WO2024193113A1 - 一种连续培养恒浊器及其在测量细菌生长和细菌细胞周期中的应用 - Google Patents

一种连续培养恒浊器及其在测量细菌生长和细菌细胞周期中的应用 Download PDF

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WO2024193113A1
WO2024193113A1 PCT/CN2023/137655 CN2023137655W WO2024193113A1 WO 2024193113 A1 WO2024193113 A1 WO 2024193113A1 CN 2023137655 W CN2023137655 W CN 2023137655W WO 2024193113 A1 WO2024193113 A1 WO 2024193113A1
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turbidostat
bacterial
bacteria
continuous culture
culture
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French (fr)
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傅雄飞
程松涛
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • C12M27/04—Stirrer or mobile mixing elements with introduction of gas through the stirrer or mixing element
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/22—Transparent or translucent parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C12Q1/08—Quantitative determination using multifield media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245—Escherichia (G)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of microbiology, and in particular relates to a continuous culture turbidostat and an application thereof in measuring bacterial growth and bacterial cell cycle.
  • a turbidostat is a continuous culture system that maintains a constant cell density by constantly adjusting the flow rate of fresh medium into the culture vessel and removing an equal amount of spent medium. This allows for stable bacterial growth conditions, and cell density is monitored in real time using a turbidity meter, which measures the optical density of the culture.
  • existing turbidostat technology uses complex optical measurement techniques to measure the optical density of the culture, such as passing the bacterial solution through a microfluidic chip to observe the bacterial concentration under a microscope, which may be more error-prone and require more calibration than simple turbidimetry.
  • PID Proportional-Integral-Derivative Controller.
  • PID stands for "Proportional-Integral-Derivative" and is a process control system used to regulate and stabilize process output. Control algorithm. It is one of the most widely used control algorithms and is used in various applications such as temperature control, speed control, level control, etc.
  • the PID algorithm consists of three parts: proportional control, integral control, and derivative control.
  • Proportional control involves adjusting the output based on the difference between the desired set point and the current process value multiplied by the proportional gain factor.
  • Integral control integrates the error over time to help reduce steady-state error. It involves summing the error over time and multiplying it by the integral gain factor.
  • Derivative control calculates the rate of change of the error and adjusts the output accordingly to help reduce overshoot and oscillation. It involves taking the derivative of the error and multiplying it by the derivative gain factor.
  • the PID algorithm can effectively regulate and stabilize the output of the process by adjusting the control signal based on the error between the desired setpoint and the actual process value.
  • the present invention aims to address these limitations and deficiencies of the above-mentioned prior art by providing a unique and simple tank structure that allows effective stirring without disturbing the cell culture, as well as having a hollow stirring shaft to provide fresh air to the culture.
  • the bacterial concentration detection device of the present invention also provides a more accurate and reliable method for real-time monitoring of cell density.
  • the present invention aims to provide a more efficient and effective method for maintaining stable bacterial growth and research topics related to the bacterial cell cycle.
  • the purpose of the present invention is to design and provide a continuous culture turbidostat and its application in measuring bacterial growth and bacterial cell cycle.
  • the present invention successfully solves the above-mentioned technical problems by designing a unique tank structure and a bacteria concentration detection device.
  • the tank structure avoids the influence of friction on bacteria, while providing fresh air, so that bacteria can grow in a constant environment.
  • the bacteria concentration detection device ensures the accurate detection and control of the bacterial concentration, thereby ensuring the accuracy of the experimental data.
  • the combination of these functions provides a more effective method to maintain stable bacterial growth and study topics related to the bacterial cell cycle, providing optimal growth conditions for bacteria and accurately measuring bacterial concentrations, so that more accurate experiments and research can be carried out in the field of microbiology.
  • the present invention provides a continuous culture turbidostat, comprising an interconnected tank structure, a bacteria concentration detection device, and a control system;
  • the tank structure includes a tank container, a stirrer arranged in the tank container, a feed pipe, a bacteria outlet pipe and a ventilation pipe connecting the tank container with the outside world; the tank structure can effectively stir without interfering with cell culture.
  • the bacteria concentration detection device comprises a bacteria concentration detector connected to a current-voltage conversion module; and can accurately monitor the growth of bacteria.
  • the control system includes a microcontroller development board connected to a current-to-voltage conversion module. One end of the microcontroller development board is connected to a feed tube through a second peristaltic pump, which can accurately adjust the growth conditions of bacteria.
  • the microcontroller development board is programmed to adjust the bacterial nutrient supply based on the input from the sensor.
