centrifugal force is controlled based on the relative centrifugal force and the centrifuge rotor diameter. The centrifuge speed is determined with reference to Formula I, which is as follows:

[0025] RCF = 1.18 × 10 -5 × r × (rpm) 2 ;

[0026] In the formula, RCF is the relative centrifugal force (xg), r is the rotor radius (cm), and rpm is the centrifuge speed (revolutions / s).

[0027] Secondly, this invention provides a method for detecting algal chlorophyll a in a complex algal symbiotic system. The method employs the aforementioned method for separating algal chlorophyll a in a complex algal symbiotic system to extract chlorophyll. The homogeneous supernatant obtained after high-speed centrifugation is reconstituted with 95 wt.% ethanol solution to obtain the sample to be tested. The absorbance at 630 nm, 647 nm, 664 nm, and 750 nm is then measured using a spectrophotometer. The chlorophyll a content in the sample is determined according to Formula II, which is as follows:

[0028] Chl-a=[11.85×(D664-D750)-1.54×(D647-D750)-0.08×(D630-D750)]VE / (Vs×δ);

[0029] In the formula: VE is the final volume of the extract in the centrifuge tube, mL; Vs is the volume of the water sample, L; δ is the optical path of the cuvette, cm; D630, D647, D664, and D750 are the absorbance values ​​of the sample at wavelengths of 630 nm, 647 nm, 664 nm, and 750 nm, respectively.

[0030] Advantages and effects of the present invention:

[0031] This invention can effectively separate chlorophyll from algae in different types of algal symbiotic systems, and the target for chlorophyll extraction is small-sized microalgae. It is suitable for chlorophyll extraction in complex environments. The main equipment is a centrifuge and an ultrasonic generator, and the main reagent is 95 wt.% ethanol. The operation is simple, the relevant equipment and reagents are readily available, and the reagents are non-toxic, harmless, and inexpensive. The separation and detection method of this invention can effectively shorten the detection time, and the test time can be controlled within 2.5 hours, improving the timeliness of the detection data. In addition, this invention solves the problem of chlorophyll separation effect differences caused by differences in centrifugation equipment. It has wide applicability and broad application prospects. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the process flow of the "hierarchical dissociation-synergistic cell disruption-spectrophotometer detection" chlorophyll separation and detection method in the complex environment algae symbiotic system constructed by the present invention;

[0033] Figure 2 The figure shows the Bland-Altman consistency analysis of the algal chlorophyll a separation and detection method of the present invention and the detection results of high performance liquid chromatography in the suspended algal symbiotic system of Example 1.

[0034] Figure 3 The scatter plot shows the linear regression correlation analysis of the algal chlorophyll a separation and detection method of the present invention and the detection results of high performance liquid chromatography in the suspended algal symbiotic system of Example 1.

[0035] Figure 4 The figure shows the Bland-Altman consistency analysis of the algal chlorophyll a separation and detection method of the present invention and the detection results of high performance liquid chromatography in the attached algal symbiotic system of Example 2.

[0036] Figure 5 The scatter plot shows the linear regression correlation analysis of the algal chlorophyll a separation and detection method of the present invention and the detection results of high performance liquid chromatography in the attached algal symbiotic system of Example 2. Detailed Implementation

[0037] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples.

[0038] A method for separating chlorophyll a from algae in a complex algal symbiotic system includes the following steps:

[0039] Classification of microbial-algae symbiotic systems: Based on the differences in the growth morphology of microorganisms and algae in microbial-algae symbiotic systems, they are divided into two categories: suspended type and attached type. Among them, the suspended type microbial-algae symbiotic system consists of microbial communities and algae suspended in water in the form of flocs or granular sludge, while the attached type microbial-algae symbiotic system consists of microbial communities and algae attached to the surface of packing materials and physical membrane components.

[0040] Pretreatment is carried out according to the classification of bacterial-algae symbiotic systems: For suspended bacterial-algae symbiotic systems, pretreatment involves extracting a uniformly mixed bacterial-algae solution from the reactor during the system stirring or aeration stage, and adding C... 10 H 14 Prepare N2O8Na2·2H2O to a concentration of 1mM-10mM, mix with gentle shaking, control the reaction time to 30min-60min, the temperature to 15℃-25℃, and the pH value to 6-9.

[0041] The pretreatment of the attached algae-bacteria symbiotic system involves draining the water from the packing material and membrane module, and then uniformly cutting them into 0.5cm pieces. 3 -1cm 3 Small pieces of the mixture were repeatedly rinsed in a dilute alkaline solution at 20℃-25℃ and pH 8-9. The mixture containing inorganic packing material and the bacterial-algae mixture was then dispensed into centrifuges. Under a relative centrifugal force (RCF) of 300xg-1000xg, the mixture was centrifuged for 5-15 minutes until the bacteria and algae were separated from the inorganic packing material fragments. The separated bacterial-algae mixture was then diluted to volume with pure water, and C was added. 10 H 14 Prepare a solution of N2O8Na2·2H2O to a concentration of 1mM-5mM. Mix the solution with gentle shaking, and control the reaction time to 15min-45min, the temperature to 15℃-25℃, and the pH value to 6-9.

[0042] Filtration and separation of bacterial-algae mixtures: After pretreatment according to the classification of bacterial-algae symbiotic systems, 10 mL-50 mL of bacterial-algae mixtures were pipetted and filtered through a 0.45 μm fiber membrane at a pressure of 0.05 MPa-0.2 MPa using a vacuum pump. The filtered fiber membrane, which retained the bacterial-algae mixture, was then divided into 3 cm sections. 2 -5cm 2 The sample.

