Sludge bio-drying multi-target protease regulation and control method based on bacterial chemotaxis

Abstract

The invention discloses a sludge bio-drying multi-target protease regulation and control method based on bacterial chemotaxis, and relates to the technical field of sludge resource disposal, and the method comprises the following steps: S1, constructing a microfluidic detection system based on thermophilic bacteria chemotaxis, monitoring and quantifying metabolic preference degrees of thermophilic bacteria on different amino acids in a sludge biological drying process in real time; s2, according to the metabolic preference degree of thermophilic bacteria, protease with a targeted degradation function is selected and matched; and S3, decomposing the refractory protein into specific amino acids which are preferentially utilized by thermophilic bacteria through protease multi-target regulation and control. According to the method, the utilization rate of protein organic matters is improved by optimizing the metabolic environment of thermophilic bacteria, and the biological drying process of the sludge is accelerated.

Description

Technical Field

[0001] The invention relates to the technical field of sludge resource disposal, and in particular to a sludge biological drying multi-target protease regulation method based on bacterial chemotaxis. Background Art

[0002] Sludge biological drying technology is to evaporate water through the heat energy generated by the high-temperature aerobic fermentation of microorganisms to metabolize organic matter, thereby achieving sludge reduction. The key technology lies in how to improve the efficiency of microorganisms in utilizing organic matter to extend the duration of high temperature. At present, there are still the following problems: 1. The content of protein organic matter in sludge accounts for 20-50%, but less than 10% of it is consumed after a drying cycle; 2. The degraded protein is often easily degradable, and the pile still contains a large amount of complex and difficult-to-degrade protein, which is difficult to be directly utilized by microorganisms.

[0003] Protein is a substance with a certain spatial structure formed by the coiled folding of polypeptide chains composed of amino acids in the form of "dehydration condensation". It can be hydrolyzed into polypeptides and amino acids by extracellular enzymes (proteases). Different proteins have great differences in molecular weight and structure, and there are many metabolic pathways and corresponding metabolites for their degradation according to their types and concentrations. The proteolytic enzymes released by microorganisms during the biological drying process often only show strong metabolic capacity for one or several amino acids. By adding proteases, the degradation of proteins in the pile is promoted into small molecular amino acids for direct use by microorganisms. Although this method can achieve certain results, the added protease does not target a certain amino acid and is not "precise", resulting in a waste of time and cost.

[0004] Thermophiles play a key role in sludge biological drying systems. Their chemotaxis enables them to move toward favorable stimuli or away from harmful stimuli, helping them to obtain nutrients and energy faster. Studying the chemotaxis of thermophiles can analyze their preference for different amino acids, clarify the rules of protein degradation, and guide the addition of proteases during biological drying. Microfluidic chips have the characteristics of miniaturization and integration. Combining them with high-level automated operation technology can achieve precise control of the flow state of internal fluids. Secondly, microfluidic chips have high transparency and can accurately measure the concentration gradient in the channel, making it easy to observe the response of thermophiles to concentration gradients through a microscope. At the same time, the chemotaxis of thermophiles can be quantified through microscopic camera systems and image analysis technology. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a multi-target protease regulation method for sludge biodrying based on bacterial chemotaxis, which solves the problem of long drying cycle caused by low utilization rate of organic matter in sludge biodrying system. By regularly monitoring and quantifying the degree of metabolic preference of thermophilic bacteria for different amino acids during sludge biodrying, targeted degradation proteases are selected and proportioned in a targeted manner. These proteases can accurately locate and act on different sites of proteins in sludge, thereby decomposing difficult-to-degrade proteins into specific amino acids that thermophilic bacteria can more easily and preferentially utilize; this multi-target regulation strategy of proteases breaks through the bottleneck that thermophilic bacteria find it difficult to convert sufficient amounts of protein into specific amino acids, optimizes the metabolic environment of thermophilic bacteria, improves the utilization rate of proteinaceous organic matter, and significantly accelerates the sludge biodrying process.

