In-situ detection method for methane production activity of anaerobic sludge based on front surface fluorescence

Abstract

The invention discloses an anaerobic sludge methanogenesis activity in-situ detection method based on front surface fluorescence, and belongs to the technical field of environmental engineering and water treatment monitoring. Firstly, a front surface cuvette with the width being only 2 mm is adopted, optical path matching is optimized, interference of high-turbidity sludge on scattering and absorption is reduced, and therefore the signal-to-noise ratio and stability in a complex sludge system are guaranteed. Secondly, anaerobic sludge cells are crushed through ultrasonic treatment before detection, intracellular coenzyme F420 is fully released, the signal intensity is enhanced, and the detection sensitivity is improved. And finally, the whole detection process is carried out in an anaerobic environment, so that the problem that anaerobic methanogens and enzyme systems thereof are inactivated due to the fact that traditional cuvette detection is exposed to air is solved. According to the method, the F420 fluorescence signal can be rapidly obtained without complex pretreatment, the detection period is less than 3 minutes, compared with a traditional SMA experiment, the detection period is greatly shortened, the detection result is more stable, the sensitivity is higher, and the method has a good engineering application prospect.

Description

Technical Field

[0001] This invention relates to the field of environmental engineering and water treatment monitoring technology, and in particular to an in-situ detection method for methanogenic activity in anaerobic sludge based on front surface fluorescence. Background Technology

[0002] Anaerobic biological treatment is an important pathway for wastewater resource recovery and energy recycling, with methanogenesis being a key step in the final conversion of organic matter into methane. The activity of methanogenic bacteria directly determines the stability of anaerobic reactors and the efficiency of methane recovery. However, the methanogenesis process is highly susceptible to fluctuations in influent load, substrate composition, temperature, pH, toxic pollutants, and operating conditions, leading to an imbalance between acidification and methanogenesis in the system. When methanogenesis activity is insufficient, reactor operation often experiences problems such as acid accumulation, rapid increase in transmembrane pressure, and decreased gas production, which can severely lead to system instability or even complete collapse. Therefore, how to accurately characterize and rapidly monitor the methanogenesis activity of anaerobic sludge has always been a key scientific and engineering problem in the fields of environmental engineering and energy recovery.

[0003] Currently, commonly used detection methods mainly include specific methanogenic activity (SMA) assays, gas production methods, and molecular biological assays. SMA assays require substrate addition in a sealed bottle and continuous monitoring of the gas production process, typically taking 3–7 days to complete. This long detection cycle makes it difficult to meet the real-time requirements of practical engineering operations. Gas production methods rely on cumulative methanogenesis to infer bacterial activity, exhibiting significant lag, often reflecting activity decline only after reactor performance has deteriorated, lacking predictive power. Molecular biological assays such as qPCR and metagenomic sequencing can reveal the abundance of functional genes, but the experimental cycle usually requires 24–48 hours and is costly, making high-frequency or online applications difficult. In contrast, coenzyme F420 fluorescence detection is considered a promising method for characterizing methanogenic activity due to its high specificity and rapid response. F420 is a characteristic coenzyme of methanogenic bacteria, producing a significant fluorescence signal under specific wavelength excitation, which is highly correlated with methanogenic performance and is therefore often used as a rapid detection indicator.

[0004] However, traditional F420 fluorescence detection methods still have significant shortcomings in engineering applications. The commonly used 10mm thick-walled cuvettes are prone to scattering and absorption interference in high-turbidity sludge systems, resulting in low signal-to-noise ratios and large fluctuations in detection data. Simultaneously, sludge samples are not effectively treated, making it difficult to fully release intracellular F420 coenzymes, leading to insufficient detection sensitivity and often underestimating results, failing to accurately reflect the actual activity of methanogenic bacteria. Furthermore, in traditional detection processes, samples are easily exposed to air during cuvette loading and measurement. Methanogenic bacteria, as strict anaerobic bacteria, readily inactivate their enzyme systems and coenzymes under aerobic conditions, resulting in distorted and unreproducible detection results. These problems limit the widespread application of F420 fluorescence detection in practical operational monitoring.