[0043] Ultrasonic cavitation treatment: At a temperature below 4°C, place each sample in a 5mL centrifuge tube and add 4mL of ethanol solution (95wt.%) to the centrifuge tube. Then, oscillate the sample at an ultrasonic frequency of 30kHz-60kHz for 0.5min-3min.

[0044] High-speed centrifugation: At a temperature below 4°C, place the centrifuge tubes after ultrasonic cavitation treatment in a centrifuge and centrifuge for 10-20 minutes under the condition of RCF of 4500xg-20000xg. Depending on the centrifugation effect, repeat the centrifugation treatment 1-3 times. Take out the supernatant of all centrifuge tubes, mix them evenly and set them aside for later use.

[0045] The above-mentioned control of centrifugal force is based on the relative centrifugal force and the centrifuge rotor diameter. The centrifuge speed is determined with reference to Formula I, which is as follows:

[0046] RCF = 1.18 × 10 -5 × r × (rpm) 2 ;

[0047] In the formula, RCF is the relative centrifugal force (xg), r is the rotor radius (cm), and rpm is the centrifuge speed (revolutions / s).

[0048] A method for detecting chlorophyll a in algae in a complex environment involves separating and extracting chlorophyll a from algae in a complex environment using the aforementioned method for separating chlorophyll a in algae in a complex environment. The homogeneous supernatant obtained after high-speed centrifugation is reconstituted with 95 wt.% ethanol solution to obtain the sample. The absorbance at 630 nm, 647 nm, 664 nm, and 750 nm is then measured using a spectrophotometer. The chlorophyll a content in the sample is determined according to Formula II, as follows:

[0049] Chl-a=[11.85×(D664-D750)-1.54×(D647-D750)-0.08×(D630-D750)]VE / (Vs×δ);

[0050] In the formula: VE is the final volume of the extract in the centrifuge tube, mL; Vs is the volume of the water sample, L; δ is the optical path of the cuvette, cm; D630, D647, D664, and D750 are the absorbance values ​​of the sample at wavelengths of 630 nm, 647 nm, 664 nm, and 750 nm, respectively.

[0051] like Figure 1As shown, this invention constructs a method for chlorophyll separation and detection in a fungal-algal symbiotic system under mixed environments, employing a "graded dissociation-synergistic cell disruption-spectrophotometer detection" process. This invention can control the separation and detection time to within 2.5 hours. Based on the differences in the growth morphology of the fungal-algal symbiotic system, this invention classifies it into "suspension type" and "attached type." Based on the differences in their microstructure and algal attachment growth, different pretreatment separation methods are used for each type, avoiding the defects such as reduced extraction rate or over-separation caused by simply using a uniform separation method without considering sample characteristics. In the pretreatment process of the suspended algae-bacteria system, this invention, by rationally controlling the dosage of EDTA, allows it to react with calcium and magnesium ions in EPS, weakening the structural strength of the EPS encapsulating the algae and increasing the negative charge on the surface of the algae and bacteria. This weakens the structural connections and mechanical strength between the algae and bacteria, improving chlorophyll separation efficiency and shortening the separation reaction time without damaging the algae and chlorophyll. This provides a foundation for reducing the mechanical strength and time of separation and improving the separation effect in subsequent operations. In the pretreatment process of the attached algae-bacteria system, the attached algae-bacteria symbiotic system contains higher protein content and a denser physical structure. By penetrating the packing material and membrane module pores with a mild dilute alkaline solution, it can selectively induce EPS protein denaturation and increase the repulsion of negative charges on the surface of the algae and bacteria, thereby effectively weakening the adhesion between the algae and bacteria and the carrier, achieving the dissociation of the internal biofilm and EPS encapsulation, and achieving the gentle release of algae. The alkaline solution with a pH of 8-9 is relatively mild, effectively promoting dissociation without eroding or damaging chlorophyll and cell structure, forming a mild dissociation method for extracellular polymers suitable for the non-biological carrier-biofilm composite interface.

[0052] This invention presents a complete chlorophyll separation and extraction technology for algae symbiotic systems, combining chemical dissociation, physical separation, and organic extraction. The process involves sequentially using EDTA to chemically dissociate EPS, loosening the extracellular polymeric substances (EPS) encapsulating the algae; then, using non-toxic ethanol as the extraction solvent, multiple short-duration ultrasonic treatments and high-speed centrifugation are employed to break down the cell walls and extract chlorophyll from the algae. Each treatment stage has minimal impact on chlorophyll production, the entire detection process is short, and the reaction temperature is low, reducing the risk of chlorophyll oxidation and denaturation and improving the accuracy of detection results. Furthermore, by controlling the centrifugal force rather than the centrifuge speed, the centrifugation process parameters are controlled, eliminating differences in centrifugation results at the same speed caused by variations in centrifuge specifications.

[0053] Example 1

[0054] A method for separating chlorophyll a from algae in a complex algal symbiotic system, used in a suspended algal symbiotic system formed by co-cultivation of activated sludge and microalgae, wherein the algae and microorganisms in the system are suspended in water in the form of flocs or small granular sludge, including the following steps:

[0055] Pretreatment of the suspended algae-bacterial symbiotic system: During the system stirring stage, 100 mL of uniformly mixed algae-bacterial solution was extracted from the reactor and placed into a clean beaker, and C was added. 10 H 14 N2O8Na2·2H2O was prepared to a concentration of 5mM. The mixture was gently shaken to weaken the structural strength of the bacterial and algal EPS. The reaction time was controlled at 45min, the temperature at 20℃, and the pH at 8.

[0056] Filtration separation of bacterial-algae mixture: 30 mL of pretreated bacterial-algae mixture was pipetted and filtered through a 0.45 μm pore size and 5 cm diameter fiber membrane using a vacuum pump at 0.1 MPa pressure, trapping the bacterial-algae mixture on the membrane surface. The filtered fiber membrane containing the bacterial-algae mixture was then evenly divided into four portions, each with a sample area of ​​4.9 cm². 2 The sample.