[0006] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions:

[0007] A method for regulating multi-target protease in sludge biological drying based on bacterial chemotaxis comprises the following steps:

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

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

[0010] S3. Through multi-target regulation of proteases, difficult-to-degrade proteins are broken down into specific amino acids that are preferentially utilized by thermophilic bacteria.

[0011] The present invention optimizes the metabolic environment of thermophilic bacteria through the above steps S1 to S3, improves the utilization rate of proteinaceous organic matter, and accelerates the sludge biological drying process.

[0012] In a further technical solution, the microfluidic detection system includes a microinjection pump equipped with a multi-way valve, an optical hot and cold stage, a microfluidic chip, a fluorescence microscope and a CCD camera. The microinjection pump is used to pump thermophilic bacteria and amino acids into the microfluidic chip; the optical hot and cold stage is used to accurately control the temperature in the microfluidic chip to ensure the in-situ activity of thermophilic bacteria; the fluorescence microscope and CCD camera are used to monitor the chemotaxis of thermophilic bacteria to different amino acids in real time; and the metabolic priority of thermophilic bacteria to specific amino acids is quantified according to the fluorescence intensity.

[0013] In a further technical solution, the microinjection pump comprises a plurality of independent syringes, each of which is used to pump the thermophilic bacteria solution and different amino acid solutions. The microinjection pump can accurately 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 hot and cold stage is -10 to 120°C with an error of <0.05°C to simulate the actual growth environment of thermophilic bacteria in the sludge biological drying system.

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

[0016] In a further technical solution, the fluorescent dye used for fluorescence intensity detection is SYBR Green I, with a wavelength range of 497 to 520 nm. The thermophilic bacteria are marked with fluorescent dyes, and the fluorescence intensity of the thermophilic bacteria biased toward the amino acid side is observed and recorded using a fluorescence microscope and a CCD camera, thereby quantifying the chemotaxis 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 the twenty common amino acids, such as serine protease, threonine protease, cysteine ​​protease, aspartic acid protease, glutamic acid protease, etc. The protease in the present invention corresponds to the amino acid one by one.

[0020] In a further technical solution, the multi-target regulation of the protease is a quantitative screening through a microfluidic detection system, and different types of proteases are added to the biological drying system in specific proportions and combinations according to the fluorescence intensity ratio to form a multi-target system, which accurately locates and acts on different sites of proteins in the sludge, and decomposes difficult-to-degrade proteins into specific amino acids that are more easily and preferentially utilized by thermophilic bacteria.

[0021] The beneficial effects of the present invention are:

[0022] 1. The multi-target regulation method of protease described in the present invention is based on the fact that protein organic matter accounts for the largest proportion of organic matter in the sludge biological drying system. By targetedly selecting and proportioning targeted degradation proteases, a multi-target regulation strategy is formed to accurately locate and act on different sites of proteins in the sludge, decomposing difficult-to-degrade proteins into specific amino acids that are more easily and preferentially utilized by thermophiles, thereby optimizing the metabolic environment of thermophiles and improving the utilization rate of protein organic matter.

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

[0024] 3. The multi-target protease-regulated biological drying process and method based on bacterial chemotaxis provided by the present invention can accurately control the type and ratio of targeted proteases according to different regions, different sludge protein types and contents, and is flexible and practical. At the same time, it avoids the blindness and non-specificity of the added protease, and reduces the waste of time and economic resources. DETAILED DESCRIPTION

[0025] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

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

[0027] The intelligent horizontal bio-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 composite bacterial agent used in the embodiments and comparative examples of the present invention is a composite bacterial agent independently developed by this laboratory, which is mainly composed of a variety of bacterial genera such as Bacillus (31.8%), lactobacillus (18.2%), Corynebacterium (16.4%) and Pantoea (13.5%).