[0005] To address these issues, researchers attempted to employ front-face fluorescence optics (FEM) to reduce scattering and absorption interference, and improved signal quality through mathematical decomposition and correction. However, existing cuvette specifications are largely unsatisfactory for front-face fluorescence detection, failing to fully leverage the advantages of this method, lacking generalization predictive ability regarding methanogenesis activity under long-term operating conditions, and failing to establish a stable online detection and early warning system. Therefore, current technologies still have significant shortcomings in terms of real-time performance, accuracy, and applicability.

[0006] In view of this, the present invention proposes an improved method for detecting the methanogenic activity of front-surface fluorescent anaerobic sludge. Summary of the Invention

[0007] The purpose of this invention is to provide an in-situ detection method for methanogenic activity in anaerobic sludge based on front-surface fluorescence, overcoming three major bottlenecks in existing anaerobic process methanogenic activity detection: traditional methods have excessively long cycles, alternative solutions are costly and have time lags, and existing fluorescence methods suffer from insufficient stability and sensitivity due to signal interference and oxidative inactivation, thus failing to meet the requirements for rapid, in-situ, and engineering applications. This invention achieves rapid, stable, and highly sensitive characterization of methanogenic activity through the organic combination of cuvette structure optimization, ultrasonic disruption to release F420, and full-process anaerobic detection.

[0008] To achieve the above objectives, this invention discloses an in-situ detection method for methanogenic activity in anaerobic sludge based on front surface fluorescence, comprising the following steps: S1, Sample loading The sludge sample was filled into a fully enclosed front-surface cuvette. The cuvette has openings on only two sides for sample entry and exit, avoiding exposure of the sample to an aerobic environment. The optical path of the cuvette is 2 mm. Within this short optical path, the internal filtration effect can be significantly reduced, thus eliminating the need for additional internal standard correction.

[0009] S2, Ultrasonic disruption to release F420 enzyme Before testing, the sludge sample was subjected to low-power ultrasonic treatment to prevent the sludge from flocculating and causing uneven light path propagation, and to fully release intracellular coenzyme F420. S3, Front Surface Fluorescence Detection The sample was placed in the front surface fluorescence configuration sample stage, and the incident light and emitted light were set at 30° to keep the sludge in the anaerobic membrane bioreactor in a circulating state. The fluorescence spectrum signal was rapidly acquired and the fluorescence intensity was recorded within 3 minutes using the front surface fluorescence geometric optical path. The methanogenic activity of coenzyme F420 in the sludge was characterized by the fluorescence signal. After the test, the sludge was output from the cuvette and flowed back into the anaerobic membrane bioreactor.

[0010] Preferably, in S1, the cuvette has dimensions of 10mm × 2mm × 50mm.

[0011] Preferably, in S2, the low-power ultrasonic treatment of the sludge sample has an ultrasonic power of 2W and an action time of 1 minute.

[0012] Preferably, in S3, the front surface fluorescence configuration sample stage is placed in a three-dimensional fluorescence spectrometer, and both it and the anaerobic membrane bioreactor are placed in a completely anaerobic detection closed loop, which also includes an ultrasonic device. In the closed loop of the fully anaerobic detection, the sludge flow direction is: anaerobic membrane bioreactor - ultrasonic device - front surface fluorescent configuration sample stage - anaerobic membrane bioreactor.

[0013] Preferably, in the three-dimensional fluorescence spectrometer, the scanning speed is set to 30000 nm / min, the excitation wavelength is 250 nm-500 nm with 5 nm intervals, and the emission wavelength is 250 nm-550 nm with 1 nm intervals.

[0014] Preferably, after high-throughput sequencing results have confirmed that the dominant bacterial community is hydrogen-type methanogens, the fluorescence signal is used as an effective indicator to characterize the sludge's methane production performance and potential methanogenic activity.

[0015] Preferably, the closed loop of the fully anaerobic detection system is under nitrogen protection to prevent methanogens and their coenzyme F420 from being inactivated by air exposure.

