Multi-target protease regulation method for sludge bio-drying based on bacterial chemotaxis

Abstract

The application discloses a sludge biological drying multi-target proteinase regulation method based on bacterial chemotaxis, relates to the technical field of sludge resource disposal, and comprises the following steps: S1, constructing a microfluidic detection system based on the chemotaxis of thermophilic bacteria, and monitoring and quantifying the metabolic preference degree of the thermophilic bacteria to different amino acids in a sludge biological drying process; S2, selecting and proportioning proteinases with a targeted degradation function according to the metabolic preference degree of the thermophilic bacteria; and S3, decomposing refractory proteins into specific amino acids preferred by the thermophilic bacteria through multi-target regulation of the proteinases. The application optimizes the metabolic environment of the thermophilic bacteria to improve the utilization rate of protein organic matter and accelerate the sludge biological drying process.

Description

A Multi-Target Protease Regulation Method for Sludge Bio-drying Based on Bacterial Chemotaxis Technical Field

[0001] This invention relates to the field of sludge resource utilization and treatment technology, specifically to a multi-target protease regulation method for sludge bio-drying based on bacterial chemotaxis. Background Technology

[0002] Sludge biological drying technology uses the heat energy generated by microbial high-temperature aerobic fermentation of organic matter to evaporate water, thereby reducing sludge volume. The key technology lies in how to improve the efficiency of microbial utilization of organic matter to extend the duration of high-temperature drying. Currently, the following problems exist: 1. Protein-based organic matter accounts for 20-50% of the sludge, but less than 10% is consumed after one drying cycle; 2. The degraded proteins are often easily degradable, and the sludge pile still contains a large amount of complex, recalcitrant proteins that are difficult for microorganisms to directly utilize.

[0003] Proteins are substances with a specific spatial structure formed by polypeptide chains composed of amino acids through dehydration condensation and folding. They can be hydrolyzed into polypeptides and amino acids by extracellular enzymes (proteases). Different proteins vary greatly in molecular weight and structure, and their degradation involves multiple metabolic pathways and corresponding metabolites depending on the type and concentration. The proteolytic enzymes released by microorganisms during bio-drying often exhibit strong metabolic capabilities for only one or a few amino acids. Adding proteases can promote the degradation of proteins in the biomass into smaller amino acids for direct use by microorganisms. While this method can achieve some results, the added proteases do not target specific amino acids, lacking "precision" and resulting in a waste of time and money.

[0004] Thermophilic bacteria play a crucial role in sludge bio-drying systems. Their chemotaxis enables them to move towards beneficial stimuli or away from harmful ones, facilitating faster acquisition of nutrients and energy. Studying the chemotaxis of thermophilic bacteria can elucidate their preferences for different amino acids, clarify protein degradation patterns, and guide the addition of proteases during bio-drying. Microfluidic chips, characterized by miniaturization and integration, combined with high-level automated operation technologies, enable precise control of the internal fluid flow. Furthermore, the high transparency of microfluidic chips allows for accurate measurement of concentration gradients within the channels, facilitating microscopic observation of the thermophilic bacteria's response to concentration gradients. Simultaneously, microscopic imaging systems and image analysis techniques facilitate the quantification of thermophilic bacteria's chemotaxis. Summary of the Invention

[0005] The technical problem this invention aims to solve is to provide a multi-target protease regulation method for sludge bio-drying based on bacterial chemotaxis, which addresses the problem of long drying cycles caused by low organic matter utilization in sludge bio-drying systems. By periodically monitoring and quantifying the metabolic preferences of thermophilic bacteria for different amino acids during sludge bio-drying, targeted degradation proteases are selected and proportioned. These proteases can precisely locate and act on different sites of proteins in the sludge, thereby decomposing recalcitrant proteins into specific amino acids that are more readily utilized by thermophilic bacteria. This multi-target protease regulation strategy overcomes the bottleneck of thermophilic bacteria's inability to convert sufficient amounts of protein into specific amino acids, optimizes the metabolic environment of thermophilic bacteria, improves the utilization rate of protein-based organic matter, and significantly accelerates the sludge bio-drying process.

[0006] The technical problem to be solved by this invention is achieved by the following technical solution:

[0007] A method for regulating multi-target proteases in sludge bio-drying based on bacterial chemotaxis includes the following steps:

[0008] S1. A microfluidic detection system was constructed based on the chemotaxis of thermophilic bacteria to monitor and quantify the metabolic preference of thermophilic bacteria for different amino acids during sludge bio-drying in real time.

[0009] S2. Select and formulate proteases with targeted degradation functions according to the metabolic preferences of thermophilic bacteria;

[0010] S3. Through multi-target regulation of proteases, recalcitrant proteins are broken down into specific amino acids that thermophilic bacteria preferentially utilize.

