Efficient targeted separation device and method for environmental functional microorganisms

Abstract

The invention relates to the technical field of microbial separation, in particular to an efficient targeted separation device and method for environmental functional microorganisms. The device comprises an environmental sample preset culture module and an intelligent detection and feedback regulation module which are arranged in a box body, the preset culture module is used for culturing a pretreated target environmental microorganism sample, the intelligent detection and feedback regulation module is used for detection every certain period of culture, and the sample is obtained through automatic separation until the sample is completely cultured. The device can realize efficient culture and separation of target environmental functional microorganisms, avoids sample loss and pollution risk, remarkably improves the standardization degree and efficiency of the culture process, and solves the problems of low efficiency, poor accuracy, limited separation quantity and insufficient standardization of environmental functional microorganisms in the prior art.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial separation technology, in particular to an efficient targeted separation device and method of environmental functional microorganisms. BACKGROUND

[0002] Environmental functional microorganisms refer to those microbial groups that play specific ecological functions (such as pollutant degradation, nutrient cycling, environmental remediation, etc.) in natural or artificial environments. Environmental functional microorganisms in nature have important application value in the fields of agriculture, energy, medicine, etc. Screening environmental functional microorganisms, efficiently exploring and utilizing new functional gene and functional enzyme biomass resources can improve human living standards and is an important guarantee for the sustainable development of society.

[0003] Currently, the identification and resource utilization of environmental functional microorganisms mainly include traditional culture method and molecular biology method. However, both methods have technical bottlenecks. Although the traditional culture method can directly obtain active microorganisms, its process relies on a large amount of manual operation, such as the need for regular sampling and determination of OD 600 to judge the concentration of the bacterial solution. This process not only causes sample loss and introduces pollution risk, but also causes uncertain culture cycle and limited throughput due to low proportion of cultivable microorganisms, complex pretreatment, etc. The molecular biology method (such as metagenomics) avoids the culture bias, directly extracts nucleic acids in the environmental sample for analysis, but it highly depends on the integrity of the existing database, and cannot obtain living microorganisms, making it difficult to realize the separation, expansion and practical application of the strain.

[0004] In order to efficiently obtain target environmental functional microorganisms, the present application designs an efficient targeted separation device for environmental functional microorganisms, which is used to receive microencapsulated pretreated microbial samples, provides an in-situ culture mode for target environmental functional microorganisms, realizes automatic continuation of high-efficiency culture process without manual sampling operation, and provides reliable technical support for efficient mining and utilization of microbial resources. SUMMARY

[0005] The present application provides an efficient targeted separation device and method for environmental functional microorganisms, which can realize efficient culture and separation of target environmental functional microorganisms, avoid sample loss and pollution risk, significantly improve the standardization degree and efficiency of the culture process, and solve the problems of low separation efficiency, poor accuracy, limited separation quantity and insufficient standardization of environmental functional microorganisms in the prior art.

[0006] One of the technical solutions adopted by the present application is:

[0007] An efficient targeted separation device for environmental functional microorganisms is provided, comprising:

[0008] An environmental sample pre-set culture module includes a rotating culture mechanism, a temperature regulating component, and / or a light source; the rotating culture mechanism includes a drive motor and a turntable assembly for carrying multiple sample tubes.

[0009] The intelligent detection and feedback adjustment module includes a turbidity emitting unit, a turbidity receiving unit, a controller, a timer, and an alarm. The turbidity receiving unit receives the light signal from the sample tube placed on the turntable assembly through the turbidity emitting unit and transmits it to the controller. When the set target turbidity is reached, the alarm is activated to remind the user.

[0010] The temperature regulating component, light source, drive motor, turbidity emitting unit, turbidity receiving unit, timer, and alarm are all electrically connected to the controller.

[0011] The housing contains all the modules mentioned above.

[0012] Furthermore, the enclosure is a transparent or semi-transparent enclosure, with a base supported inside the enclosure and a fixed column fixed on the base. The turntable assembly is movably fitted onto the fixed column, and the turbidity emitting unit is fixed on the fixed column above the turntable assembly. The turbidity receiving unit is disposed on the inner wall of the enclosure corresponding horizontally to the turbidity emitting unit. The turntable assembly is driven to rotate horizontally circumferentially on the fixed column by a drive motor located below it. The drive motor is a servo motor.

[0013] Furthermore, the turntable assembly includes a rotating shaft sleeved on the fixed column, and two turntables are arranged at intervals on the upper and lower sides of the rotating shaft. Several sets of limiting holes are correspondingly arranged on each turntable. Every two upper and lower corresponding limiting holes on the two turntables are used to fix the sample tube. The rotating shaft is driven by a servo motor located below it to reciprocate around the fixed column or rotate continuously around the column at certain intervals.

[0014] Furthermore, the aforementioned rotating shaft is a hollow rotating shaft; the turntable and the hollow rotating shaft are integrally formed.

[0015] Furthermore, the limiting holes are evenly spaced circumferentially on each turntable.

[0016] Furthermore, a bearing is provided below the rotating shaft, and a groove is provided in the base. The bearing is fitted in the groove, and the bottom end of the fixing column is fixedly connected to the middle groove wall. An opening is provided on the groove wall, and a first gear is fixedly sleeved on the bottom of the bearing. The first gear partially protrudes out of the aforementioned opening. A second gear is sleeved on the output shaft of the servo motor, and the first and second gears mesh with each other.