  • the peristaltic pump is used to supply fresh nutrient solution to the culture when necessary and provide flowing bacterial solution to the cuvette.
  • the material of the tank container is transparent material, which is convenient for observing the growth of bacteria.
  • the tank structure includes a sealing cover, which can easily contact the bacterial culture without introducing contaminants.
  • the stirrer is suspended in the tank container to avoid rubbing the bacteria on the bottom of the tank like a magnetic stirring bar. This suspension structure design suspends the stirring bar in the air for stirring, rather than at the bottom of the tank, which can avoid rubbing the bacteria and causing damage.
  • the continuous culture turbidostat described in the invention comprises a stirrer including a magnetic stirrer, a hollow tube and a stainless steel shaft for fixing the magnetic stirrer and the hollow tube, wherein the hollow tube is connected to an air compressor and a precision pressure controller.
  • the hollow tube is arranged so that the fixed shaft of the stirrer is hollow, which can simultaneously have the functions of stirring and providing fresh air for bacteria. This design helps to maintain the optimal growth conditions for bacteria.
  • the airflow is controlled by an air compressor and a precision pressure controller, and the air supply can be accurately adjusted.
  • the bacteria concentration detector includes an LED coaxial light source, a flow cuvette with a fine channel connected to the LED coaxial light source, and a silicon photodiode.
  • the bacteria concentration detector draws the bacteria liquid out of the culture container through a capillary and enters the micro cuvette with a fine channel.
  • the bacteria concentration can be accurately measured in real time through the LED coaxial light source and the silicon photodiode.
  • the current-voltage conversion module When the light source voltage is constant, if the bacterial concentration reaches a certain value, the current generated by the silicon photodiode will reach a value. Then the current-voltage conversion module generates a corresponding voltage and transmits it to the MCU development board to determine whether the bacterial concentration is above or below the target value.
  • the MCU development board can also transmit data to the computer through its supporting software, and the user can view the voltage value on the computer interface.
  • the software interface displays real-time data about bacterial growth and allows the user to adjust growth conditions and change program parameters as needed.
  • the flow cuvette provides flowing bacterial solution through the control of the first peristaltic pump.
  • the single-chip development board is connected to a computer, and the single-chip development board transmits data to the computer through supporting software.
  • control system controls the bacteria concentration in the tank container according to a PID algorithm.
  • the present invention provides a method for measuring bacterial cell cycle, using any one of the continuous culture turbidostat to measure bacterial cell cycle.
  • the present invention provides a use of the continuous culture turbidostat described in any one of the items in determining the bacterial cell cycle;
  • the bacteria is Escherichia coli.
  • the present invention provides use of the continuous culture turbidostat described in any one of the items in detecting bacterial sensitivity to antibiotics, producing microbial products, and monitoring bacterial contamination in the environment.
  • the present invention has the following beneficial effects:
  • the present invention provides a more accurate, reliable and cheaper method for studying topics related to the bacterial cell cycle. It can achieve bacterial growth in a stable state so as to conduct research related to the bacterial cell cycle. It can achieve accurate detection and control of bacterial concentration to ensure the stability of the bacterial growth environment and the accuracy of experimental data. These advantages can bring more consistent and reliable results, which is beneficial for research and other applications. Specifically:
  • the unique suspended stirring rod tank structure avoids rubbing bacteria when stirring at the bottom of the tank, which is conducive to the stable growth of bacteria, prevents damage to bacterial cells during stirring, and makes the results more consistent and reliable. Compared with the tank body of traditional bioreactor, the cost is much lower. All the parts needed for the reaction tank can be obtained by ordinary wide-mouth reagent bottles and simple machine tool processing of tetrafluoroethylene.
  • the fixed shaft of the stirring shaft is hollow, so that the stirring bar can simultaneously realize the dual functions of stirring and providing fresh air for bacteria, which helps bacteria to fully utilize the nutrients in the culture solution for growth.
  • Bacteria concentration detection device A unique bacteria concentration detection device is designed, including components such as micro-cuvettes, LED light sources, and silicon photodiodes, which can monitor the bacteria concentration in real time and accurately, so as to better control the culture conditions and facilitate the study of bacterial growth and life cycle.