[0011] This invention optimizes the metabolic environment of thermophilic bacteria through the above steps S1 to S3, improves the utilization rate of protein-based organic matter, and accelerates the bio-drying process of sludge.

[0012] In a further technical solution, the microfluidic detection system includes a microinjection pump equipped with a multi-directional valve, an optical heating and cooling stage, a microfluidic chip, a fluorescence microscope, and a CCD camera. The microinjection pump injects thermophilic bacteria and amino acids into the microfluidic chip; the optical heating and cooling stage precisely controls the temperature within the microfluidic chip to ensure the in-situ activity of the thermophilic bacteria; the fluorescence microscope and CCD camera monitor the chemotactic behavior of the thermophilic bacteria towards different amino acids in real time; and the metabolic priority of the thermophilic bacteria for specific amino acids is quantified based on fluorescence intensity.

[0013] In a further technical solution, the microinjection pump comprises multiple independent syringes, each for pumping in thermophilic bacterial solutions and different amino acid solutions. The microinjection pump can precisely pump the liquid in the syringe into the microfluidic chip at a flow rate of less than 100 μL / h.

[0014] In a further technical solution, the working temperature of the optical heating and cooling stage is -10 to 120°C with an error of <0.05°C, in order to simulate the actual growth environment of thermophilic bacteria in the sludge biological drying system.

[0015] In a further technical solution, the microfluidic chip is made of PDMS and has "Y"-shaped channels inside.

[0016] In a further technical solution, the fluorescent dye used for fluorescence intensity detection is SYBR Green I, with a wavelength range of 497–520 nm. Thermophilic bacteria are labeled with the fluorescent dye, and the fluorescence intensity of the bacteria biased towards the amino acid side is observed and recorded using a fluorescence microscope and a CCD camera, thereby quantifying the chemotactic behavior data of the thermophilic bacteria.

[0017] In a further technical solution, the thermophilic bacteria are extracted by density gradient centrifugation, including but not limited to at least one of Thermaerobacte, Calditerricola, Pseudomonas, and Methylophilaceae.

[0018] In a further technical solution, the amino acid is at least one of the twenty common amino acids, such as serine, threonine, cysteine, aspartic acid, and glutamic acid.

[0019] In a further technical solution, the protease is at least one of the proteases corresponding to twenty common amino acids, such as serine protease, threonine protease, cysteine ​​protease, aspartic protease, and glutamate protease. In this invention, the protease and amino acid correspond one-to-one.

[0020] In a further technical solution, the protease multi-target regulation is achieved through quantitative screening using a microfluidic detection system. Different types of proteases are added to the biological drying system in specific proportions and combinations based on fluorescence intensity ratios to form a multi-target system. This system precisely locates and acts on different sites of proteins in sludge, decomposing recalcitrant proteins into specific amino acids that are more readily utilized by thermophilic bacteria.

[0021] The beneficial effects of this invention are:

[0022] 1. The protease multi-target regulation method of the present invention is based on the fact that protein-based organic matter accounts for the highest proportion of organic matter in sludge biological drying system. By selectively selecting and proportioning proteases to target degradation, a multi-target regulation strategy is formed, which accurately locates and acts on different sites of proteins in sludge, decomposing recalcitrant proteins into specific amino acids that are more easily utilized by thermophilic bacteria, thus optimizing the metabolic environment of thermophilic bacteria and improving the utilization rate of protein-based organic matter.

[0023] 2. The protease multi-target regulation process and method described in this invention are based on the chemotaxis of thermophilic bacteria and are quantified using microfluidic technology. It has advantages such as small size, high sensitivity, low sample consumption, and easy integration. It can monitor and quickly and accurately distinguish the preference of thermophilic bacteria for amino acids in real time.

[0024] 3. The multi-target protease-regulated bio-drying process and method based on bacterial chemotaxis provided by this invention can precisely control the type and ratio of target proteases according to different regions and different types and contents of sludge proteins, making it flexible, versatile, and highly practical. At the same time, it avoids the blind and non-specific nature of adding exogenous proteases, reducing the waste of time and economic resources. Detailed Implementation

[0025] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0026] In the embodiments and comparative examples of the present invention, municipal sludge with a water content of 80% from a sewage treatment plant in an urban area was used, with sawdust as an auxiliary material, to verify the effect of the multi-target protease regulation method for sludge bio-drying based on bacterial chemotaxis provided by the present invention on the efficiency of sludge bio-drying.

[0027] The intelligent horizontal biological drying reactor used in the embodiments and comparative examples of the present invention is equipped with oxygen and temperature and humidity detection devices, which can automatically perform operations such as aeration, material turning and ventilation dehydration based on real-time monitoring data such as oxygen concentration and humidity in the container.