[0016] The above method has been validated in the operation of the AnMBR anaerobic reactor, and it can identify the decline trend of methanogenic activity 3-5 days in advance, providing real-time early warning and control basis for engineering operation. Compared with traditional methods, the method provided by this invention shortens the detection cycle by about 95%, and significantly improves signal sensitivity and stability, making it more suitable for promotion and application in practical engineering, especially for the operation and control of processes such as anaerobic digestion, anaerobic membrane bioreactor (AnMBR), and upflow anaerobic sludge blanket (UASB).

[0017] Therefore, the present invention employs the above-mentioned in-situ detection method for methanogenic activity in anaerobic sludge based on front surface fluorescence, and the specific beneficial effects are as follows: 1. Improved Detection Speed ​​and Engineering Applicability: This invention, by employing a front-surface fluorescence configuration and an optimized short-path cuvette, achieves a single detection cycle of no more than 3 minutes, reducing the time by approximately 95% compared to traditional SMA experiments (3-7 days), and significantly improving efficiency compared to molecular detection methods. This near-real-time detection capability changes the previous passive situation of relying on long-cycle, offline experiments, meeting the urgent needs of engineering sites for high-frequency, rapid feedback, and providing technical support for real-time control and intelligent management of anaerobic processes.

[0018] 2. Significantly Enhanced Signal Sensitivity and Accuracy: This invention utilizes low-power ultrasonic pretreatment to effectively break down sludge flocs and cell walls, promoting the full release of intracellular coenzyme F420 into the liquid phase. Compared to untreated samples, the fluorescence signal intensity is increased by 30-50%, allowing previously masked activity information to be accurately captured. This enhancement significantly improves the correlation between the fluorescence signal and actual methanogenic activity, enabling the detection results to more accurately and sensitively reflect the transient microbial state and metabolic potential of the anaerobic system.

[0019] 3. Improved Stability and Result Authenticity: This invention solves the problem of result distortion caused by oxygen exposure in traditional methods by constructing a fully anaerobic detection environment. The use of a fully sealed cuvette and a closed loop purged with nitrogen ensures that the sample is isolated from air throughout the entire process from loading to measurement, fundamentally avoiding the oxidative inactivation of methanogens and their coenzyme F420. This not only effectively prevents signal attenuation but also ensures that the measured signal originates from a fully active microbial enzyme system, thereby greatly improving the authenticity, reliability, and repeatability of the detection data across different batches.

[0020] 4. Early Warning Capability and Cost Advantage: This invention combines excellent early warning capabilities with significant cost advantages. It can detect the trend of activity decline 3-5 days in advance, providing a critical early warning window for process control. Simultaneously, this method eliminates the need for complex reagents and expensive consumables, reducing manual operation and lowering the overall detection cost by 40-60% compared to traditional methods. This high efficiency and low cost, coupled with the ease of online retrofitting of the device, makes it suitable for widespread application in municipal wastewater treatment plants, industrial wastewater treatment, and energy recovery systems.

[0021] 5. Breakthrough in Overall System Performance: This invention is not merely an improvement on a single technology, but a comprehensive systemic solution integrating "optical path optimization, sample pretreatment, and environmental control." The innovative cuvette structure overcomes turbidity interference, ultrasonic treatment ensures signal strength, and the entire anaerobic process guarantees biological activity. These three elements work synergistically to overcome long-standing technical bottlenecks such as signal attenuation, insufficient sensitivity, and oxidative inactivation. Ultimately, this invention represents a substantial leap from laboratory methods to stable and reliable engineering applications, providing a novel technical approach for the stable operation, performance evaluation, and widespread adoption of anaerobic processes.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of the front surface fluorescent cuvette of the present invention; Figure 2 The above are comparison diagrams of the front surface fluorescence configuration sample stage and the conventional right-angle fluorescence configuration sample stage in the embodiments of the present invention. (a) is a schematic diagram of the front surface fluorescence configuration sample stage, and (b) is a schematic diagram of the conventional right-angle fluorescence configuration sample stage. Figure 3 Schematic diagram of a closed-loop device for detecting complete anaerobic conditions; Figure 4 F420 fluorescence score values ​​of the front surface fluorescence of cuvettes of different sizes; Figure 5 F420 fluorescence score values ​​were obtained for sludge under different ultrasonic powers. Figure 6The graph shows the F420 fluorescence score values ​​of the front surface fluorescence, where (a) shows the change of F420 fluorescence score values ​​over time under the anaerobic front surface fluorescence detection method; and (b) shows the F420 fluorescence score values ​​of sludge detected by the conventional method, front surface fluorescence method 1, and anaerobic front surface fluorescence method 2. Figure 7 This is a graph showing the correlation between F420 fluorescence score and methane production after 30 days of continuous operation on AnMBR. Detailed Implementation