  • Fig. 1 is a schematic diagram of the structure of a turbidostat
  • FIG2 is a light source-cuvette-photoelectric sensor bacteria concentration detection device
  • Figure 3 shows the voltage value corresponding to the bacterial concentration displayed in the software supporting the single-chip development board
  • FIG4 is a schematic diagram of a flow chart of a PID algorithm
  • Figure 5 is a picture of the turbidostat in operation
  • FIG6 is a table showing the change of bacterial concentration over time obtained by sampling the bacterial solution in the turbidostat for multiple times and measuring the optical density of the bacteria in a spectrophotometer;
  • 1-tank structure 101-tank container, 102-agitator, 1021-magnetic stirring bar, 1022-hollow tube, 1023-stainless steel shaft, 103-feeding tube, 104-bacteria outlet tube, 105-venting tube, 106-sealing cover, 107-air compressor, 108-precision pressure controller, 2-bacteria concentration detection device, 201-bacteria concentration detector, 2011-LED coaxial light source, 2012-flow cuvette, 2013-silicon photodiode, 202-current-voltage conversion module, 203-first peristaltic pump, 3-control system, 301-single-chip microcomputer development board, 302-second peristaltic pump, 303-computer.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • a continuous culture turbidostat comprising a tank structure 1, a bacteria concentration detection device 2 and a control system 3 connected to each other;
  • the tank structure 1 includes a tank container 101, an agitator 102 arranged in the tank container 101, a feed pipe 103, a bacteria outlet pipe 104 and a vent pipe 105 connecting the tank container 101 to the outside; the material of the tank container 101 is a transparent material; the tank structure 1 includes a sealing cover 106; the agitator 102 is suspended in the tank container 101.
  • the agitator 102 includes a magnetic stirrer 1021, a hollow tube 1022 and a stainless steel shaft 1023 for fixedly connecting the magnetic stirrer 1021 and the hollow tube 1022, and the hollow tube 1022 is connected to an air compressor 107 and a precision pressure controller 108. It has the functions of stirring and providing fresh air for bacteria at the same time, which helps to maintain the best growth conditions for bacteria.
  • the bacteria concentration detection device 2 includes a bacteria concentration detector 201 connected to a current-voltage conversion module 202; the bacteria concentration detector 201 includes an LED coaxial light source 2011, a flow cuvette 2012 with a fine channel connected to the LED coaxial light source 2011, and a silicon photodiode 2013.
  • the flow cuvette 2012 is controlled by a first peristaltic pump 203 to provide flowing bacterial liquid.
  • the bacterial liquid is drawn out of the tank container 101 through the ventilation tube 105 and enters the flow cuvette 2012 with a fine channel to accurately measure the bacterial concentration in real time.
  • the control system 3 includes a single-chip microcomputer development board 301 connected to the current-voltage conversion module 202, and one end of the single-chip microcomputer development board 301 is connected to the feed pipe 103 through the second peristaltic pump 302.
  • the single-chip microcomputer development board 301 is connected to the computer 303, and the single-chip microcomputer development board 301 transmits data to the computer 303 through supporting software.
  • the control system controls the bacterial concentration in the tank container according to the PID algorithm.
  • FIG1 it is a schematic diagram of the structure of the turbidostat of the present invention.
  • the turbidostat of the present invention can provide optimal growth conditions for bacteria and accurately measure the concentration of bacteria.
  • the tank structure 1 is designed to allow the stirrer 102 to suspend for stirring, avoiding rubbing of bacteria on the bottom of the tank like the magnetic stirring bar in the prior art.
  • the tank container 101 is made of transparent material, which is convenient for observing the growth of bacteria. It is also equipped with a sealing cover 106, which allows easy access to the bacterial culture without introducing contaminants.
  • the stirrer 102 of the turbidostat adopts a fixed hollow shaft design, which has the function of stirring and providing fresh air for the bacteria. Dual function of air.
  • Agitator 102 consists of a magnetic stirrer 1021, a stainless steel shaft 1023 and a hollow tube 1022 that supplies air to the bacteria.
  • the air flow is controlled by an air compressor 107 and a precision pressure controller 108, which can accurately regulate the air supply.
  • the bacterial concentration detection device 201 provided in the turbidostat can accurately monitor bacterial growth.
  • the detection device is composed of a flow cuvette 2012 with a fine hole channel, an LED coaxial light source 2011, and a silicon photodiode 2013.
  • the light source voltage is constant, if the bacterial concentration reaches a certain value, the current generated by the silicon photodiode 2013 will reach a value.
  • the current-voltage conversion module 202 generates a corresponding voltage and transmits it to the single-chip development board 301 to determine whether the bacterial concentration is higher or lower than the target value.
  • the single-chip development board 301 can also transmit data to the computer 303 through its supporting software, and the user can view the voltage value on the computer interface.
  • the voltage displayed in the interface divided by 2058 and multiplied by 3.3 is the actual voltage value.