[0028] The compound microbial agents used in the embodiments and comparative examples of this invention are compound microbial agents independently developed by our laboratory, mainly composed of multiple bacterial genera such as Bacillus (31.8%), lactobacillus (18.2%), Corynebacterium (16.4%), and Pantoea (13.5%).

[0029] Example 1

[0030] 75 kg of municipal sludge and 25 kg of sawdust were mixed evenly, and 1 kg of compound bacterial agent was added. After thorough mixing, the mixture was added to an intelligent horizontal bio-drying reactor. After 15 hours, the reactor temperature reached 50°C. Thermophilic bacterial solution was extracted using density gradient centrifugation. Glutamic acid, serine, and cysteine ​​were selected to quantify the metabolic priority of thermophilic bacteria. The extracted thermophilic bacterial solution was stained with the fluorescent dye SYBR Green I. Thermophilic bacterial solution was pumped into the central channel of a "Y"-shaped microfluidic chip using a microinjection pump at a flow rate of 40 μL / h. A 1 mmol / L amino acid solution and a buffer solution were pumped into the two side channels, respectively, at a flow rate of 80 μL / h. The microarray was photographed at a distance of 2000 μm from the inlet using a fluorescence microscope, and the fluorescence intensity was analyzed using ImageJ software. According to fluorescence microscopy recordings, the thermophilic bacteria in the middle channel all shifted towards the amino acid side. The fluorescence intensity quantification results showed glutamic acid (6500), serine (5600), and cysteine ​​(5200). Therefore, the metabolic priority of thermophilic bacteria for amino acids was glutamic acid > serine > cysteine. The proteases that targeted the degradation of the three amino acids were threonine protease, serine protease, and cysteine ​​protease, respectively. Based on chemotaxis quantification results, glutamic acid protease (65g), serine protease (26g), and cysteine ​​protease (9g) were added, with a total protease addition of 100g. After 32 hours, the pile reached a maximum temperature of 70.7℃, with the high-temperature period (>50℃) lasting for 8 days. After 10 days, the water content decreased from 67.53% to 40.62%, with 23.23% of the protein consumed.

[0031] Example 2

[0032] 75 kg of municipal sludge and 25 kg of sawdust were mixed evenly, and 1 kg of compound bacterial agent was added. After thorough mixing, the mixture was added to an intelligent horizontal biological drying reactor. The thermophilic bacterial solution was extracted at 50 °C using the same method as in Example 1 to quantify its metabolic priority for three amino acids. According to fluorescence microscopy recordings, the thermophilic bacteria in the middle channel all shifted towards the amino acid side. The fluorescence intensity quantification results were aspartic acid (7100), threonine (6300), and histidine (5400). Therefore, the metabolic priority of the thermophilic bacteria for amino acids was aspartic acid > threonine > histidine. The proteases that targeted the degradation of the three amino acids were aspartic protease, threonine protease, and histidine protease, respectively. Based on the chemotaxis quantification results, aspartic protease (55 g), threonine protease (34 g), and histidine protease (11 g) were added, respectively, for a total protease addition of 100 g. After 30 hours, the pile reached a maximum temperature of 71.4℃, with the high-temperature period (>50℃) lasting for 9 days. After 10 days, the moisture content dropped from 66.27% to 40.18%, with 25.32% of the protein consumed.

[0033] Example 3

[0034] 75 kg of municipal sludge and 25 kg of sawdust were mixed evenly, and 1 kg of compound bacterial agent was added. After thorough mixing, the mixture was added to an intelligent horizontal biological drying reactor. The thermophilic bacterial solution was extracted at 50 °C using the same method as in Example 1 to quantify its metabolic priority for three amino acids. According to fluorescence microscopy recordings, the thermophilic bacteria in the middle channel all shifted towards the amino acid side. The fluorescence intensity quantification results were aspartic acid (7000), glutamic acid (6550), and threonine (6200). Therefore, the metabolic priority of the thermophilic bacteria for amino acids was aspartic acid > glutamic acid > threonine. The proteases that targeted the degradation of the three amino acids were aspartic protease, glutamic acid protease, and threonine protease, respectively. Based on the chemotactic quantification results, aspartic protease (42 g), glutamic acid protease (33 g), and threonine protease (25 g) were added, respectively, for a total protease addition of 100 g. After 27 hours, the pile reached a maximum temperature of 74.5℃. The high-temperature period (>50℃) lasted for 10 days. After 8 days, the moisture content dropped from 66.85% to 39.43%, of which 27.54% of the protein was consumed.

[0035] Comparative Example 1

[0036] The difference between Comparative Example 1 and Example 1 is that a non-targeted protease was added.