[0025] This invention discloses an in-situ detection method for methanogenic activity in anaerobic sludge based on front surface fluorescence, comprising the following steps: S1, Sample loading The sludge sample was filled into a fully enclosed front-surface cuvette. The cuvette has openings on only two sides for sample entry and exit, avoiding exposure of the sample to an aerobic environment. The optical path of the cuvette is 2 mm. Within this short optical path, the internal filtration effect can be significantly reduced, thus eliminating the need for additional internal standard correction.

[0026] S2, Ultrasonic disruption to release F420 enzyme Before testing, the sludge samples were subjected to low-power ultrasonic treatment to prevent sludge flocculation and uneven light path propagation, and to fully release intracellular coenzyme F420.

[0027] S3, Front Surface Fluorescence Detection The sample was placed in the front surface fluorescence configuration sample stage, and the incident light and emitted light were set at 30° to keep the sludge in the anaerobic membrane bioreactor in a circulating state. The fluorescence spectrum signal was rapidly acquired and the fluorescence intensity was recorded within 3 minutes using the front surface fluorescence geometric optical path. The methanogenic activity of coenzyme F420 in the sludge was characterized by the fluorescence signal. After the test, the sludge was output from the cuvette and flowed back into the anaerobic membrane bioreactor.

[0028] In S1, the cuvette measures 10mm × 2mm × 50mm.

[0029] In S2, the sludge sample underwent low-power ultrasonic treatment with an ultrasonic power of 2W for 1 minute.

[0030] In S3, the front surface fluorescence configuration sample stage is placed in a three-dimensional fluorescence spectrometer, and both it and the anaerobic membrane bioreactor are placed in a completely anaerobic detection closed loop, which also includes an ultrasonic device. In the closed loop of the fully anaerobic detection, the sludge flow direction is: anaerobic membrane bioreactor - ultrasonic device - front surface fluorescent configuration sample stage - anaerobic membrane bioreactor.

[0031] In the three-dimensional fluorescence spectrometer, the scanning speed was set to 30,000 nm / min, the excitation wavelength was 250 nm-500 nm with 5 nm intervals, and the emission wavelength was 250 nm-550 nm with 1 nm intervals.

[0032] After high-throughput sequencing results revealed that the dominant bacterial group was hydrogen-type methanogens, fluorescence signals were used as an effective indicator to characterize the sludge's methane production performance and potential methanogenic activity.

[0033] The closed loop of the fully anaerobic detection system is under nitrogen protection to prevent methanogens and their coenzyme F420 from being inactivated by air exposure.

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0035] The present invention will be further described below through specific embodiments. However, it should be understood that these embodiments are only for more detailed description and should not be construed as limiting the present invention in any way, that is, not intended to limit the scope of protection of the present invention.

[0036] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0037] Example 1 This embodiment constructs a front surface fluorescence detection system, including: AnMBR reactor equipped with a heating device is used for sampling; Anaerobic serum bottles placed in a water bath ultrasonic cooker are used for sludge sample pretreatment and coenzyme F420 release. And a front surface fluorescence configuration sample stage placed in a three-dimensional fluorescence spectrometer for detecting coenzyme F420 signals.

[0038] The sample stage with the front surface fluorescence configuration contains a modified front surface cuvette, arranged as follows: Figure 2 As shown in (a), in the three-dimensional fluorescence spectrometer, the sample stage adopts a front-surface fluorescence configuration to fix and support the modified front-surface cuvette. The cuvette is positioned in the center of the sample stage, forming an angle of approximately 30° with the incident and emitted light, ensuring that the light can be reflected multiple times within a short optical path to enhance signal acquisition sensitivity. The cuvette is coupled to the spectrometer via a quartz fiber optic interface, achieving sealed optical transmission and preventing the entry of external air. Figure 2 In the middle (b), a conventional right-angle fluorescent sample stage is shown, with an angle of 90° between the incident light and the emitted light.