[0057] Ultrasonic cavitation treatment: At 4℃, each filter membrane sample was placed in a 5mL centrifuge tube, and 4mL of ethanol solution (95wt.%) was added to the centrifuge tube. The centrifuge tube was then placed in an ultrasonic oscillator and oscillated at an ultrasonic frequency of 40kHz for 0.9min.

[0058] High-speed centrifugation: At 4℃, centrifuge tubes treated with ultrasonic cavitation were placed in a centrifuge. Based on this, microalgae and activated sludge in the sample aggregated into hard, small particles. The RCF was set to 10000 x g. According to Formula I: RCF = 1.18 × 10⁻⁶ -5 × r × (rpm) 2 The centrifuge rotor has a rotation radius of r=6.5cm and a theoretical speed of 11418rpm. Therefore, the centrifuge speed is set to 12000rpm and centrifuged for 10min. Based on the centrifugation effect, the centrifugation process is repeated twice. The supernatants from the two centrifugations are combined for later use to ensure that chlorophyll is fully extracted.

[0059] A method for detecting chlorophyll a in algae in a complex algal symbiotic system is disclosed. The chlorophyll is separated and extracted using the separation method described in Example 1. The homogeneous supernatant obtained after high-speed centrifugation is reconstituted with 95 wt.% ethanol solution to a final volume of 20 ml as the sample to be tested. The absorbance at 630 nm, 647 nm, 664 nm, and 750 nm is then measured using a spectrophotometer. The chlorophyll a content in the sample is determined according to Formula II, which is as follows:

[0060] Chl-a=[11.85×(D664-D750)-1.54×(D647-D750)-0.08×(D630-D750)]VE / (Vs×δ);

[0061] In the formula: VE is the final volume of the extract in the centrifuge tube, 20 mL; Vs is the volume of the water sample, 0.03 L; δ is the optical path length of the cuvette, 1 cm;

[0062] The suspended bacterial-algae symbiotic system of Example 1 was sampled 20 times continuously, and the above separation and detection methods were used. Simultaneously, parallel detection was performed with high performance liquid chromatography (HPLC). The absorbance values ​​and detection results are shown in Table 1.

[0063] Table 1. Absorbance values ​​and detection results of each sample in Example 1;

[0064] .

[0065] According to the tests and calculations, the average concentration of chlorophyll a detected by spectrophotometry was 5.91 mg / L, and the average concentration of chlorophyll a detected by high performance liquid chromatography (HPLC) was 6.07 mg / L.

[0066] Bland-Altman and linear regression analyses were performed on the above data, and the results are as follows:

[0067] according to Figure 2 As shown in the Bland-Altman plot, analysis reveals that the average difference between the two methods is -0.152 mg / L (95% CI: -0.162 mg / L to -0.143 mg / L), indicating a stable negative systematic bias in the spectrophotometric method of this invention compared to the HPLC control method. Specifically, the average measured values ​​using this method are lower than those using the HPLC method. This bias is due to inherent characteristics of the method and is not a random error; Method-HPMC exhibits a stable negative systematic bias. The consensus threshold (LoA) is -0.194 mg / L to -0.111 mg / L, and 19 out of 20 samples (95%) of the differences fall within this threshold; the correlation between the two methods is high (R = 0.963). Figure 3 As shown in the scatter plot, the linear regression equation is y = 0.965x + 0.059 (R²). 2 =0.928), further confirming the closeness of the values, with a slope slightly less than 1; indicating that after the suspended bacterial-algal symbiotic sample is dissociated and broken by the method of this invention, the chlorophyll a result detected by spectrophotometer is basically consistent with the result detected by HPLC. Moreover, the total detection time of this method, including sampling, pipetting, and volume adjustment, is 2 hours, which is much shorter than that of HPLC, and the data timeliness is stronger.

[0068] Example 2

[0069] A method for separating chlorophyll a from algae in a complex algal symbiotic system, comprising an attached algal symbiotic system in which microalgae attach and grow on a soft packing material, wherein algae and microorganisms attach and grow on the surface of the packing material, including the following steps:

[0070] Pretreatment of the attached algae-bacterial symbiotic system: Select a soft packing material with uniform biomass attachment within the reactor, gently squeeze to remove excess water. The soft packing material should be approximately 2cm × 2cm × 2cm in size. Divide it into 8 equal portions, each with a sample volume of 1cm³. 3 The sample was placed in 100 mL of dilute alkaline solution at 20°C and pH 8.5 and gently rinsed six times to soften the attached structure and preliminarily elute the bacterial-algae mixture. The mixture, containing packing debris and preliminarily eluted bacteria and algae, was then transferred and aliquoted into centrifuge tubes. The centrifuge tubes were set to an RCF of 800 x g, and calculated using Formula I: RCF = 1.18 × 10⁻⁶ g. -5 × r × (rpm) 2 The centrifuge rotor's rotation radius r = 6.5 cm, and the theoretical rotation speed is 3230 rpm. Therefore, the centrifuge speed was set to 4000 rpm, and centrifugation was performed for 10 minutes. Eight portions of the supernatant from the bacterial-algae mixture were collected, and the volume was adjusted to 100 mL with pure water. Then, C was added. 10 H 14 Prepare a solution of N2O8Na2·2H2O to a concentration of 3mM, mix by gentle shaking, control the reaction time to 30min, the temperature to 25℃, and the pH value to 8.