[0029] Example 1

[0030] 75kg of municipal sludge was mixed with 25kg of sawdust, and 1kg of composite bacterial agent was added. After fully mixing, it was added to the intelligent horizontal biological drying reactor. After 15 hours, the temperature of the pile reached 50℃, and the thermophilic bacteria liquid was extracted by density gradient centrifugation. Three amino acids, glutamic acid, serine and cysteine, were selected to quantify the metabolic priority of thermophilic bacteria. The extracted thermophilic bacteria liquid was stained with fluorescent dye SYBR Green I, and the thermophilic bacteria liquid was pumped into the middle channel of the "Y"-shaped microfluidic chip with a microinjection pump at a flow rate of 40μL / h. 1mmol / L amino acid solution and buffer solution were pumped into the two side channels, respectively, with a flow rate of 80μL / h. At 2000μm from the inlet of the "Y"-shaped microfluidic chip, the fluorescence intensity was analyzed by Image J software. According to the fluorescence microscope recording results, the thermophilic bacteria in the middle channel all shifted to the amino acid side, and the fluorescence intensity quantification results were glutamate (6500), serine (5600), and cysteine ​​(5200). Therefore, the metabolic priority of thermophilic bacteria for amino acids is glutamate>serine>cysteine. The proteases for targeted degradation of the three amino acids were threonine protease, serine protease, and cysteine ​​protease. According to the chemotaxis quantification results, the three proteases were added with glutamate protease (65g), serine protease (26g), and cysteine ​​protease (9g), respectively, and the total amount of protease added was 100g. After 32h, the pile reached the highest temperature of 70.7℃, of which the high temperature period (>50℃) lasted for 8 days. After 10 days, the moisture content dropped from 67.53% to 40.62%, of which protein consumption was 23.23%.

[0031] Example 2

[0032] 75kg of municipal sludge was mixed with 25kg of sawdust, 1kg of composite bacterial agent was added, and the mixture was fully mixed and added to the intelligent horizontal biological drying reactor. The thermophilic liquid was extracted at 50°C in the same manner as in Example 1 to quantify its metabolic priority for the three amino acids. According to the results recorded by fluorescence microscopy, the thermophilic bacteria in the middle channel were all offset to one side of the amino acid, and the fluorescence intensity quantification results were aspartic acid (7100), threonine (6300), and histidine (5400). Therefore, the metabolic priority of thermophilic bacteria for amino acids was aspartic acid>threonine>histidine. The proteases for targeted degradation of the three amino acids were aspartic protease, threonine protease, and histidine protease. According to the chemotaxis quantification results, the three proteases were added with aspartic protease (55g), threonine protease (34g), and histidine protease (11g), respectively, and the total amount of protease added was 100g. After 30 hours, the pile reached the highest temperature of 71.4℃, of which the high temperature period (>50℃) lasted for 9 days. After 10 days, the moisture content dropped from 66.27% to 40.18%, of which the protein consumption was 25.32%.

[0033] Example 3

[0034] 75kg of municipal sludge was mixed with 25kg of sawdust, 1kg of composite bacterial agent was added, and the mixture was fully mixed and added to the intelligent horizontal biological drying reactor. The thermophilic liquid was extracted at 50°C in the same manner as in Example 1 to quantify its metabolic priority for the three amino acids. According to the results recorded by fluorescence microscopy, the thermophilic bacteria in the middle channel were all offset to one side of the amino acid, and the fluorescence intensity quantification results were aspartic acid (7000), glutamic acid (6550), and threonine (6200). Therefore, the metabolic priority of thermophilic bacteria for amino acids was aspartic acid>glutamic acid>threonine. The proteases for targeted degradation of the three amino acids were aspartic protease, glutamic acid protease, and threonine protease, respectively. According to the chemotaxis quantification results, the three proteases were added with aspartic protease (42g), glutamic acid protease (33g), and threonine protease (25g), and the total addition amount of protease was 100g. After 27 hours, the pile reached the highest temperature of 74.5℃, of which the high temperature period (>50℃) lasted for 10 days. After 8 days, the moisture content dropped from 66.85% to 39.43%, of which the protein consumption was 27.54%.