[0039] A schematic diagram of the improved front surface cuvette is shown below. Figure 1 As shown, to achieve a fully sealed interface design, openings for the inlet and outlet of the mixed liquor sludge are provided only on two sides, with the two openings located at different heights to ensure anaerobic conditions during the detection process. The cuvette measures 10 mm × 2 mm × 50 mm (length × width × height).

[0040] The above-mentioned device constitutes as follows Figure 3 The closed-loop detection system shown is connected as follows: A circulation pipeline is connected from the laboratory-scale AnMBR reactor to introduce the mixed liquor sludge into a 100 mL anaerobic serum bottle, which is sealed throughout and maintained in an anaerobic environment; the outlet of the anaerobic serum bottle is connected to a modified front-surface cuvette, which is connected to the AnMBR reactor via a pipeline. The sludge sample can be automatically returned after detection, forming a closed-loop detection system of "AnMBR → anaerobic serum bottle → front-surface cuvette → AnMBR", thus avoiding sample waste and air exposure.

[0041] In the three-dimensional fluorescence spectrometer, fluorescence detection is performed using a Shimadzu RF-6000 fluorescence spectrophotometer, equipped with a front surface optical path assembly, and coupled to the cuvette through a quartz fiber optic interface to ensure a sealed and leak-free detection process.

[0042] Example 2 This embodiment is based on a pre-surface fluorescence detection system provided in Example 1. The sludge sample is pretreated and coenzyme F420 is released. The entire process is completed in a sealed serum bottle. The top of the serum bottle is kept airtight by a butyl rubber stopper and a metal seal. Nitrogen gas is purged during pretreatment to ensure an anaerobic environment inside the bottle and to prevent air from entering and causing F420 signal inactivation. The entire detection circuit is strictly sealed. Before pretreatment, the entire detection circuit is purged with nitrogen gas to ensure an anaerobic environment.

[0043] The process is as follows: (1) After the AnMBR sludge flows into the 100 mL anaerobic serum bottle, it is immediately treated in an ultrasonic water bath. The ultrasonic conditions are 35℃, 2 W, and 1 minute.

[0044] (2) After treatment, the sludge flocs and some cells were broken, the release of intracellular F420 coenzyme increased significantly, the signal intensity increased by about 30-50%, and the sludge after ultrasonic treatment was easier to homogenize and less likely to deposit in cuvettes.

[0045] Example 3 This embodiment is based on a front surface fluorescence detection system of Embodiment 1, and performs front surface fluorescence detection on sludge samples that have been ultrasonically treated in Embodiment 2 and automatically flowed into the front surface cuvette. The front surface cuvette adopts a fully sealed design, with both ends connected to the anaerobic serum bottle and AnMBR through pipelines to prevent air from seeping in.

[0046] During detection, the incident angle was set to 30° and the optical path length to approximately 2 mm to reduce the impact of sample turbidity on scattering and absorption. The excitation wavelength range was 200–500 nm (5 nm step), and the emission wavelength range was 250–550 nm (1 nm step). The single scan time did not exceed 3 minutes. Therefore, compared with the traditional right-angle fluorescence method, this method improves the signal-to-noise ratio by approximately 40% in high-turbidity sludge systems and shortens the detection cycle to 1 / 20.

[0047] Example 4 This embodiment, based on the front surface fluorescence detection system provided in Embodiment 1, and the pretreatment methods and front surface fluorescence detection methods provided in Embodiments 2 and 3, constitutes an engineered closed-loop operation. The sludge sample after fluorescence detection in Embodiment 3 is directly returned to the AnMBR body through the outlet pipeline of the front surface cuvette. The entire process forms a closed-loop operation of "sampling-ultrasound-detection-return", ensuring that the sample is not exposed to the outside air. The system can automatically perform a detection once according to the set cycle (e.g., 2-4 hours) to achieve semi-online or quasi-online monitoring.