[0017] Further, the bearing is a ball bearing, the inner ring of the bearing is fixedly sleeved on the outer side of the bottom of the fixed column, the outer ring of the bearing is fixedly connected with the bottom end of the rotating shaft, and a vertical gap is left between the bearing and the inner bottom wall of the groove. In this way, under the driving of the servo motor, the outer ring of the bearing drives the rotating shaft to rotate on the fixed column.

[0018] Further, the outer ring of the ball bearing is detachably fixedly connected with the bottom end of the rotating shaft through the connecting plate.

[0019] Further, the outer ring of the ball bearing is detachably fixedly connected with the bottom end of the rotating shaft through the connecting plate.

[0020] Further, the temperature adjusting member comprises a heater, a refrigerator and an auxiliary temperature control member; and the light source is natural light.

[0021] Further, the auxiliary temperature control member is a heat dissipation fan.

[0022] Further, the heater and the refrigerator are respectively arranged on two opposite inner walls of the box body; and the heater and the refrigerator are respectively a semiconductor heater and a semiconductor refrigerator.

[0023] Further, the heat dissipation fan is arranged on the inner bottom wall of the box body.

[0024] Further, a control panel is further arranged on the outer side of the box body. The environment sample preset culture module, the intelligent detection and feedback adjustment module in the box body are controlled by the controller under the control of the control panel.

[0025] Further, the control panel integrates a controller, a timer, an alarm, a turbidity detector, a panel touch screen and a power module, supports parameter setting, and the parameters include culture temperature, culture light intensity, rotating shaft rotating speed, rotating angle, culture period, turbidity threshold and the like.

[0026] Further, the culture temperature is set in the range of 0-40 DEG C, the culture light intensity is set in the range of 0-10000 lux, the rotating shaft rotating speed is set in the range of 0-60 rpm, the rotating angle of the rotating shaft in the culture stage is set in the range of 0-180 DEG (reciprocating rotation left and right), the culture time is set in the range of 0-9999 s, the rotating angle of the rotating shaft in the detection stage after each culture period is determined by the number of limit holes on the rotating disc, and the stay time after rotating the corresponding angle is set in the range of 0-50 s, and the stay time is the time interval of turbidity detection.

[0027] Further, the number of limit holes on the rotating disc is 8, and when the rotating shaft rotates clockwise in the detection stage, the rotating shaft stops for 0-50 s after rotating 45 DEG.

[0028] Further, a support plate is arranged in the box, the support plate is integrally formed by a left plate, a bottom plate and a right plate; the rotating culture mechanism is supported on the bottom plate; and the temperature control member is arranged on the inner wall of the box outside the support plate.

[0029] Further, the heating semiconductor is arranged between the left plate and the inner wall of the box, the refrigeration semiconductor is arranged between the right plate and the inner wall of the box, and the heat dissipation fan is arranged on the bottom wall of the box below the bottom plate. Through the above arrangement, the heating semiconductor and the refrigeration semiconductor exposed on the side wall of the box are separated from the rotating culture mechanism by the support plate, so that the arrangement and layout of each structural member are more reasonable and more beautiful on the basis of ensuring good temperature control effect in the box.

[0030] Further, the support plate is a hollow support plate, which is a support plate provided with strip-shaped openings or a support plate provided with hollow circular holes.

[0031] Further, a box cover and a handle are arranged at the top end of the box, and supporting legs are arranged at the bottom end of the box.

[0032] Further, the sample tube comprises a test tube rack fixedly arranged on the rotating disc assembly, and a test tube is arranged in the test tube rack.

[0033] Further, the test tube rack is clamped and fixed in cooperation with the upper and lower limiting holes on the two rotating discs.

[0034] Further, eight limiting holes arranged on each rotating disc can be used for fixing test tube racks of three specifications of 10 mL, 20 mL and 50 mL and test tubes of corresponding capacities.

[0035] Further, the test tube rack and the test tube can be specially manufactured based on the clamping and fixing effect in cooperation with the limiting hole.

[0036] Further, the environmental functional microorganism is a functional microorganism in an environment such as river water, seawater, sludge and soil.

[0037] The second technical solution adopted in the application is:

[0038] An environmental functional microorganism efficient targeted separation method is provided, and the device described above is used for culture separation, and the steps include:

[0039] S1, the microencapsulated pretreated environmental functional microorganism sample and the in-situ environmental culture solution are placed in a sterilized sample test tube, a target environmental functional microorganism carbon source is added, the sample test tube is placed in a test tube rack and transferred to the rotating culture mechanism in the box;

[0040] S2, the culture parameters are set: culture temperature, culture light intensity, rotating mode of the rotating culture mechanism, culture period, turbidity detection time interval and turbidity target value.

[0041] The above culture temperature and culture light intensity parameters are set based on the in-situ environment of different environmental functional microorganisms; the above rotating culture mechanism rotating mode includes a culture stage rotating mode and a detection stage rotating mode, the culture stage rotating mode is set as horizontal circumferential reciprocating rotation at a certain angle, and a single culture period is set as 1 h; the detection stage rotating mode is set as continuous rotation at a certain turbidity detection time interval in the horizontal circumferential direction; the turbidity detection time interval is set as 0-50 s; and the above turbidity target value is set as OD 600 = 1.

[0042] S3, taking the turbidity target value detected in the detection stage as a standard, a control operation of restarting the culture stage or ending the culture stage is performed: if after the first culture period ends, the turbidity detection value does not reach the target value, the culture stage is restarted after the detection ends, and the “culture-detection” process is repeated until the turbidity detection values of all samples reach the standard; during the detection process, for the samples whose turbidity detection values reach the standard, an alarm is started to remind, and the corresponding sample tube is taken out after the corresponding detection stage ends, and the rest of the sample tubes restart the above “culture-detection” process.