[0071] Filtration separation of bacterial-algae mixture: 30 mL of pretreated bacterial-algae mixture was pipetted and filtered through a 0.45 μm pore size and 5 cm diameter fiber membrane using a vacuum pump at 0.1 MPa pressure, trapping the bacterial-algae mixture on the membrane surface. The filtered fiber membrane containing the bacterial-algae mixture was then evenly divided into four portions, each with a sample area of ​​4.9 cm². 2 The sample.

[0072] Ultrasonic cavitation treatment: At 4℃, each filter membrane sample was placed in a 5mL centrifuge tube, and 4mL of ethanol solution (95wt.%) was added to the centrifuge tube. The centrifuge tube was then placed in an ultrasonic oscillator and oscillated at an ultrasonic frequency of 40kHz for 0.9min.

[0073] High-speed centrifugation: At 4℃, centrifuge tubes treated with ultrasonic cavitation were placed in a centrifuge. Based on this, microalgae and activated sludge in the sample aggregated into hard, small particles. The RCF was set to 14000 x g. According to Formula I: RCF = 1.18 × 10⁻⁶ -5 × r × (rpm) 2 The centrifuge rotor has a rotation radius of r = 6.5 cm and a theoretical rotation speed of 13510 rpm. Therefore, the centrifuge speed is set to 14000 rpm, and the centrifugation time is 15 min. The supernatant from all centrifuge tubes is then combined for later use.

[0074] A method for detecting chlorophyll a in algae in a complex algal symbiotic system is disclosed. The chlorophyll is separated and extracted using the separation method described in Example 2. The homogeneous supernatant obtained after high-speed centrifugation is reconstituted with 95 wt.% ethanol solution to a final volume of 12 ml as the sample to be tested. The absorbance at 630 nm, 647 nm, 664 nm, and 750 nm is then measured using a spectrophotometer. The chlorophyll a content in the sample is determined according to Formula II, which is as follows:

[0075] Chl-a=[11.85×(D664-D750)-1.54×(D647-D750)-0.08×(D630-D750)]VE / (Vs×δ);

[0076] In the formula: VE is the final volume of the extract in the centrifuge tube, 12 mL; Vs is the volume of the water sample, 0.03 L; δ is the optical path length of the cuvette, 1 cm;

[0077] The attached algal symbiotic system of Example 2 was sampled 20 times continuously, and the above separation and detection methods were used. Simultaneously, parallel detection was performed with high performance liquid chromatography (HPLC). The absorbance values ​​and detection results are shown in Table 2.

[0078] Table 2. Absorbance values ​​and detection results of each sample in Example 2;

[0079] .

[0080] According to the test results, the average concentration of chlorophyll a detected by spectrophotometry was 3.38 mg / L, and the average concentration of chlorophyll a detected by high performance liquid chromatography (HPLC) was 3.51 mg / L.

[0081] Bland-Altman and linear regression analyses were performed on the above data, and the results are as follows:

[0082] according to Figure 4 As shown in the Bland-Altman plot, analysis revealed an average difference of -0.135 mg / L between the two methods (95% CI: -0.138 mg / L to -0.131 mg / L), indicating a stable negative systematic bias in the spectrophotometric method compared to the HPLC method. The agreement limit (LoA) was -0.151 mg / L to -0.118 mg / L, with the differences in 20 samples (100%) falling within this limit; the correlation between the two methods was extremely high (R = 0.992). Figure 5 As shown in the scatter plot, the linear regression equation is y = 0.994x + 0.113 (R²). 2=0.984), indicating that after the attached bacterial-algal symbiotic sample was dissociated and broken by the method of the present invention, the chlorophyll a result detected by spectrophotometer was basically consistent with the result detected by HPLC. Moreover, the total detection time of the method of the present invention, including sampling, pipetting, and volume adjustment, is 2.5 hours, which is much shorter than that of HPLC, and the data timeliness is stronger.

[0083] Example 3

[0084] A method for separating chlorophyll a from algae in a complex algal symbiotic system, used in a suspended algal symbiotic system formed by co-cultivation of activated sludge and microalgae, wherein the algae and microorganisms in the system are suspended in water in the form of flocs or small granular sludge, including the following steps:

[0085] Pretreatment of the suspended algae-bacterial symbiotic system: During the system stirring stage, 100 mL of uniformly mixed algae-bacterial solution was extracted from the reactor and placed into a clean beaker, and C was added. 10 H 14 N2O8Na2·2H2O was prepared to a concentration of 1mM. The mixture was gently shaken to weaken the structural strength of the bacterial and algal EPS. The reaction time was controlled at 30 min, the temperature at 15℃, and the pH at 6.

[0086] Filtration separation of bacterial-algae mixture: 10 mL of the pretreated bacterial-algae mixture was pipetted and filtered through a 0.45 μm pore size and 5 cm diameter fiber membrane using a vacuum pump at a pressure of 0.05 MPa, so that the bacterial-algae mixture was retained on the membrane surface. The filtered fiber membrane containing the bacterial-algae mixture was then evenly divided into 4 portions, each with a sample area of ​​3 cm². 2 The sample.

[0087] Ultrasonic cavitation treatment: At 4℃, each filter membrane sample was placed in a 5mL centrifuge tube, and 4mL of ethanol solution (95wt.%) was added to the centrifuge tube. The centrifuge tube was then placed in an ultrasonic oscillator and oscillated at an ultrasonic frequency of 30kHz for 0.5min.

[0088] High-speed centrifugation: At 4℃, centrifuge tubes treated with ultrasonic cavitation were placed in a centrifuge. Based on this, microalgae and activated sludge in the sample aggregated into hard, small particles. The RCF was set to 4500 x g. According to Formula I: RCF = 1.18 × 10⁻⁶ -5 × r × (rpm) 2 The centrifuge rotor has a rotation radius of r=6.5cm and a theoretical speed of 7660rpm. Therefore, the centrifuge speed is set to 8000rpm and centrifuged for 10min. Based on the centrifugation effect, the centrifugation process is repeated twice. The supernatants from the two centrifugations are combined for later use to ensure that chlorophyll is fully extracted.