[0048] Result Validation To verify the influence of the cuvette optical path structure on the front surface fluorescence detection results, comparative experiments were conducted using quartz cuvettes with dimensions of 10 mm × 2 mm, 10 mm × 4 mm, and 10 mm × 10 mm, respectively. All cuvettes were made of the same material and had the same transmittance. The sample used was a mixture from the same AnMBR reactor (MLSS approximately 6 g / L), sampled under nitrogen protection and pretreated with 2 W sonication for 1 min before direct detection. Fluorescence acquisition conditions were: excitation wavelength 250–500 nm (5 nm step), emission wavelength 250–550 nm (1 nm step), incident and emission angles both 30°, and a scan rate of 30,000 nm / min. -1 The result is as follows Figure 4 As shown, the shorter the optical path, the less interference from scattering and absorption. The 10 mm × 2 mm cuvette has the strongest F420 peak, with a signal-to-noise ratio improvement of about 40%, and its fluorescence score is significantly higher than that of the 10 mm × 4 mm and 10 mm × 10 mm specifications.

[0049] To investigate the effect of ultrasonic pretreatment intensity on coenzyme F in anaerobic sludge 420To investigate the effect of fluorescence signal, comparative experiments were conducted using different ultrasonic power conditions (0 W, 0.5 W, 2 W, 10 W). The experimental sludge was taken from the mixed liquor of a stably operating AnMBR reactor, with MLSS maintained at approximately 6 g / L. Equal volumes of sludge (10 mL) from each group were placed in centrifuge tubes with an ice-water bath and treated with a probe-type ultrasonic instrument (20 kHz) at a constant temperature for 1 min to prevent enzyme activity degradation due to temperature rise. Immediately after treatment, nitrogen gas was introduced for protection, followed by direct frontal surface fluorescence detection (excitation range 250–500 nm, emission range 250–550 nm, steps of 5 nm and 1 nm, respectively). Results are as follows: Figure 5 As shown, moderate ultrasonic disruption significantly promotes the release of intracellular coenzyme F420. At 0 W (untreated), the F420 fluorescence signal is weak, indicating insufficient release of intracellular components. At 0.5 W, the signal slightly increases but remains limited by the cell wall. When the ultrasonic power is increased to 2 W, the peak intensity of F420 significantly increases, the signal-to-noise ratio improves by approximately 35%, and the fluorescence score is about 50% higher than the untreated sample, indicating that under this condition, the cell structure is moderately disrupted without damage to the enzyme molecules. However, at 10 W, due to excessive ultrasonic cavitation, some cells rupture and enzyme structure is destroyed, leading to a decrease in the F420 signal. In summary, 2 W is the optimal ultrasonic intensity, maximizing signal extraction efficiency while maintaining enzyme activity, providing the best pretreatment parameter for rapid fluorescence detection of anaerobic sludge.

[0050] To verify the influence of cuvette structure and sealing on the front surface fluorescence detection results, a comparative experiment was conducted using a conventional transmission cuvette, an unsealed front surface fluorescence cuvette, and an improved sealed front surface fluorescence cuvette. All three cuvettes were made of optical-grade quartz, with a path length of 10 mm and an incident angle of 30°, differing only in the upper opening and sealing structure. The experimental sludge was taken from the same AnMBR reactor mixed liquor (MLSS approximately 6 g / L), pretreated with 2 W sonication for 1 min before sampling to ensure sufficient release of intracellular coenzyme F420. Before detection, an equal volume (2 mL) of sample was injected into each cuvette. The sealed cuvette used a silicone gasket and threaded cap design. After sample loading, nitrogen gas was purged for 3 min to replace oxygen and maintain an anaerobic state. All detections were performed at 35 ± 2 °C, with an excitation wavelength range of 250–500 nm (5 nm step) and an emission wavelength range of 250–550 nm (1 nm step), at a scan rate of 30,000 nm min. -1 .