[0043] S4, for the samples whose turbidity detection values reach the standard, the culture is considered to be ended, and the functional microorganisms separated from the sample tubes are obtained.

[0044] Further, a single period of the culture detection stage is timed by a timer and transmitted to the controller for corresponding culture and detection conversion control.

[0045] Further, the sample tubes in step S1 are respectively fixed on the rotating disc of the rotating culture mechanism, and the sample tubes are labeled with numbers on the rotating disc, and the corresponding number sequence corresponds to each turbidity detection value in the subsequent detection; the microencapsulated preprocessed environmental functional microorganism sample is a sample microencapsulated by an emulsification solidification method, and the carbon source is a carbon source for the target functional environmental microorganism.

[0046] Further, the carbon source added in the in-situ environment culture solution in S1 is determined by the target environmental functional microorganism to be separated, such as the target functional microorganism for degrading the target pollutant taking the target pollutant as the carbon source.

[0047] Further, the sample tube obtained in step S4, wherein the functional microorganism is in the upper part of the sample liquid based on gravity, so as to realize separation from non-environmental microorganisms.

[0048] Further, after the culture of the sample tube in S4 is ended, the target environmental functional microorganism in the sample tube is in the upper layer of the sample liquid of the sample tube due to the gravity difference with the microencapsulated bacterial microspheres.

[0049] Further, the method is divided into culture, detection and separation stages, and the specific operation steps of each stage are as follows:

[0050] The culture stage: open the box of the separation device, mix the microencapsulated pretreated environmental functional microorganism and the corresponding in-situ environmental culture solution, and place them in a sterilized disposable test tube, fix the test tube on the test tube rack and insert it into the limiting hole on the rotating culture mechanism rotating disc, and a number is arranged on the rotating disc beside the limiting hole, which synchronously corresponds to the sample detection order of the detection stage.

[0051] In combination with the in-situ environment where the cultured environmental functional microorganism is located, the culture temperature and light intensity are set through the control panel; at the same time, the single culture cycle is set to 1h, the rotating shaft of the rotating culture mechanism is set to horizontal reciprocating rotation of 90° during the culture cycle, and the rotating speed is 60rpm; the turbidity detection time interval and the turbidity target value are set; after the above parameter setting, the rotating culture mechanism is started, and the rotating shaft drives the test tube on the rotating disc to rotate back and forth by 90° during the culture stage, at this time, the temperature control part and the cooling fan are selectively opened according to the set culture temperature; the light source is selectively opened based on the in-situ environment;

[0052] The detection stage: after the single culture cycle of the culture stage ends, the turbidity detection is continued by the controller, at this time, the rotating culture mechanism is reset, and the rotating shaft thereon continues to rotate by 360° every certain angle interval of 0-50s, the interval time is the turbidity detection time, and the turbidity detection values of the sample liquids in the test tubes are recorded in turn, so that each test tube is detected, and after the detection is completed, all the test tubes return to the original position: if the turbidity detection value does not reach the target turbidity in this detection, the controller controls the restart of the aforementioned culture stage, and the culture-detection process is repeated until all the sample liquids in the test tubes reach the standard and the culture is completed; if there is a test tube sample with a turbidity detection value reaching the standard in a certain detection stage, the corresponding test tube is taken out after detection, and the next round of culture is continued for other sample liquids.

[0053] The separation stage: for the sample liquid in the culture test tube with a turbidity detection value reaching the standard, the target environmental functional microorganism in the sample liquid and the microencapsulated non-target environmental microorganism are separated due to gravity.

[0054] The beneficial effects of the present application are:

[0055] 1. The device of the present application realizes the precise and intelligent enrichment culture screening and automatic separation of the target environmental functional microorganism in the microencapsulated pretreated environmental microorganism sample. Compared with the traditional culture method, the sample loss is reduced, no pollution risk is introduced, the culture cycle is more easily determined, and the proportion of cultivable target environmental functional microorganisms is improved.

[0056] 2. The device of this invention integrates the cultivation and screening of target environmental functional microorganisms into a sealed chamber. The entire cultivation process simulates the in-situ environment of the target functional microorganisms, achieving a high degree of automation. The OD of the sample solution is detected after periodic cultivation. 600 This device determines the growth status of target functional microorganisms within the culture medium and achieves more efficient separation. The cultivation stage employs reciprocating rotation to promote the rapid growth of the target functional microorganisms in the in-situ culture medium. The detection stage involves turbidity testing of the cultured microbial sample to confirm its logarithmic growth phase. When the pre-set target turbidity value is reached, the enrichment of the target functional microorganisms is achieved. Through periodic monitoring, the device promptly tracks the growth status of microorganisms in the sample. If the cultivation target is not reached, the cultivation-detection process is repeated until all microbial samples are fully cultured.