[0089] A method for detecting chlorophyll a in algae in a complex algal symbiotic system is disclosed. The chlorophyll is separated and extracted using the separation method described in Example 3. The homogeneous supernatant obtained after high-speed centrifugation is reconstituted with 95 wt.% ethanol solution to a final volume of 10 ml as the sample to be tested. The absorbance at 630 nm, 647 nm, 664 nm, and 750 nm is then measured using a spectrophotometer. The chlorophyll a content in the sample is determined according to Formula II, which is as follows:

[0090] Chl-a=[11.85×(D664-D750)-1.54×(D647-D750)-0.08×(D630-D750)]VE / (Vs×δ);

[0091] In the formula: VE is the final volume of the extract in the centrifuge tube, 10 mL; Vs is the volume of the water sample, 0.01 L; δ is the optical path length of the cuvette, 1 cm;

[0092] The suspended bacterial-algae symbiotic system of Example 3 was sampled 20 times continuously, and the above separation and detection methods were used. Simultaneously, parallel detection was performed with high performance liquid chromatography (HPLC). The absorbance values ​​and detection results are shown in Table 3.

[0093] Table 3. Absorbance values ​​and detection results of each sample in Example 3;

[0094] .

[0095] According to the tests and calculations, the average concentration of chlorophyll a detected by spectrophotometry was 8.47 mg / L, and the average concentration of chlorophyll a detected by high performance liquid chromatography (HPLC) was 8.34 mg / L.

[0096] Example 4

[0097] A method for separating chlorophyll a from algae in a complex algal symbiotic system, comprising an attached algal symbiotic system in which microalgae attach and grow on a soft packing material, wherein algae and microorganisms attach and grow on the surface of the packing material, including the following steps:

[0098] Pretreatment of the attached algae-bacterial symbiotic system: Select a soft packing material with uniform biomass attachment within the reactor, gently squeeze to remove excess water. The soft packing material should be approximately 1cm × 2cm × 2cm in size. Divide it into 8 equal portions, each with a sample volume of 0.5cm³. 3 The sample was placed in 100 mL of dilute alkaline solution at 20°C and pH 8, and gently rinsed five times to soften the attached structure and preliminarily elute the bacterial-algae mixture. The mixture, containing packing debris and preliminarily eluted bacteria and algae, was then transferred and aliquoted into centrifuge tubes. The centrifuge tubes were set to an RCF of 300 x g, and calculated using Formula I: RCF = 1.18 × 10⁻⁶ g. -5× r × (rpm) 2 The centrifuge rotor's rotation radius r = 6.5 cm, and the theoretical rotation speed is 1798 rpm. Therefore, the centrifuge speed was set to 2000 rpm, and centrifugation was performed for 5 minutes. Eight portions of the supernatant from the bacterial-algae mixture were collected, and the volume was adjusted to 100 mL with pure water. Then, C was added. 10 H 14 Prepare a solution of N2O8Na2·2H2O to a concentration of 1 mM, mix by gentle shaking, control the reaction time to 15 min, the temperature to 15℃, and the pH value to 6.

[0099] Filtration separation of bacterial-algae mixture: 10 mL of the pretreated bacterial-algae mixture was pipetted and filtered through a 0.45 μm pore size and 5 cm diameter fiber membrane using a vacuum pump at a pressure of 0.05 MPa, so that the bacterial-algae mixture was retained on the membrane surface. The filtered fiber membrane containing the bacterial-algae mixture was then evenly divided into 4 portions, each with a sample area of ​​3 cm². 2 The sample.

[0100] Ultrasonic cavitation treatment: At 4℃, each filter membrane sample was placed in a 5mL centrifuge tube, and 4mL of ethanol solution (95wt.%) was added to the centrifuge tube. The centrifuge tube was then placed in an ultrasonic oscillator and oscillated at an ultrasonic frequency of 30kHz for 0.5min.

[0101] High-speed centrifugation: At 4℃, centrifuge tubes treated with ultrasonic cavitation were placed in a centrifuge. Based on this, microalgae and activated sludge in the sample aggregated into hard, small particles. The RCF was set to 4500 x g. According to Formula I: RCF = 1.18 × 10⁻⁶ -5 × r × (rpm) 2 The centrifuge rotor has a rotation radius of r = 6.5 cm and a theoretical rotation speed of 7660 rpm. Therefore, the centrifuge speed is set to 8000 rpm, and the centrifugation time is 10 min. The supernatant from all centrifuge tubes is then combined for later use.

[0102] A method for detecting chlorophyll a in algae in a complex algal symbiotic system is disclosed. The chlorophyll is separated and extracted using the separation method described in Example 4. The homogeneous supernatant obtained after high-speed centrifugation is reconstituted with 95 wt.% ethanol solution to a final volume of 10 ml as the sample to be tested. The absorbance at 630 nm, 647 nm, 664 nm, and 750 nm is then measured using a spectrophotometer. The chlorophyll a content in the sample is determined according to Formula II, which is as follows:

[0103] Chl-a=[11.85×(D664-D750)-1.54×(D647-D750)-0.08×(D630-D750)]VE / (Vs×δ);

[0104] In the formula: VE is the final volume of the extract in the centrifuge tube, 10 mL; Vs is the volume of the water sample, 0.01 L; δ is the optical path length of the cuvette, 1 cm;

[0105] The attached algae-bacteria symbiotic system of Example 4 was sampled 20 times continuously. The above separation and detection methods were used, and the detection was performed in parallel with high performance liquid chromatography (HPLC). The absorbance values ​​and detection results are shown in Table 4.