[0051] The results are as follows Figure 6As shown in (b), neither the conventional cuvettes nor the unsealed cuvettes could maintain a stable anaerobic environment. During the detection process, air entered, causing partial oxidation and inactivation of coenzyme F420, resulting in rapid signal intensity decay and fluctuations exceeding 25% within the detection period. In contrast, the sealed cuvettes, under anaerobic conditions created by nitrogen replacement, exhibited stable F420 signal intensity, with variations not exceeding 5% within the detection period, and the signal-to-noise ratio remained consistently high. Further analysis results are as follows... Figure 6 As shown in (a), the results indicate that the fluorescence score of F420 under closed cuvettes shows a good linear relationship with the detection time, which means that the device can effectively maintain the activity of anaerobic bacteria and the stability of coenzymes, avoid external oxygen interference, and achieve the accuracy and repeatability of front surface fluorescence detection.

[0052] To verify the stability and predictive reliability of the method of this invention under long-term operating conditions, an in-situ monitoring experiment was conducted on a stably operating anaerobic membrane bioreactor (AnMBR) for 30 consecutive days. The reactor operating temperature was maintained at 35 ± 2℃, the hydraulic retention time (HRT) was 24 h, and the sludge concentration (MLSS) was approximately 6 g / L. -1 The influent substrate and operating load were kept constant. Detection was performed using a front-surface sealed fluorescent cuvette (10 mm × 2 mm path length). Samples were taken daily at regular intervals into the circulation pipeline, and FF-EEM measurements were immediately performed under nitrogen protection after sampling. Spectral acquisition parameters were: excitation wavelength 250–500 nm (5 nm step), emission wavelength 250–550 nm (1 nm step), and incident angle 30°.

[0053] Simultaneously, the daily cumulative methane production was recorded using gas chromatography (GC7900) to obtain the reactor performance change trend. The obtained fluorescence data were corrected for internal filtration effect and processed by parallel factor analysis to extract the F420 principal component signal, and the daily fluorescence score was calculated. The results are as follows: Figure 7 As shown, during the entire 30-day operating cycle, F 420 The score values ​​and the trend of methane production change are highly consistent, and the correlation coefficient between the two is R. 2 ≥ 0.90; When the system enters the load fluctuation stage, the F420 signal shows significant fluctuations 1–2 days before the change in methanogenesis rate, enabling early warning. This result demonstrates that the method of this invention possesses excellent signal stability and forward-looking predictive ability under long-term operating conditions, sustainably reflecting changes in methanogenic bacterial activity, and providing a reliable basis for the dynamic monitoring and intelligent control of AnMBR systems.

[0054] In continuous operation monitoring of AnMBR, this method can detect the decline trend of methanogenic activity 3–5 days in advance, which is more forward-looking than traditional methods; Compared with the control group that did not use ultrasonic pretreatment or fully sealed cuvettes, the signal intensity of this method was increased by 30–50%, the repeatability was significantly enhanced, and the detection stability was improved by about 40%.

[0055] By setting up comparative examples, the detection results under different experimental conditions are obtained, and the advantages of this method are described: Comparative Example 1 The sludge samples in this comparative example did not undergo the ultrasonic pretreatment disclosed in Example 2. That is, the sludge samples were directly fed into the front surface cuvette for detection after being taken from the AnMBR, without undergoing ultrasonic water bath treatment. The detection conditions were the same as in Example 3, with an excitation range of 200–500 nm and an emission range of 250–550 nm.

[0056] The detection results showed that the fluorescence signal intensity decreased by about 35% compared with the ultrasonically treated sample in Example 2, indicating that the intracellular F420 was not fully released, resulting in insufficient detection sensitivity.

[0057] Comparative Example 2 The sludge samples in this comparative example were packed into conventional 10 mm optical path, thick-walled quartz cuvettes; the pretreatment process was the same as in Example 2, and the detection conditions were the same as in Example 3. The results showed that under high-turbidity sludge conditions (MLSS≈6 g / L), the signal-to-noise ratio decreased by approximately 40%, obvious scattering bands and absorption interference appeared in the EEM spectrum, the F420 signal resolution was poor, repeatability was insufficient, and the batch-to-batch deviation exceeded 20%.

[0058] Comparative Example 3 In this comparative example, sludge samples were placed in unsealed open cuvettes and fluorescence detection was performed in air. The results showed that the F420 signal decreased by about 25% within 5 minutes after sampling and by more than 50% within 10 minutes, indicating that methanogens and their coenzymes were rapidly inactivated under the action of oxygen, resulting in distorted results.