  • the software interface displays real-time data on bacterial growth and allows the user to adjust the growth conditions and change the program parameters as needed.
  • the turbidostat is equipped with a control system 3 that can accurately adjust the growth conditions of bacteria.
  • the control system 3 includes a microcontroller development board 301 and multiple peristaltic pumps.
  • the microcontroller development board 301 is programmed to adjust the bacterial nutrient supply based on the input of the sensor, the first peristaltic pump 203 is used to provide flowing bacterial solution to the cuvette, and the second peristaltic pump 302 is used to supply fresh nutrient solution to the culture when necessary.
  • This algorithm is used to control the speed of the stepper motor of the peristaltic pump.
  • the motor adopts PWM speed control and the speed is expressed in units of revolutions per minute.
  • rin(t) is the preset speed value
  • the output rout(t) is the actual value of the motor speed
  • the actuator is a stepper motor.
  • the output rout(t) is the actual speed value (r/min);
  • the deviation is the difference between the preset value and the actual value (r/min).
  • Figure 5 is a picture of the turbidostat in operation.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • the bacteria are centrifuged in a high-speed centrifuge at 4 degrees Celsius and 3000 rpm, and then poured into the culture medium for resuspending. Then the pipeline of the reaction tank is connected and the bacterial solution is poured into the reaction tank. This operation is performed in a clean bench.
  • Figure 5 shows the status of the equipment after startup.
  • Turbidistat parameters such as medium flow rate and air flow rate, can be adjusted according to specific experimental needs and requirements.
  • Embodiment 3 is a diagrammatic representation of Embodiment 3
  • the turbidity of the bacterial culture can be monitored in real time using a spectrophotometer, and the data can be collected and analyzed using a computer software program.
  • the program can plot a bacterial growth curve and provide information about the hysteresis phase, exponential phase, and stationary phase of bacterial growth.