[0057] 3. The device of this invention, in accordance with the requirements of the corresponding culture and separation method, has high efficiency in the culture of environmental microorganisms with target functions and significantly improves the standardization of the culture process, thus providing reliable technical support for the efficient exploration and utilization of subsequent microbial resources. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of the device structure according to Embodiment 1 of the present invention;

[0059] Figure 2 This is a structural reference diagram of the test tube rack of the present invention;

[0060] Figure 3 This is a structural reference diagram of the test tube of the present invention;

[0061] Figure 4 This is a schematic diagram of the connection structure between the rotating culture mechanism, the fixed shaft, and the base in the device of the present invention;

[0062] Figure 5 for Figure 4 A schematic diagram of one embodiment of a servo motor-driven rotating shaft;

[0063] Figure 6 This is a schematic diagram of the device structure in Embodiment 2 of the present invention;

[0064] Figure 7 This is a simplified diagram of the control module of the method of the present invention;

[0065] Figure 8 This is a growth curve diagram of the thiamethoxam-degrading bacteria isolated in an embodiment of the present invention;

[0066] Figure 9 Phylogenetic tree of total thiamethoxam-degrading microorganisms identified in embodiments of the present invention;

[0067] Figure 10The fitting curves of the degradation of clothianidin of different test samples of the embodiment of the present application are compared.

[0068] Wherein, 1 rotating disc, 101 first rotating disc, 102 second rotating disc, 2 heating semiconductor, 3 servo motor, 4 heat dissipation fan, 5 natural light module, 6 fixed column, 7 turbidity transmitting unit, 8 turbidity receiving unit, 9 rotating shaft, 10 refrigeration semiconductor, 11 box cover, 12 handle, 13 operation control panel, 14 general switch, 15 limiting hole, 16 test tube, 17 test tube rack, 18 base, 19 ball bearing, 191 outer ring, 192 inner ring, 20 groove, 21 opening, 22 first gear, 23 second gear, 24 connecting plate, 25 left side plate, 26 bottom plate, 27 right side plate. DETAILED DESCRIPTION

[0069] In order to clearly illustrate the technical features of the scheme, the present application will be described in detail below with specific embodiments, combined with the drawings.

[0070] In the description of the present application, it should be understood that the terms "upper", "lower", "front", "back", "left", "right", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0071] In the description of the present application, the term "environmental functional microorganism" refers to those microorganism groups that play specific ecological functions in natural or artificial environment, such as pollutant degradation, nutrient cycling, environmental remediation, etc. The term "target environmental functional microorganism" refers to environmental functional microorganisms with specific functions such as harmful substance degradation ability, antibiotic resistance, etc. The device and method provided by the present application aims to efficiently separate and screen target environmental functional microorganisms.

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing specific embodiments only and is not intended to be limiting of the application.

[0073] The present application is to realize efficient targeted separation of target environmental functional microorganisms, and provides the following device: the device comprises an environmental sample preset culture module and an intelligent detection and feedback adjustment module, and on the basis of the above modules, the automatic separation of target functional microorganisms obtained by culture from other non-target functional microorganisms is realized. The above environmental sample preset culture module and intelligent detection and feedback adjustment module realize the automatic, standardized, precise and intelligent culture screening of the environmental sample, and the target functional microorganisms separated by screening are finally separated by gravity gradient field.

[0074] The environmental sample received by the above device is a mixed sample of microencapsulated pretreated environmental sample and in-situ environmental sample liquid. The mixed sample is added with a carbon source available to the target environmental functional microorganisms.

[0075] Specifically, referring to Figures 1-3 , the above device comprises a transparent box body, and the aforementioned environmental sample preset culture module and intelligent detection and feedback adjustment module are arranged in the box body. The environmental sample preset culture module comprises a double-layered rotating disc 1, a heating semiconductor 2, a servo motor 3, a heat dissipation fan 4, a natural light module 5, a fixed column 6, a rotating shaft 9, a refrigeration semiconductor 10, a test tube 16 for containing environmental sample, and a test tube rack 17. The rotating shaft 9 is a hollow rotating shaft sleeved on the fixed column 6; the rotating disc is arranged in two layers on the rotating shaft 9; the heating semiconductor and the refrigeration semiconductor are arranged on the two opposite inner side walls of the box body, respectively; and the heat dissipation fan is arranged on the bottom wall in the box body. A box body cover 11, a handle 12, an operation control panel 13 and a main switch 14 are arranged on the box body. The natural light module is arranged on the rear wall in the box body. Eight limiting holes 15 are uniformly and spacedly arranged on each rotating disc along the circumferential direction thereof, and each two limiting holes corresponding to each other on the two rotating discs are used for placing the test tube rack, and the test tube is placed in each test tube rack.

[0076] The intelligent detection and feedback adjustment module includes a turbidity detector, a PLC controller, a timer, and an alarm. The turbidity detector includes a turbidity emitting unit 7 and a turbidity receiving unit 8. In the initial state of the rotating shaft, the turbidity emitting unit, the test tube in the limiting hole (number 1), and the turbidity receiving unit are on the same horizontal line. The turbidity emitting unit 7 is located on the upper part of the aforementioned fixed column 6. The turbidity receiving unit 8 is arranged parallel to the turbidity emitting unit, maintaining a coaxiality error of ≤0.1mm. The turbidity receiving unit 8 can be supported and fixed on the right side wall of the box by a support plate. The output end of the turbidity receiving unit is connected to the PLC controller through a shielded cable. After receiving the light signal emitted by the turbidity emitting unit and transmitted through the sample liquid in the test tube, the turbidity receiving unit transmits the received signal to the PLC controller. The aforementioned operation control panel 13 integrates a PLC controller, a timer, an alarm, a turbidity detector, a panel touch screen, and a power module. It supports parameter setting, including incubation temperature, light intensity, rotation speed, rotation angle, incubation period, and turbidity threshold (target turbidity). The target turbidity is based on OD600=1, which means that the turbidity of the bacterial solution is characterized by measuring the absorbance (OD value) at a wavelength of 600nm, thereby indirectly reflecting the bacterial concentration.