[0059] In summary, compared with the comparative examples, the present invention significantly enhances the F420 signal intensity by introducing ultrasonic pretreatment, effectively suppresses scattering and absorption interference through cuvette structure optimization, and avoids signal inactivation caused by air exposure through anaerobic detection throughout the process. The overall results show that the detection method of the present invention is far superior to traditional methods in terms of sensitivity, stability, and accuracy, and is more suitable for widespread application under engineering conditions.

[0060] Therefore, this invention employs the aforementioned in-situ detection method for methanogenic activity in anaerobic sludge based on front surface fluorescence. By optimizing the cuvette structure, a fully sealed design with a narrow groove size of 10mm × 2mm × 50mm is adopted, thereby shortening the optical path, reducing the internal filtration effect, and preventing air ingress, effectively improving detection stability and accuracy. Before detection, the sludge undergoes low-power ultrasonic treatment to break up flocs and promote the release of intracellular F420, significantly enhancing signal intensity and improving detection sensitivity. The entire detection process is completed under nitrogen protection, ensuring a fully anaerobic environment and preventing the inactivation of methanogenic bacteria and their coenzymes due to air exposure, thus ensuring the authenticity and repeatability of the detection results.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for in-situ detection of methanogenic activity in anaerobic sludge based on front surface fluorescence, characterized in that: Includes the following steps: S1, Sample loading The sludge sample was loaded into a fully enclosed front-surface cuvette, which has openings on both sides for sample entry and exit; the optical path of the cuvette is 2 mm. S2, Ultrasonic disruption to release F420 enzyme Before testing, the sludge samples were subjected to low-power ultrasonic treatment to fully release intracellular coenzyme F420. S3, Front Surface Fluorescence Detection The sample was placed in the front surface fluorescence configuration sample stage, and the incident light and emitted light were set at 30° to keep the sludge in the anaerobic membrane bioreactor in a circulating state. The fluorescence spectrum signal was rapidly acquired and the fluorescence intensity was recorded within 3 minutes using the front surface fluorescence geometric optical path. The methanogenic activity of coenzyme F420 in the sludge was characterized by the fluorescence signal. After the test, the sludge was output from the cuvette and flowed back into the anaerobic membrane bioreactor.

2. The method for in-situ detection of methanogenic activity in anaerobic sludge based on front surface fluorescence according to claim 1, characterized in that: In S1, the cuvette measures 10mm × 2mm × 50mm.

3. The method for in-situ detection of methanogenic activity in anaerobic sludge based on front surface fluorescence according to claim 1, characterized in that: In S2, the sludge sample underwent low-power ultrasonic treatment with an ultrasonic power of 2W for 1 minute.

4. The method for in-situ detection of methanogenic activity in anaerobic sludge based on front surface fluorescence according to claim 1, characterized in that: In S3, the front surface fluorescence configuration sample stage is placed in a three-dimensional fluorescence spectrometer, and both it and the anaerobic membrane bioreactor are placed in a completely anaerobic detection closed loop, which also includes an ultrasonic device. In the closed loop of the fully anaerobic detection, the sludge flow direction is: anaerobic membrane bioreactor - ultrasonic device - front surface fluorescent configuration sample stage - anaerobic membrane bioreactor.

5. The method for in-situ detection of methanogenic activity in anaerobic sludge based on front surface fluorescence according to claim 4, characterized in that: In the three-dimensional fluorescence spectrometer, the scanning speed was set to 30,000 nm / min, the excitation wavelength was 250 nm-500 nm with 5 nm intervals, and the emission wavelength was 250 nm-550 nm with 1 nm intervals.

6. The method for in-situ detection of methanogenic activity in anaerobic sludge based on front surface fluorescence according to claim 1, characterized in that: When the dominant bacterial community in the sludge is hydrogen-type methanogens with high levels of F420, the fluorescence signal is used as an effective indicator to characterize the sludge's methane generation performance and potential methanogenic activity.

7. The method for in-situ detection of methanogenic activity in anaerobic sludge based on front surface fluorescence according to claim 4, characterized in that: All closed loops for the fully anaerobic detection are under nitrogen protection and are pre-purged with nitrogen.

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