  • the bacteria were taken out several times and the optical density of the bacteria at 600 nm wavelength was measured using a spectrophotometer. The results are shown in Figure 6, which shows that the turbidostat can stably control the bacterial concentration.
  • the equipment After each use, the equipment should be thoroughly cleaned with 70% ethanol and allowed to dry completely before storage. Regular maintenance of the peristaltic pump and air compressor is also necessary to ensure the proper operation of the equipment.
  • the invention is feasible through experiments, simulations and use.
  • the feasibility test shows that the turbidostat can provide stable bacterial growth and maintain a constant bacterial concentration, and the bacterial concentration detection device will provide accurate and precise bacterial concentration measurement.
  • the design of the suspended stirring rod, the dual function of the stirring shaft, the bacterial concentration detection device and the like of the invention enables bacteria to maintain activity and growth for a long time and monitor the bacterial concentration in real time.

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Abstract

提供了一种连续培养恒浊器及其在测量细菌生长和细菌细胞周期中的应用，属于微生物学技术领域。提供了连续培养恒浊器，包含相互连接的罐体结构、细菌浓度检测装置和控制系统；罐体结构包括罐体容器，设置于罐体容器中的搅拌器，将罐体容器与外界相连的进料管、出菌管和通气管；细菌浓度检测装置包括细菌浓度检测器与电流-电压转换模块相连；控制系统包括与电流-电压转换模块相连的单片机开发板，单片机开发板的一端通过第二蠕动泵连接进料管。该恒浊器能够实现细菌在稳定状态下的生长，以便进行细菌细胞周期相关的研究；实现对细菌浓度的精准检测和控制，以保证细菌生长环境的稳定和实验数据的准确性。

Description

一种连续培养恒浊器及其在测量细菌生长和细菌细胞周期中的应用 技术领域
本发明属于微生物学技术领域，具体涉及一种连续培养恒浊器及其在测量细菌生长和细菌细胞周期中的应用。
背景技术
测量细菌生长的传统方法包括从培养物中取样并使用分光光度法测量细胞密度或使用显微镜对细胞进行计数。然而，这些方法非常耗时，会破坏生长环境，并且可能无法准确反映培养物中的真实细胞密度。
恒浊器的开发是为了解决上述问题。恒浊器是一种连续培养系统，通过不断调整新鲜培养基进入培养容器的流速，并去除等量的废培养基来维持恒定的细胞密度。这使得其中具有稳定的细菌生长条件，使用浊度计实时监测细胞密度，浊度计可测量培养物的光密度。
然而，在恒浊器中使用的传统磁力搅拌棒会导致细胞培养中断，尤其是当细胞对剪切应力敏感时。这导致了避免损坏细胞新技术的发展，例如，气升式发酵罐和Rushton(拉什顿)叶轮，它们使用不同类型的搅拌来避免损坏细胞。例如，Cellstat(细胞状态)系统恒浊器，使用摇摆运动来提供搅动并避免干扰细胞培养，具有用于测量培养物的光密度的单独腔室。但Cellstat系统的搅动方法可能不如其他方法有效，并且可能导致整个培养容器的生长速率不一致。此外，Cellstat系统不能为培养物提供新鲜空气，这可能会限制其为某些细菌物种维持最佳生长条件的能力。
另一方面，现有的恒浊器技术采用复杂的光学测量技术来测量培养物的光密度，如菌液通过微流控芯片下显微观察细菌浓度等，与简单的比浊法相比，这可能更容易出错并且需要更多的校准。