[0077] Furthermore, the above-mentioned culture temperature parameters are set within a range of 0-40℃, determined by the optimal culture temperature of the sample. This temperature is based on the growth temperature of the in-situ environment where the target environmental functional microorganisms reside. After setting the culture temperature, the internal temperature uniformity is regulated through the combined use of heating and cooling semiconductors and a cooling fan within the chamber. The culture stage is the growth stage of the target environmental functional microorganisms. During this stage, the rotating shaft rotates 90° left and right. The detection stage involves detecting the turbidity of the sample liquid after each culture cycle (generally set to 1 hour), based on a 360° circumferential rotation, with a 0-50s pause at each rotation angle. The rotation angle at this point is determined by the number of limiting holes dispersed on the turntable, with a 0-50s pause at each 45° rotation. The turbidity threshold is expressed as OD. 600 The target value of 1 is the culture endpoint. The light intensity is switched on and off and adjusted according to the light requirements of the target environmental functional microorganisms.

[0078] Furthermore, the eight limiting holes on the turntable are used to fix 10ml, 20ml, and 50ml test tube racks and corresponding capacity test tubes. At the end of the above detection stage, the rotating shaft pauses for 0-50 seconds at each time it rotates to the next limiting hole position to perform turbidity detection.

[0079] The power supply lines of the temperature control module composed of the heating semiconductor and the refrigeration semiconductor, the natural light module, the servo motor, the turbidity detector, the timer, the alarm and the like are uniformly connected to the power module of the operation control panel, and the signal lines are connected to the I / O interface of the PLC controller through the terminal block; the communication protocol is adopted to realize the real-time transmission of the servo motor speed, the culture temperature, the culture period, the light intensity and the turbidity detection data.

[0080] The application also provides a method for high-efficiency targeted separation of environmental functional microorganisms by using the device.

[0081] S1, preparing the environmental functional microorganism sample to be separated and performing microencapsulation pretreatment:

[0082] The sample containing the target environmental functional microorganism in different environments (such as river water, seawater, activated sludge, soil and the like) is first wrapped by the microencapsulation treatment technology, and then the microencapsulation treatment microorganism sample is transferred into the sterile test tube 16 together with the original environmental culture solution of the sample and placed on the test tube rack on the rotating disc in the device, the test tube rack is fixed in the eight limiting holes on the rotating disc, and the numbers 1-8 are marked on the rotating disc beside the limiting holes (not shown in the figure), which correspond to the turbidity results of each test tube recorded in the subsequent detection stage; the target degradable substance needs to be added as the carbon source in the aforementioned original environmental culture solution;

[0083] S2, the following culture parameters are set through the control panel on the box of the device: culture temperature, culture light intensity, rotating mode of the rotating culture mechanism, culture period, turbidity detection time interval and turbidity target value;

[0084] The culture temperature and culture light intensity parameters are set based on the original environment of different environmental functional microorganisms: culture temperature: 0-40℃, culture light intensity: 0-10000lux; the rotating mode of the rotating culture mechanism includes the rotating mode in the culture stage and the rotating mode in the detection stage, the rotating mode in the culture stage is set as horizontal circumferential reciprocating rotation at a certain angle (such as 90°), the rotating angle adjustment range is 0-180°, and the single culture period is set as 1h; the rotating mode in the detection stage is set as continuous rotation at a certain time interval (such as 20-50s); the turbidity target value is set as OD600=1;

[0085] S3, after the parameters are set, the total switch is started to start the culture, which is specifically divided into a periodic culture stage and a detection stage, and the turbidity of the sample liquid in each test tube is detected every 1h to obtain the value;

[0086] S4, before detection, ensure that the rotating shaft is reset, control the rotating shaft to rotate 360° circumferentially, and stop and stay for 50S when each test tube in the 1 limiting hole on the turntable is parallel to the turbidity detector; the determination method is infrared light detection; the turbidity emission unit and the turbidity receiving unit are initially parallel to the test tube in the No. 1 limiting hole on the turntable, the controller controls the turbidity emission unit to start, infrared light is observed through the sample liquid in the test tube in the No. 1 limiting hole, and the turbidity receiving unit receives the infrared light, the output end transmits the signal to the controller, and the turbidity detection value of the No. 1 test tube is recorded, then the turntable is rotated clockwise to the No. 2 limiting hole, and the turbidity of the No. 2 test tube is detected, and the process is repeated to ensure that the eight test tubes are detected;

[0087] When the first round of turbidity detection value does not reach the set turbidity target value, the culture stage is started again, and the environmental microorganisms are cultured for 1h, when the second round of culture time is over, the detection stage is started, and the process is repeated, when the turbidity target value is reached, the culture is successful by default, and the detection stage is completed;

[0088] In the above detection process, if the turbidity detection value in a test tube meets the standard, the controller will control the alarm to remind, and record the test tube number of the completed culture, and the corresponding test tube can be taken out in time after the detection of this round is completed; the test tubes that do not meet the culture requirements continue to be detected in the next round;

[0089] S5, the test tubes taken out above are actually separated after the detection is completed, and the separation is mainly realized by gravity, that is, the microorganisms in the sample liquid are target environmental functional microorganisms, and the non-target microorganisms in the microcapsules are in the lower part of the sample liquid in the test tube due to gravity.

[0090] The environmental functional microorganism high-efficiency targeted separation device and method provided by the present application will be further described in detail through specific examples.