[0106] Table 4. Absorbance values ​​and detection results of each sample in Example 4;

[0107] .

[0108] According to the tests and calculations, the average concentration of chlorophyll a detected by spectrophotometry was 6.95 mg / L, and the average concentration of chlorophyll a detected by high performance liquid chromatography (HPLC) was 6.81 mg / L.

[0109] Example 5

[0110] A method for separating chlorophyll a from algae in a complex algal symbiotic system, used in a suspended algal symbiotic system formed by co-cultivation of activated sludge and microalgae, wherein the algae and microorganisms in the system are suspended in water in the form of flocs or small granular sludge, including the following steps:

[0111] Pretreatment of the suspended algae-bacterial symbiotic system: During the system stirring stage, 100 mL of uniformly mixed algae-bacterial solution was extracted from the reactor and placed into a clean beaker, and C was added. 10 H 14 N2O8Na2·2H2O was prepared to a concentration of 10mM. The mixture was gently shaken to weaken the structural strength of the bacterial and algal EPS. The reaction time was controlled at 60min, the temperature at 25℃, and the pH at 9.

[0112] Filtration separation of bacterial-algae mixture: 50 mL of the pretreated bacterial-algae mixture was pipetted and filtered through a 0.45 μm pore size and 5 cm diameter fiber membrane using a vacuum pump at 0.2 MPa pressure, so that the bacterial-algae mixture was retained on the membrane surface. The filtered fiber membrane containing the bacterial-algae mixture was then evenly divided into 4 portions, each with a sample area of ​​5 cm². 2 The sample.

[0113] Ultrasonic cavitation treatment: At 4℃, each filter membrane sample was placed in a 5mL centrifuge tube, and 4mL of ethanol solution (95wt.%) was added to the centrifuge tube. The centrifuge tube was then placed in an ultrasonic oscillator and oscillated at an ultrasonic frequency of 60kHz for 3min.

[0114] High-speed centrifugation: At 4℃, centrifuge tubes treated with ultrasonic cavitation were placed in a centrifuge. Based on this, microalgae and microbial activated sludge in the sample aggregated into hard, small particles. The RCF was set to 20000 x g. According to Formula I: RCF = 1.18 × 10⁻⁶ -5 × r × (rpm) 2 The centrifuge rotor has a rotation radius of r=6.5cm and a theoretical speed of 16148rpm. Therefore, the centrifuge speed is set to 17000rpm and centrifuged for 20min. Based on the centrifugation effect, the centrifugation process is repeated twice. The supernatants from the two centrifugations are combined for later use to ensure that chlorophyll is fully extracted.

[0115] A method for detecting chlorophyll a in algae in a complex algal symbiotic system is disclosed. The chlorophyll is separated and extracted using the separation method described in Example 5. The homogeneous supernatant obtained after high-speed centrifugation is reconstituted with 95 wt.% ethanol solution to a final volume of 15 ml as the sample to be tested. The absorbance at 630 nm, 647 nm, 664 nm, and 750 nm is then measured using a spectrophotometer. The chlorophyll a content in the sample is determined according to Formula II, which is as follows:

[0116] Chl-a=[11.85×(D664-D750)-1.54×(D647-D750)-0.08×(D630-D750)]VE / (Vs×δ);

[0117] In the formula: VE is the final volume of the extract in the centrifuge tube, 15 mL; Vs is the volume of the water sample, 0.05 L; δ is the optical path length of the cuvette, 1 cm;

[0118] The suspended bacterial-algae symbiotic system of Example 5 was sampled 20 times continuously, and the above separation and detection methods were used. Simultaneously, parallel detection was performed with high performance liquid chromatography (HPLC). The absorbance values ​​and detection results are shown in Table 5.

[0119] Table 5. Absorbance values ​​and detection results of each sample in Example 5;

[0120] .

[0121] According to the test results, the average concentration of chlorophyll a detected by spectrophotometry was 2.65 mg / L, and the average concentration of chlorophyll a detected by high performance liquid chromatography (HPLC) was 2.60 mg / L.

[0122] Example 6

[0123] A method for separating chlorophyll a from algae in a complex algal symbiotic system, comprising an attached algal symbiotic system in which microalgae attach and grow on a soft packing material, wherein algae and microorganisms attach and grow on the surface of the packing material, including the following steps:

[0124] Pretreatment of the attached algae-bacterial symbiotic system: Select a soft packing material with uniform biomass attachment within the reactor, gently squeeze to remove excess water. The soft packing material should be approximately 2cm × 2cm × 2cm in size. Divide it into 8 equal portions, each with a sample volume of 1cm³. 3 The sample was placed in 100 mL of dilute alkaline solution at 25°C and pH 9, and gently rinsed 8 times to soften the attached structure and preliminarily elute the bacterial-algae mixture. The mixture, containing packing debris and preliminarily eluted bacteria and algae, was then transferred and aliquoted into centrifuge tubes. The centrifuge tubes were set to an RCF of 1000 x g, and calculated using Formula I: RCF = 1.18 × 10⁻⁶ g. -5 × r × (rpm) 2 The centrifuge rotor's rotation radius r = 6.5 cm, and the theoretical rotation speed is 3611 rpm. Therefore, the centrifuge speed was set to 4000 rpm, and centrifugation was performed for 15 minutes. Eight portions of the supernatant from the bacterial-algae mixture were collected, and the volume was adjusted to 100 mL with pure water. Then, C was added. 10 H 14 Prepare a solution of N2O8Na2·2H2O to a concentration of 5 mM, mix by gentle shaking, control the reaction time to 45 min, the temperature to 25℃, and the pH value to 9.