PID全称比例(proportion)-积分(integral)-微分(derivative)控制器。PID代表“比例-积分-微分”，它是过程控制系统中用于调节和稳定过程输出的 控制算法。它是使用最广泛的控制算法之一，用于各种应用，例如温度控制、速度控制、液位控制等。
PID算法由三部分组成：比例控制、积分控制和微分控制。比例控制涉及根据所需设定点与当前过程值之间的差异乘以比例增益系数来调整输出。积分控制对随时间变化的误差进行积分，以帮助减少稳态误差。它涉及对随时间的误差求和并将其乘以积分增益因子。微分控制计算误差的变化率并相应地调整输出以帮助减少超调和振荡。它涉及对误差求导并将其乘以微分增益因子。
通过结合这三个组成部分，PID算法可以根据所需设定值与实际过程值之间的误差调整控制信号，从而有效地调节和稳定过程的输出。
本发明旨在通过提供一种独特且简单的罐结构来解决上述现有技术的这些局限性和缺陷，本发明罐结构允许在不干扰细胞培养的情况下进行有效搅拌，以及具有空心搅拌轴为培养物提供新鲜空气。本发明的细菌浓度检测装置还提供了一种更加准确可靠的实时监测细胞密度的方法。总的来说，本发明旨在提供一种更高效和有效的方法来维持稳定的细菌生长和与细菌细胞周期相关的研究课题。
发明内容
针对上述现有技术中存在的问题，本发明的目的在于设计提供一种连续培养恒浊器及其在测量细菌生长和细菌细胞周期中的应用。本发明通过设计独特的罐体结构和细菌浓度检测装置，成功地解决了上述技术问题。罐体结构避免了摩擦对细菌的影响，同时提供新鲜空气，使得细菌在恒定的环境下进行生长。而细菌浓度检测装置则保证了细菌浓度的精准检测和控制，从而保证了实验数据的准确性。这些功能的组合提供了一种更有效的方法来维持稳定的细菌生长和研究与细菌细胞周期相关的主题，为细菌提供最佳生长条件并准确测量细菌浓度，从而可在微生物学领域进行更精确的实验和研究。
为了实现上述目的，本发明采用以下技术方案：
一方面，本发明提供了一种连续培养恒浊器，包含相互连接的罐体结构、细菌浓度检测装置和控制系统；
罐体结构包括罐体容器，设置于罐体容器中的搅拌器，将罐体容器与外界相连的进料管、出菌管和通气管；罐体结构可以在不干扰细胞培养的情况下进行有效搅拌。
细菌浓度检测装置包括细菌浓度检测器与电流-电压转换模块相连；可以准确监测细菌生长。
控制系统包括与电流-电压转换模块相连的单片机开发板，单片机开发板的一端通过第二蠕动泵连接进料管，可以准确调节细菌的生长条件。单片机开发板被编程为根据传感器的输入调节细菌营养供应，蠕动泵用于在必要时向培养物供应新鲜的营养液以及为比色皿提供流动的菌液。
所述的一种连续培养恒浊器，所述罐体容器的材料为透明材料，便于观察细菌生长情况。所述罐体结构包括密封盖，可以方便地接触细菌培养物而不会引入污染物。所述搅拌器为悬浮设置于罐体容器中，避免像磁力搅拌棒那样摩擦罐底的细菌。这种悬浮结构设计将搅拌棒悬浮在空中进行搅拌，而不是在罐底，可以避免摩擦细菌而造成损坏。
所述的一种连续培养恒浊器，所述搅拌器包括磁力搅拌子、中空管及用于固定连接磁力搅拌子和中空管的不锈钢轴，中空管与空气压缩机和精密压力控制器相连。设置中空管，使搅拌器的固定轴是空心的，可以同时具有搅拌和为细菌提供新鲜空气的功能。这种设计有助于保持细菌的最佳生长条件。气流由空气压缩机和精密压力控制器控制，可以精确调节空气供应。
所述的一种连续培养恒浊器，所述细菌浓度检测器包括LED同轴光源、与LED同轴光源相连的具有细孔道的流动比色皿和硅光电二极管。该细菌浓度检测器通过毛细管将细菌液体从培养容器中抽出，并进入带有细通道的微量比色皿中。并且，通过LED同轴光源和硅光电二极管，可以实时准确测量细菌浓度。
当光源电压一定时，如果细菌浓度达到一定值，硅光电二极管产生的电流 就会达到一个值。然后电流-电压转换模块产生相应的电压传输给单片机开发板以确定细菌浓度是高于还是低于目标值。另外，单片机开发板还可以通过其配套的软件将数据传输给电脑，用户可以在电脑界面上查看电压值。软件界面显示有关细菌生长的实时数据，并允许用户根据需要调整生长条件，更改程序参数。
所述的一种连续培养恒浊器，所述流动比色皿通过第一蠕动泵控制提供流动的菌液。
所述的一种连续培养恒浊器，所述单片机开发板与计算机相连，单片机开发板通过配套软件将数据传输给计算机。
所述的一种连续培养恒浊器，所述控制系统根据PID算法控制罐体容器中的细菌浓度。
第二方面，本发明提供了一种测定细菌细胞周期的方法，利用任一项所述的连续培养恒浊器进行测定细菌细胞周期。
第三方面，本发明提供了任一项所述的连续培养恒浊器在细菌细胞周期测定中的用途；
优选，细菌为大肠杆菌。
第四方面，本发明提供了任一项所述的连续培养恒浊器在检测细菌对抗生素的敏感性、生产微生物产品、监测环境中细菌污染中的用途。
与现有技术相比，本发明具有以下有益效果：
本发明为研究细菌细胞周期相关主题提供了一种更精确和可靠且更廉价的方法。能够实现细菌在稳定状态下的生长，以便进行细菌细胞周期相关的研究。实现对细菌浓度的精准检测和控制，以保证细菌生长环境的稳定和实验数据的准确性。这些优势可以带来更一致、更可靠的结果，有利于研究和其他应用。具体地：