[0037] 75 kg of municipal sewage sludge was mixed evenly with 25 kg of sawdust, and 1 kg of compound microbial agent and 100 g of non-targeted protease (produced by deep fermentation of Bacillus) were added. After thorough mixing, the mixture was added to an intelligent horizontal bio-drying reactor. After 35 hours, the pile reached a maximum temperature of 70.4℃, with the high-temperature period (>50℃) lasting for 8 days. After 11 days, the moisture content decreased from 67.56% to 41.15%, and the protein consumption was 19.43%.

[0038] Comparative Example 2

[0039] The difference between Comparative Example 2 and Example 1 is that no protease was added.

[0040] 75 kg of municipal sludge was mixed evenly with 25 kg of sawdust, and 1 kg of compound microbial agent was added. After thorough mixing, the mixture was added to an intelligent horizontal biological drying reactor. After 46 hours, the pile reached a maximum temperature of 66.5℃, with the high-temperature period (>50℃) lasting for 4 days. Subsequent moisture removal mainly relied on physical methods such as turning the pile and forced ventilation. After 14 days, the moisture content decreased from 67.43% to 41.50%, and the protein consumption was 9.5%.

[0041] As can be seen from Examples 1-3 and Comparative Examples 1-2, the present invention monitors and quantifies the metabolic preferences of thermophilic bacteria for different amino acids during the sludge biological drying process at regular intervals, and selects and proportions targeted degradation proteases to form a multi-target system. This system accurately locates and acts on different sites of proteins in sludge, decomposing recalcitrant proteins into specific amino acids that thermophilic bacteria can preferentially utilize, thereby improving the utilization rate of protein organic matter.

[0042] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for multi-target regulation of proteases in sludge bio-drying based on bacterial chemotaxis, characterized in that, Includes the following steps: S1. A microfluidic detection system was constructed based on the chemotaxis of thermophilic bacteria during the sludge biological drying process to monitor and quantify the metabolic preference of thermophilic bacteria for different amino acids in real time. S2. Select and formulate proteases with targeted degradation functions according to the metabolic preferences of thermophilic bacteria; S3. During the sludge biological drying process, the protease is used to decompose recalcitrant proteins in the sludge into specific amino acids that are preferentially utilized by thermophilic bacteria through multi-target regulation.

2. The method for multi-target regulation of protease in sludge bio-drying based on bacterial chemotaxis according to claim 1, characterized in that: The microfluidic detection system includes a microinjection pump equipped with a multi-directional valve, an optical heating and cooling stage, a microfluidic chip, a fluorescence microscope, and a CCD camera. The microinjection pump injects thermophilic bacteria and amino acids into the microfluidic chip. The optical heating and cooling stage precisely controls the temperature within the microfluidic chip. The fluorescence microscope and CCD camera monitor the chemotactic behavior of thermophilic bacteria towards different amino acids in real time. The metabolic priority of thermophilic bacteria towards amino acids is quantified based on fluorescence intensity. The amino acid is at least one selected from serine, threonine, cysteine, aspartic acid, and glutamic acid.

3. The method for multi-target regulation of protease in sludge bio-drying based on bacterial chemotaxis according to claim 2, characterized in that: The microinjection pump contains multiple independent syringes, each for pumping in thermophilic bacterial solutions and different amino acid solutions.

4. The method for multi-target regulation of protease in sludge bio-drying based on bacterial chemotaxis according to claim 2, characterized in that: The operating temperature of the optical heating and cooling stage is -10~120℃, with an error of <0.05℃.

5. The method for multi-target regulation of protease in sludge bio-drying based on bacterial chemotaxis according to claim 2, characterized in that: The microfluidic chip is made of PDMS and has "Y"-shaped channels inside.

6. The method for multi-target regulation of protease in sludge bio-drying based on bacterial chemotaxis according to claim 2, characterized in that: The fluorescent dye used for fluorescence intensity detection is SYBR Green I, with a wavelength range of 497~520 nm.

7. The method for multi-target regulation of protease in sludge bio-drying based on bacterial chemotaxis according to claim 1 or 2, characterized in that: In step S1, the thermophilic bacteria are extracted by density gradient centrifugation and include at least one of Thermaerobacte, Calditerricola, Pseudomonas, and Methylophilaceae.

8. The method for multi-target regulation of protease in sludge bio-drying based on bacterial chemotaxis according to claim 1, characterized in that: The protease is at least one of serine protease, threonine protease, cysteine ​​protease, aspartic protease, and glutamate protease.

9. The method for multi-target regulation of protease in sludge bio-drying based on bacterial chemotaxis according to claim 1, characterized in that: The protease multi-target regulation is achieved through quantitative screening using a microfluidic detection system. Different types of proteases are added to the sludge biological drying system in specific proportions and combinations based on fluorescence intensity ratios to form a multi-target system. This system precisely locates and acts on different sites of proteins in the sludge, breaking down recalcitrant proteins into specific amino acids that are more readily utilized by thermophilic bacteria.