[0125] Filtration separation of bacterial-algae mixture: 50 mL of the pretreated bacterial-algae mixture was pipetted and filtered through a 0.45 μm pore size and 5 cm diameter fiber membrane using a vacuum pump at 0.2 MPa pressure, so that the bacterial-algae mixture was retained on the membrane surface. The filtered fiber membrane containing the bacterial-algae mixture was then evenly divided into 4 portions, each with a sample area of ​​5 cm². 2 The sample.

[0126] Ultrasonic cavitation treatment: At 4℃, each filter membrane sample was placed in a 5mL centrifuge tube, and 4mL of ethanol solution (95wt.%) was added to the centrifuge tube. The centrifuge tube was then placed in an ultrasonic oscillator and oscillated at an ultrasonic frequency of 60kHz for 3min.

[0127] High-speed centrifugation: At 4℃, centrifuge tubes treated with ultrasonic cavitation were placed in a centrifuge. Based on this, microalgae and microbial activated sludge in the sample aggregated into hard, small particles. The RCF was set to 20000 x g. According to Formula I: RCF = 1.18 × 10⁻⁶ -5 × r × (rpm) 2 The centrifuge rotor has a rotation radius of r = 6.5 cm and a theoretical rotation speed of 16148 rpm. Therefore, the centrifuge speed is set to 17000 rpm, and the centrifugation time is 20 min. The supernatant from all centrifuge tubes is then combined for later use.

[0128] A method for detecting chlorophyll a in algae in a complex algal symbiotic system is disclosed. The chlorophyll is separated and extracted using the method described in Example 6. The homogeneous supernatant obtained after high-speed centrifugation is reconstituted with 95 wt.% ethanol solution to a final volume of 15 ml as the sample to be tested. The absorbance at 630 nm, 647 nm, 664 nm, and 750 nm is then measured using a spectrophotometer. The chlorophyll a content in the sample is determined according to Formula II, which is as follows:

[0129] Chl-a=[11.85×(D664-D750)-1.54×(D647-D750)-0.08×(D630-D750)]VE / (Vs×δ);

[0130] In the formula: VE is the final volume of the extract in the centrifuge tube, 15 mL; Vs is the volume of the water sample, 0.05 L; δ is the optical path length of the cuvette, 1 cm;

[0131] The attached algae-bacteria symbiotic system of Example 6 was sampled 20 times continuously. The above separation and detection methods were used, and the detection was performed in parallel with high performance liquid chromatography (HPLC). The absorbance values ​​and detection results are shown in Table 6.

[0132] Table 6. Absorbance values ​​and detection results of each sample in Example 6;

[0133] .

[0134] According to the test results, the average concentration of chlorophyll a detected by spectrophotometry was 2.58 mg / L, and the average concentration of chlorophyll a detected by high performance liquid chromatography (HPLC) was 2.53 mg / L.

[0135] Comparative Example 1

[0136] According to the "Water Quality - Determination of Chlorophyll a - Spectrophotometry" (HJ 897-2017), 20 parallel tests were performed on the suspended bacterial-algae symbiotic system sample formed by the co-cultivation of activated sludge and microalgae in Example 1. The average concentration was 3.55 mg / L, which was significantly lower than the chlorophyll a concentration after graded dissociation and synergistic cell disruption by the present invention. This indicates that the separation method of the present invention can effectively improve the separation and extraction effect of chlorophyll a in the suspended bacterial-algae symbiotic system.

[0137] Comparative Example 2

[0138] According to the "Water Quality - Determination of Chlorophyll a - Spectrophotometry" (HJ 897-2017), 20 parallel tests were performed on the samples of the attached algae-bacterial symbiotic system in Example 2, in which microalgae attached to and growing on soft packing material. The average concentration was 2.15 mg / L, which was significantly lower than the concentration of chlorophyll a after graded dissociation and synergistic cell disruption by the present invention. This indicates that the separation method of the present invention can effectively improve the separation and extraction effect of chlorophyll a in the attached algae-bacterial symbiotic system.

[0139] As demonstrated by the above examples and comparative examples, the detection results of this invention in the separation and detection of chlorophyll a in suspended and attached algal symbiotic systems are basically consistent with those of the HPLC method, but the separation and detection time is shorter, within 2.5 hours, which is much faster than the HPLC method. The cost of detection instruments and reagents is much lower than that of the HPLC method. While ensuring data quality, it significantly improves detection efficiency and is more suitable for rapid monitoring and evaluation of algal systems in practical engineering. Compared with the method in "Water Quality - Determination of Chlorophyll a - Spectrophotometry" (HJ 897-2017) which uses acetone as the extractant, the separation method of this invention has a better fragmentation effect and uses 95wt.% ethanol as the extractant, which is non-toxic and lower in cost. This invention effectively solves the problems of low chlorophyll extraction rate, poor repeatability, and cumbersome operation in complex algal systems through key technologies such as classification pretreatment, EDTA-assisted ultrasonic cell disruption, and standardized control of centrifugal force. It is an accurate, rapid, safe, and economical method for chlorophyll detection.