1、独特的悬浮式搅拌棒罐体结构，避免了在罐底搅拌时摩擦细菌，这有利于细菌的稳定生长，防止搅拌过程中对细菌细胞的损伤，使结果更加一致可靠。 相比于传统生物反应器的罐体，成本更加低廉，只需要普通的广口试剂瓶以及对四聚氟乙烯进行简单的机床加工就可以得到反应罐所需的所有零部件。
2、搅拌轴的固定轴是中空的，使得搅拌子同时实现搅拌和为细菌提供新鲜空气的双重功能，有助于细菌的充分利用培养液中的营养成分进行生长。
3、细菌浓度检测装置设计了独特的细菌浓度检测装置，包括微量比色皿、LED光源以及硅光电二极管等组件，可实时、精确地监测细菌浓度，从而更好地控制培养条件，有利于研究细菌的生长和生命周期。
4、在检测装置中使用微量比色皿、LED光源和硅光电二极管是一种比现有技术中使用的其他方法更精确和有效的方法。
附图说明
图1为恒浊器结构示意图；
图2为光源-比色皿-光电传感器细菌浓度检测装置；
图3为单片机开发板配套软件中显示的细菌浓度对应的电压值；
图4为PID算法的流程示意图；
图5为恒浊器正在运行的图片；
图6为对恒浊器的菌液进行多次取样，并在分光光度计中测量细菌的光密度值，得到细菌浓度随时间的变化表；
其中，1-罐体结构，101-罐体容器，102-搅拌器，1021-磁力搅拌子，1022-中空管，1023-不锈钢轴，103-进料管，104-出菌管，105-通气管，106-密封盖，107-空气压缩机，108-精密压力控制器，2-细菌浓度检测装置，201-细菌浓度检测器，2011-LED同轴光源，2012-流动比色皿，2013-硅光电二极管，202-电流-电压转换模块，203-第一蠕动泵，3-控制系统，301-单片机开发板，302-第二蠕动泵，303-计算机。
具体实施方式
以下将通过附图和实施例对本发明作进一步说明。
实施例1：
一种连续培养恒浊器，包含相互连接的罐体结构1、细菌浓度检测装置2和控制系统3；
罐体结构1包括罐体容器101，设置于罐体容器101中的搅拌器102，将罐体容器101与外界相连的进料管103、出菌管104和通气管105；罐体容器101的材料为透明材料；罐体结构1包括密封盖106；搅拌器102为悬浮设置于罐体容器101中。搅拌器102包括磁力搅拌子1021、中空管1022及用于固定连接磁力搅拌子1021和中空管1022的不锈钢轴1023，中空管1022与空气压缩机107和精密压力控制器108相连。具有同时搅拌和为细菌提供新鲜空气的功能，有助于保持细菌的最佳生长条件。
细菌浓度检测装置2包括细菌浓度检测器201与电流-电压转换模块202相连；细菌浓度检测器201包括LED同轴光源2011、与LED同轴光源2011相连的具有细孔道的流动比色皿2012、硅光电二极管2013。流动比色皿2012通过第一蠕动泵203控制提供流动的菌液。通过通气管105将细菌液体从罐体容器101中抽出，并进入带有细通道的流动比色皿2012中，实时准确测量细菌浓度。
控制系统3包括与电流-电压转换模块202相连的单片机开发板301，单片机开发板301的一端通过第二蠕动泵302连接进料管103。单片机开发板301与计算机303相连，单片机开发板301通过配套软件将数据传输给计算机303。控制系统根据PID算法控制罐体容器中的细菌浓度。
如图1所示，为本发明恒浊器的结构示意图，本发明恒浊器能够为细菌提供最佳生长条件并准确测量细菌浓度。
实际使用时，罐体结构1设计让搅拌器102悬浮进行搅拌，避免像现有技术磁力搅拌棒那样摩擦罐底的细菌。罐体容器101由透明材料制成，便于观察细菌生长情况。还配备了一个密封盖106，可以方便地接触细菌培养物而不会引入污染物。恒浊器的搅拌器102采用固定空心轴设计，具有搅拌和为细菌提供新鲜 空气的双重功能。搅拌器102由磁力搅拌子1021、不锈钢轴1023和向细菌提供空气的中空管1022组成。气流由空气压缩机107和精密压力控制器108控制，可以精确调节空气供应。
恒浊器设置的细菌浓度检测装置201，可以准确监测细菌生长。如图2所示，检测装置由带细孔道的流动比色皿2012、LED同轴光源2011、硅光电二极管2013。当光源电压一定时，如果细菌浓度达到一定值，硅光电二极管2013产生的电流就会达到一个值。然后电流-电压转换模块202产生相应的电压传输给单片机开发板301以确定细菌浓度是高于还是低于目标值。另外，单片机开发板301还可以通过其配套的软件将数据传输给计算机303，用户可以在计算机界面上查看电压值。如图3所示，界面中显示的电压除以2058乘以3.3即为实际电压值。软件界面显示有关细菌生长的实时数据，并允许用户根据需要调整生长条件，更改程序参数。
恒浊器配备了一个控制系统3，可以准确调节细菌的生长条件。控制系统3包括单片机开发板301和多个蠕动泵。单片机开发板301被编程为根据传感器的输入调节细菌营养供应，第一蠕动泵203用于为比色皿提供流动的菌液，第二蠕动泵302用于在必要时向培养物供应新鲜的营养液。
本发明利用的PID原理如图4所示，如下式(1)，其中规定(在t时刻)；输入量为r(t)；输出量为c(t)；偏差量为e(t)＝rin(t)-rout(t)；