[0091] Example 1

[0092] As Figures 1-5, show a kind of environmental functional microorganism high-efficiency targeted separation device, the device is based on the foregoing introduction to the structure of the device, the specific size is, the size of the box, i.e. the shell is set to 55cm × 45cm × 45cm, the radius of rotating disc 1 is 12cm, and the limiting hole 15 is 8, and each radius is 1.8cm. A base 18 is supported by a support rod at the bottom of the box, and the aforementioned fixed column 6 is fixedly arranged on the upper middle part of the base, a hollow rotating shaft 9 is movably arranged on the outer side of the fixed column, and a first rotating disc 101 and a second rotating disc 102 are fixedly arranged on the upper and lower parts of the rotating shaft 9 respectively, 8 limiting holes 15 are arranged on each rotating disc, and the first rotating disc is provided with labels numbered 1-8 on the outer side of the limiting hole. The turbidity emitting unit 7 is arranged on the top of the fixed column 6 above the first rotating disc 101, and the turbidity receiving unit 8 is arranged on the right inner wall of the box flush with the turbidity emitting unit. The turbidity receiving unit is supported and fixed by the support frame plate fixed on the side wall of the box. The heating semiconductor 2 and the refrigeration semiconductor 10 are fixed on the left and right side walls of the box respectively, and the heat dissipation fan 4 is arranged on the bottom wall of the box. The operation control panel 13 is arranged on the upper part of the outer side of the box. The supporting leg is arranged on the bottom of the box.

[0093] Referring to Figure 2 、 3 , show the structure reference diagram of test tube rack and test tube, the test tube rack of the structure is matched with the top tube cover of test tube in direction through the concave through slot in upper part, can guarantee the stability of test tube in the process of rotating disc rotation. The above test tube and test tube rack are divided into three different specifications: 10ml, 20ml, 50ml, and different amounts of sample liquid can be placed. The test tube rack and limiting hole 5 can be connected and fixed by clamping; the clamping connection and fixing structure can be frictionally clamped and inserted by the tubular frame body of test tube rack and limiting hole, or additional structure can be arranged between test tube rack and rotating disc, such as clamping convex is arranged on the top of the outer side of test tube rack, and clamping groove is arranged on the first rotating disc outside the limiting hole, so that the test tube rack placed in the limiting hole is more effectively clamped on the rotating disc by the cooperation of clamping convex and clamping groove.

[0094] Referring to Figure 4 、 5In one implementation, the drive connection structure for the aforementioned rotating shaft 9, driven by the servo motor 3 and rotating horizontally circumferentially along the fixed column 6 on the base 18, is as follows: A ball bearing 19 is provided at the bottom of the rotating shaft 9, and the outer ring 191 of the ball bearing is fixedly connected to the rotating shaft 9. Specifically, the outer ring of the ball bearing is fixedly connected to the bottom end of the rotating shaft 9 through a connecting plate 24, and the connecting plate is fixed to the outer ring of the ball bearing by bolts. The inner ring 192 of the ball bearing is fixedly sleeved with the fixed rod 6. A groove 20 is provided in the base 18, and the ball bearing is fitted into the groove. An opening 21 is provided on one side of the groove, and a first gear 22 is fixedly sleeved at the bottom of the outer ring of the ball bearing. The first gear protrudes out of the aforementioned opening, and a second gear 23 is sleeved on the output shaft of the servo motor. The first and second gears mesh with each other. A vertical gap is left between the aforementioned ball bearing and the inner bottom wall of the groove.

[0095] Example 2

[0096] Another structure of the highly efficient targeted separation device for environmental functional microorganisms of the present invention is provided. See also Figure 6 Similar to the device structure in Embodiment 1, except that the rotating culture mechanism is supported and fixed by a support plate located inside the box. Specifically, the support plate is integrally formed by a left side plate 25, a bottom plate 26, and a right side plate 27; the turntable assembly is supported on the bottom plate; the heating semiconductor is provided on the left side plate and the left side wall inside the box, and the cooling semiconductor is provided on the right side wall and the right side wall inside the box; the heat dissipation fan is located on the bottom wall inside the box below the bottom plate; and perforations are provided on the support plate to help to ensure uniform temperature inside the box.

[0097] Example 3

[0098] A method for culturing and isolating target functional microorganisms using the apparatus described in the above embodiments is provided, specifically including three steps: "culturing, detection, and isolation". Before isolation, environmental samples need to be pretreated by microencapsulation.

[0099] This embodiment uses the cultivation and isolation of target environmental functional microorganisms—thiamethoxam-degrading functional microorganisms—as an example. These target environmental functional microorganisms are derived from sludge in river water, and the specific isolation method is carried out according to the following steps:

[0100] S1. Microencapsulation pretreatment:

[0101] Microencapsulation treatment of activated sludge in river water using emulsification and solidification method:

[0102] A. In a 50mL centrifuge tube, take an appropriate amount of mineral oil, add 1.0mL of ABIL EM 90 (cetyl polyethylene glycol / polypropylene glycol-10 / 1 dimethylsiloxane alcohol, water-in-oil emulsifier) ​​and 25μL of Triton X-100, and then make up the volume to 50mL with mineral oil to prepare an emulsion system. Preheat the emulsion system in a water bath at 65℃.

[0103] B, take 1 mL activated sludge supernatant (after standing and settling), centrifuged at 4000 rpm for 5 minutes, washed twice with sterile water, resuspended to 5 mL with sterile water, preheated at 65℃. Dissolve 1 g agar powder in 50 mL distilled water to make a 2% agar solution, mix with an equal volume of pre-treated sludge sample at 65℃ to obtain an agar sludge mixture.