Claims

1. A method for separating chlorophyll a from algae in a complex algal symbiotic system, characterized in that, Includes the following steps: Classification of bacterial-algal symbiotic systems: Based on the differences in the growth morphology of bacteria and algae in bacterial-algal symbiotic systems, they are divided into two categories: suspended and attached. Pretreatment is carried out according to the classification of bacterial-algae symbiotic systems: the pretreatment of suspended bacterial-algae symbiotic systems involves extracting a uniformly mixed bacterial-algae mixture from the reactor during the system stirring or aeration stage, adding ethylenediaminetetraacetic acid (EDTA), and gently shaking to mix and react. The pretreatment of the attached bacterial-algae symbiotic system involves draining the water from the packing material and membrane module, cutting them into small pieces, and rinsing them repeatedly in a dilute alkaline solution. Then, the mixture containing the inorganic packing material and bacterial-algae mixture is packaged and centrifuged. The separated bacterial-algae mixture is then diluted with pure water, and ethylenediaminetetraacetic acid is added and gently shaken to mix. Filtration and separation of bacterial-algae mixture: After pretreatment according to the classification of bacterial-algae symbiotic system, a certain amount of bacterial-algae mixture is transferred and filtered through a fiber membrane. The fiber membrane that retains the bacterial-algae mixture after filtration is divided into samples of the target area. Ultrasonic cavitation treatment: Place each sample in a centrifuge tube, add ethanol solution, control the ultrasonic frequency, and perform oscillation treatment; High-speed centrifugation: After ultrasonic cavitation treatment, the centrifuge tubes are placed in a centrifuge, and the RCF is controlled to perform centrifugation separation. The supernatant of all centrifuge tubes is taken out, mixed evenly, and then used for later use.

2. The method for separating algal chlorophyll a in a complex algal symbiotic system as described in claim 1, characterized in that, In the pretreatment of suspended algal symbiotic systems, which are classified according to the type of algal symbiotic system, the pretreatment of ethylenediaminetetraacetic acid (EDTA) is C. 10 H 14 N2O8Na2·2H2O, the concentration of ethylenediaminetetraacetic acid salt added is 1mM-10mM.

3. The method for separating algal chlorophyll a in a complex algal symbiotic system as described in claim 1, characterized in that, In the pretreatment of suspended bacterial-algae symbiotic systems, which are classified according to the type of bacterial-algae symbiotic system, the reaction time is controlled at 30-60 minutes, the temperature at 15-25℃, and the pH at 6-9.

4. The method for separating algal chlorophyll a in a bacterial-algal symbiotic system under complex conditions as described in claim 1, characterized in that, In the pretreatment of attached algal symbiotic systems, which are classified according to their type, the pretreatment involves uniformly cutting small pieces with a volume of 0.5 cm³. 3 -1cm 3 Control the temperature of the dilute alkaline solution to 20℃-25℃, the pH value to 8-9, and rinse repeatedly 5-8 times; control the relative centrifugal force to 300xg-1000xg, and the centrifugation time to 5min-15min; EDTA-1-acetate is C 10 H 14 N2O8Na2·2H2O, the concentration of ethylenediaminetetraacetic acid salt added is 1mM-5mM.

5. The method for separating algal chlorophyll a in a complex algal symbiotic system as described in claim 1, characterized in that, In the pretreatment of attached algal symbiotic systems, which are classified according to the type of algal symbiotic system, the reaction time is controlled at 15 min-45 min, the temperature at 15℃-25℃, and the pH value at 6-9.

6. The method for separating algal chlorophyll a in a bacterial-algal symbiotic system under complex conditions as described in claim 1, characterized in that, In the filtration separation of bacterial-algae mixture, the volume of bacterial-algae mixture drawn by pipette is 10mL-50mL; the pore size of the fiber membrane is controlled at 0.45μm with a diameter of 5cm; the filtration pressure is controlled at 0.05MPa-0.2MPa; and the target area of ​​the fiber membrane that retains the bacterial-algae mixture after filtration is 3cm². 2 -5cm 2 .

7. The method for separating algal chlorophyll a in a complex algal symbiotic system as described in claim 1, characterized in that, In the ultrasonic cavitation treatment, the effective volume of the centrifuge tube is 5 mL, the mass fraction of the ethanol solution is 95%, and the amount of ethanol solution added is 4 mL. The ultrasonic frequency is controlled at 30kHz-60kHz, the oscillation time is 0.5min-3min, and the reaction temperature is ≤4℃.

8. The method for separating algal chlorophyll a in a bacterial-algal symbiotic system under complex conditions as described in claim 1, characterized in that, During high-speed centrifugation, the centrifugation process is repeated 1 to 3 times depending on the centrifugation effect; the relative centrifugal force is controlled at 4500xg-20000xg, the centrifugation time is 10min-20min, and the reaction temperature is ≤4℃.

9. The method for separating algal chlorophyll a in a bacterial-algal symbiotic system under complex conditions as described in claim 1, characterized in that, In the pretreatment and high-speed centrifugation of the attached algae-bacterial symbiotic system, the centrifugal force is controlled based on the relative centrifugal force and the centrifuge rotor diameter. The centrifuge speed is determined with reference to Formula I, which is as follows: RCF = 1.18 × 10 -5 × r × (rpm) 2 ; In the formula, RCF is the relative centrifugal force (xg), r is the rotor radius (cm), and rpm is the centrifuge speed (revolutions / s).

10. A method for detecting chlorophyll a in algae in a complex algal symbiotic system, characterized in that, The chlorophyll a separation method for algal symbiotic systems under complex environments, as described in claim 1, was used to separate and extract chlorophyll. The homogeneous supernatant obtained after high-speed centrifugation was reconstituted with 95 wt.% ethanol solution to obtain the sample to be tested. The absorbance at 630 nm, 647 nm, 664 nm, and 750 nm was then measured using a spectrophotometer. The chlorophyll a content in the sample was determined according to Formula II, as follows: Chl-a=[11.85×(D664-D750)-1.54×(D647-D750)-0.08×(D630-D750)]VE / (Vs×δ); In the formula: VE is the final volume of the extract in the centrifuge tube, mL; Vs is the volume of the water sample, L; δ is the optical path of the cuvette, cm; D630, D647, D664, and D750 are the absorbance values ​​of the sample at wavelengths of 630 nm, 647 nm, 664 nm, and 750 nm, respectively.

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