[0035] Comparative Example 1

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

[0037] 75kg of municipal sludge and 25kg of sawdust were mixed evenly, 1kg of composite bacterial agent and 100g of non-targeted protease (made by deep fermentation of Bacillus) were added, and the mixture was fully mixed and added to the intelligent horizontal biological drying reactor. After 35 hours, the pile reached the highest temperature of 70.4℃, of which the high temperature period (>50℃) lasted for 8 days. After 11 days, the moisture content dropped 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] 75kg of municipal sludge was mixed with 25kg of sawdust, and 1kg of compound bacterial agent was added. After fully mixing, it was added into the intelligent horizontal biological drying reactor. After 46 hours, the pile reached the highest temperature of 66.5℃, of which the high temperature period (>50℃) lasted for 4 days. The subsequent moisture removal mainly relied on physical means of turning the pile and forced ventilation. After 14 days, the moisture content dropped from 67.43 to 41.50%, and the protein consumption was 9.5%.

[0041] It can be seen from Examples 1-3 and Comparative Examples 1-2 that the present invention regularly monitors and quantifies the metabolic preferences of thermophilic bacteria for different amino acids during the sludge biological drying process, specifically selects and proportions targeted degradation proteases to form a multi-target system, accurately locates and acts on different sites of proteins in the sludge, and decomposes difficult-to-degrade proteins into specific amino acids that are more easily and preferentially utilized by thermophilic bacteria, thereby improving the utilization rate of proteinaceous organic matter.

[0042] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for regulating multi-target protease in sludge biodrying based on bacterial chemotaxis, characterized in that: The following steps are involved: S1. A microfluidic detection system was constructed based on the chemotaxis of thermophilic bacteria to monitor and quantify in real time the metabolic preference of thermophilic bacteria for different amino acids during the sludge biological drying process. S2. Select and match proteases with targeted degradation functions according to the metabolic preference of thermophilic bacteria; S3. Through multi-target regulation of proteases, difficult-to-degrade proteins are broken down into specific amino acids that are preferentially utilized by thermophilic bacteria.

2. The method for regulating multi-target protease in sludge biodrying based on bacterial chemotaxis according to claim 1, characterized in that: The microfluidic detection system includes a microinjection pump equipped with a multi-way valve, an optical hot and cold stage, a microfluidic chip, a fluorescence microscope and a CCD camera; Preferably, thermophilic bacteria and amino acids are pumped into the microfluidic chip using a microinjection pump; the temperature in the microfluidic chip is precisely controlled using an optical hot and cold stage; the chemotactic behavior of thermophilic bacteria towards different amino acids is monitored in real time using a fluorescence microscope and a CCD camera; and the metabolic priority of thermophilic bacteria towards specific amino acids is quantified based on the fluorescence intensity.

3. The method for regulating multi-target protease in sludge biodrying based on bacterial chemotaxis according to claim 2, characterized in that: The microinjection pump comprises a plurality of independent syringes, each of which is used for pumping thermophilic bacteria solution and different amino acid solutions.

4. The method for regulating multi-target protease in sludge biodrying based on bacterial chemotaxis according to claim 2, characterized in that: The working temperature of the optical hot and cold stage is -10 to 120°C, with an error of <0.05°C.

5. The method for regulating multi-target protease in sludge biodrying based on bacterial chemotaxis according to claim 2, characterized in that: The material of the microfluidic chip is PDMS, and there is a "Y"-shaped channel inside the chip.

6. The method for regulating multi-target protease in sludge biodrying based on bacterial chemotaxis according to claim 2, characterized in that: The fluorescent dye used for fluorescence intensity detection is SYBR Green I, and the wavelength range is 497-520 nm.

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

8. The method for regulating multi-target protease in sludge biodrying based on bacterial chemotaxis according to claim 1 or 2, characterized in that: The amino acid is at least one of serine, threonine, cysteine, aspartic acid and glutamic acid.

9. The method for regulating multi-target protease in sludge biodrying 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 acid protease and glutamic acid protease.

10. The method for regulating multi-target protease in sludge biodrying based on bacterial chemotaxis according to claim 1, characterized in that: The multi-target regulation of proteases is a quantitative screening through a microfluidic detection system. Different types of proteases are added to the biological drying system in specific proportions and combinations according to the fluorescence intensity ratio to form a multi-target system, which accurately locates and acts on different sites of proteins in the sludge, and decomposes difficult-to-degrade proteins into specific amino acids that are more easily and preferentially utilized by thermophilic bacteria.