[0104] The above activated sludge supernatant is obtained: pour the mixed activated sludge mixture into a graduated cylinder, stand and settle for a period of time, and the interface between the sludge and water can be seen to separate, the upper layer is the supernatant, use a rubber dropper to suck the supernatant from below the liquid surface.

[0105] C, put 18 mL of the aforementioned preheated emulsion system into a centrifuge tube containing a magnet, stir for 1 minute and preheat. Add 2 mL of the aforementioned agar sludge mixture, stir at room temperature for 5 minutes at 1500 rpm, and stir in an ice bath for 1 minute. Add 20 mL of n-hexane to break the emulsion, stir for 150 seconds, then centrifuge at 1000 rpm for 30 seconds, and recover the precipitate; repeat the emulsion breaking 5 times until the emulsion layer disappears. Finally, wash the precipitate with ultrapure water 3 times (centrifuge at 500 rpm for 10 minutes) to obtain bacterial microcapsules.

[0106] S2, place the bacterial microcapsules and the in-situ environmental culture solution, i.e. river water stock solution, in a test tube, at the same time, based on the target functional microbial species to be separated, add the carbon source, i.e. clothianidin, to the in-situ environmental culture solution, i.e. the culture medium for the culture, and transfer to the device of Example 1.

[0107] S3, set the culture parameters on the device operation control panel: culture temperature 37℃, light-free culture, culture stage culture rotation shaft 90° positive and negative rotation, rotation speed 60 rpm, culture cycle 1h (turbidity detection is performed once every 1h of culture), detection stage rotation shaft angle 45°, rotation mode 360° clockwise rotation, dwell time 20s, target turbidity detection value OD 600 = 1.

[0108] Figure 7 The circuit control module diagram of the method of the application is shown.

[0109] As Figure 8, the microencapsulated sample (blue) is the growth curve of the target environmental functional microorganism - thiamethoxam-degrading bacteria plotted according to the detected absorbance data during the culture process, and the blank sample is the sample without the addition of the carbon source thiamethoxam. It can be seen that the absorbance of the microencapsulated sample is relatively flat within 0-40h, the bacteria are in the lag phase, and after 40h, the absorbance increases logarithmically with time, that is, the bacteria proliferate and break through the microcapsules to grow, and after 80h, the absorbance reaches about 1.0 to enter the stable phase, proving that the culture is complete. Due to the effect of gravity, the bacterial microcapsules in the test tube are separated from the sample solution outside which contains thiamethoxam-degrading bacteria that proliferate and grow.

[0110] Example 4

[0111] The target environmental functional microorganism obtained by the culture separation of Example 3 was detected. After the culture of Example 3 was completed, the functional microorganism population in the test tube was subjected to plate streak culture, and 16S rRNA sequencing was performed to obtain the microbial community structure. The results are shown in Figure 9 , which is the composition of the microbial community structure (genus level). These strains cover the thiamethoxam-degrading bacteria known in the literature, thereby verifying that the microbial community screened from the microencapsulated sample obtained by Example 3 has environmental functionality (degrading thiamethoxam). The thiamethoxam-degradable microorganisms obtained by Example 3 involve Gordonia, Rhodococcus, Brevibacillus, Massilia, etc. (genus level).

[0112] Example 5

[0113] The thiamethoxam-degrading functional microorganism sample liquid screened from the culture of Example 3 was subjected to a degradation experiment to verify that the target environmental functional microorganism obtained by the culture separation of the device has the ability to degrade thiamethoxam.

[0114] The results are shown in Figure 10 , which is the degradation fitting curve of thiamethoxam (concentration 50mg / L). From Figure 10 It can be seen that within 30 days, the microbial community screened from the microencapsulated sample obtained by Example 3 can degrade part of thiamethoxam (degradation rate reaches 60%), and the degradation rate of thiamethoxam is faster in the early stage of culture (0-5d), and the concentration of the pollutant gradually stabilizes within 5-30d, but still maintains a downward trend.

[0115] The above specific embodiments cannot be regarded as a limitation on the protection scope of the present application. Any alternative improvement or change made by those skilled in the art to the embodiments of the present application falls within the protection scope of the present application.

[0116] .

[0117] The details not described in the present application are well-known to those skilled in the art.

Claims

1. A highly efficient targeted separation device for environmental functional microorganisms, characterized in that, include: An environmental sample pre-set culture module includes a rotating culture mechanism, a temperature regulating component, and / or a light source; the rotating culture mechanism includes a drive motor and a turntable assembly for carrying multiple sample tubes. The intelligent detection and feedback adjustment module includes a turbidity emitting unit, a turbidity receiving unit, a controller, a timer, and an alarm. The turbidity receiving unit receives the light signal from the sample tube placed on the turntable assembly through the turbidity emitting unit and transmits it to the controller. When the set target turbidity is reached, the alarm is activated to remind the user. The temperature regulating component, light source, drive motor, turbidity emitting unit, turbidity receiving unit, timer, and alarm are all electrically connected to the controller. The housing contains all the modules mentioned above.

2. The highly efficient targeted separation device for environmental functional microorganisms according to claim 1, characterized in that, The enclosure is transparent or semi-transparent, with a base supported inside and a fixed column fixed on the base. The turntable assembly is movably fitted onto the fixed column, and the turbidity emitting unit is fixed on the fixed column above the turntable assembly. The turbidity receiving unit is disposed on the inner wall of the enclosure, which is horizontally corresponding to the turbidity emitting unit. The turntable assembly is driven to rotate horizontally circumferentially on the fixed column by a drive motor located below it. The drive motor is a servo motor.