该算法用于为蠕动泵的步进电机调速，电机采用PWM调速转速用单位转/分钟表示。rin(t)为转速预定值；输出量rout(t)为电机转速实际值；执行器为步进电机。这使得控制系统的输入量rin(t)为转速预定值(转/分钟)；输出量rout(t)为转速实际值(转/分钟)；偏差量为预定值和实际值之差(转/分钟)。如下图5为恒浊器正在运行的图片。
实施例2：
1、装置的灭菌处理：
取两个贝克曼牌灭菌袋，用剪刀剪开两个洞，然后用封口膜与灭菌胶带封住剪开的口子，为了在后面的烘干时通气。将罐体容器101、进料管103，出菌管104，通气管105分别装入灭菌袋，封口。两个灭菌袋与装培养基的试剂瓶一同放入高压灭菌锅进行灭菌在121摄氏度下对设备进行15-20分钟的灭菌。灭菌完成后，放入烘箱中烘干，去除装置内的水分。
2、细菌的培养与接种：
提前一夜在培养基中接种细菌过夜培养，第二天对细菌稀释100倍活化。使用试剂瓶与滤膜将培养基进行过滤。
细菌浓度到达0.3左右后，将细菌在高速离心机中在4摄氏度，转速3000转/分钟下进行离心，然后倒入培养基重悬。随后将反应罐的管线进行连接，将菌液倒入反应罐中，此操作在超净台中进行。
3、装置启动：
将反应罐放入水浴磁力搅拌锅当中，将管线与蠕动泵、试剂瓶、空气压缩机进行连接，随后接通电源，图5显示设备启动完成后的状态。
4、调节恒浊器参数：
要调整恒浊器参数，例如培养基流速和空气流速，可根据具体实验需要和要求进行调整。
实施例3：
1、数据收集与分析
在实验过程中，可以使用分光光度计实时监测细菌培养物的浊度，并可以使用计算机软件程序收集和分析数据。该程序可以绘制细菌的生长曲线，并提供有关细菌生长的迟滞期、指数期和稳定期的信息。将细菌多次取出，用分光光度计测量细菌每次在600纳米波长光下的光密度，结果如图6所示，这显示了恒浊器能够对细菌浓度稳定控制。
2、设备维护
每次使用后，应使用70％乙醇彻底清洁设备，并在存放前使其完全干燥。定期维护蠕动泵和空气压缩机也是必要的，以确保设备的正常运行。
3、故障排除
如果设备运行不正常，可以采取几个故障排除步骤。首先，检查所有连接是否牢固，电源是否正常工作。如果菌液浓度不稳定，检查培养基，确保菌液在倒入反应槽前混合均匀。
如果细菌浓度波动，检查蠕动泵和空气压缩机是否正常运行。
如果发生污染，请再次对设备进行消毒，并使用适当的无菌技术重复实验。
如果光密度读数不一致，请检查分光光度计校准并在每次测量前清洁比色皿。
通过实验、模拟和使用证明本发明是可行的，可行性测试表明，恒浊器可以提供稳定的细菌生长并保持恒定的细菌浓度，而细菌浓度检测装置将提供准确和精确的细菌浓度测量。本发明的悬浮搅拌棒、搅拌轴双重功能、细菌浓度检测装置等设计，使得细菌可以长期维持活性并生长和实时监测细菌浓度。

Claims (10)

  1. 一种连续培养恒浊器，其特征在于，包含相互连接的罐体结构、细菌浓度检测装置和控制系统；
    罐体结构包括罐体容器，设置于罐体容器中的搅拌器，将罐体容器与外界相连的进料管、出菌管和通气管；
    细菌浓度检测装置包括细菌浓度检测器与电流-电压转换模块相连；
    控制系统包括与电流-电压转换模块相连的单片机开发板，单片机开发板的一端通过第二蠕动泵连接进料管。
  2. 如权利要求1所述的一种连续培养恒浊器，其特征在于，所述罐体容器的材料为透明材料；所述罐体结构包括密封盖；所述搅拌器为悬浮设置于罐体容器中。
  3. 如权利要求2所述的一种连续培养恒浊器，其特征在于，所述搅拌器包括磁力搅拌子、中空管及用于固定连接磁力搅拌子和中空管的不锈钢轴，中空管与空气压缩机和精密压力控制器相连。
  4. 如权利要求1所述的一种连续培养恒浊器，其特征在于，所述细菌浓度检测器包括LED同轴光源、与LED同轴光源相连的具有细孔道的流动比色皿和硅光电二极管。
  5. 如权利要求4所述的一种连续培养恒浊器，其特征在于，所述流动比色皿通过第一蠕动泵控制提供流动的菌液。
  6. 如权利要求1所述的一种连续培养恒浊器，其特征在于，所述单片机开发板与计算机相连，单片机开发板通过配套软件将数据传输给计算机。
  7. 如权利要求1所述的一种连续培养恒浊器，其特征在于，所述控制系统根据PID算法控制罐体容器中的细菌浓度。
  8. 一种测定细菌细胞周期的方法，其特征在于，利用如权利要求1-7任一项所述的连续培养恒浊器进行测定细菌细胞周期。
  9. 如权利要求1-7任一项所述的连续培养恒浊器在测定细菌细胞周期，提供稳定的细菌生长环境中的用途；
    优选，细菌为大肠杆菌。
  10. 如权利要求1-7任一项所述的连续培养恒浊器在检测细菌对抗生素的敏感性、生产微生物产品、监测环境中细菌污染中的用途。
PCT/CN2023/137655 2023-03-21 2023-12-08 一种连续培养恒浊器及其在测量细菌生长和细菌细胞周期中的应用 Pending WO2024193113A1 (zh)

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