3. The highly efficient targeted separation device for environmental functional microorganisms according to claim 2, characterized in that, The turntable assembly includes a rotating shaft sleeved on the fixed column, and two turntables are arranged at intervals on the upper and lower sides of the rotating shaft. Several sets of limiting holes are arranged on each turntable. Every two upper and lower corresponding limiting holes on the two turntables are used to fix the sample tube. The rotating shaft is driven by a servo motor located below it to reciprocate around the fixed column or rotate continuously around the column at certain intervals.

4. The highly efficient targeted separation device for environmental functional microorganisms according to claim 1, characterized in that, The temperature regulating component includes a heater, a cooler, and an auxiliary temperature control component; the light source is natural light.

5. The highly efficient targeted separation device for environmental functional microorganisms according to claim 1, characterized in that, A support plate is installed inside the chamber, the support plate being integrally formed from a left side plate, a bottom plate, and a right side plate; the rotating culture mechanism is supported on the bottom plate; and the temperature control component is installed on the inner wall of the chamber outside the support plate.

6. The highly efficient targeted separation device for environmental functional microorganisms according to claim 1, characterized in that, The sample tube includes a test tube rack that is fixed to the turntable assembly, and test tubes are placed inside the test tube rack.

7. A highly efficient targeted isolation method for environmental functional microorganisms, characterized in that, The process of culturing and separating using the apparatus described in any one of claims 1-6 includes the following steps: S1. Place the microencapsulated pretreated environmental functional microbial sample and in-situ environmental culture medium into a sterilized sample tube, add the target environmental functional microbial carbon source, place the sample tube in the test tube rack and transfer it to the rotating culture mechanism in the box. S2. Set culture parameters: culture temperature, culture light intensity, rotation mode of rotating culture mechanism, culture cycle, turbidity detection time interval, and target turbidity value; The above-mentioned incubation temperature and light intensity parameters are set based on the in-situ environment of different functional microorganisms. The rotation mode of the rotating culture mechanism includes a culture stage rotation mode and a detection stage rotation mode. The culture stage rotation mode is set to a reciprocating rotation at a certain angle in the horizontal circumferential direction, with a single culture cycle set to 1 hour. The detection stage rotation mode is set to a continuous rotation in the horizontal circumferential direction at a certain turbidity detection time interval, with the turbidity detection time interval set to 0-50 seconds. The target turbidity value is set to OD. 600 =1; S3. Based on the turbidity target value detected in the detection phase, control operations are performed to restart the culture phase or end the culture phase: If the turbidity detection value does not reach the target value after the first culture cycle, the culture phase is restarted after the detection is completed, and the "culture-detection" process is repeated until the turbidity detection value of all samples meets the standard; During the detection process, for samples whose detected turbidity detection value meets the standard, the alarm is activated to remind them, and the corresponding sample tube is removed after the corresponding detection phase is completed, while the remaining sample tubes are restarted in the aforementioned "culture-detection" process; S4. For samples whose turbidity detection values ​​meet the standards, the culture is considered complete, and the functional microorganisms isolated from the sample tube are obtained.

8. The method according to claim 7, characterized in that, In step S1, the sample tubes are fixed on the turntable of the rotating culture mechanism, and each sample tube is labeled with a number on the turntable. The corresponding number sequence corresponds to the turbidity detection values ​​in the subsequent tests. The microencapsulation pretreated environmental functional microbial samples are microencapsulated using the emulsification solidification method, and the carbon source is a carbon source that can be used by the target functional environmental microorganisms.

9. The method according to claim 7, characterized in that, The sample tube obtained in step S4 contains functional microorganisms that are located on top of the sample liquid due to gravity, thereby achieving separation from non-environmental microorganisms.

10. The method according to claim 7, characterized in that, The method is divided into three stages: culture, detection, and isolation. The specific operation steps for each stage are as follows: The culture stage is as follows: Open the box of the separation device, mix the microencapsulated pretreated environmental functional microorganisms and the corresponding in-situ environmental culture medium and place them in a sterilized disposable test tube. Fix the test tube to the test tube rack and insert it into the upper limit hole of the rotating culture mechanism. The turntable next to the limit hole is numbered, and the number corresponds synchronously to the sample detection sequence of the detection stage. Based on the in-situ environment of the functional microorganisms being cultured, the culture temperature and light intensity are set via the control panel. Simultaneously, the single culture cycle is set to 1 hour, and the rotating culture mechanism's shaft is set to rotate horizontally back and forth 90° at a speed of 60 rpm within the culture cycle. The turbidity detection time interval and target turbidity value are also set. After setting these parameters, the rotating culture mechanism is started, and its shaft drives the test tubes on the turntable to rotate back and forth 90° during the culture stage according to the set culture speed. At this time, the temperature control and cooling fans are selectively activated based on the set culture temperature, and the light source is selectively activated based on the in-situ environment. The detection process is as follows: After a single culture cycle in the culture stage, the controller restarts turbidity detection. At this time, the culture mechanism is reset, and its upper shaft rotates 360° at intervals of 0-50 seconds. The interval is the turbidity detection time. The turbidity detection values ​​of the sample liquid in each test tube are recorded sequentially to ensure that each test tube is detected. After the detection is completed, all test tubes are returned to their original positions. If the turbidity detection value in this detection does not reach the target turbidity, the controller restarts the aforementioned culture stage and repeats the "culture-detection" process until the sample liquid in all test tubes meets the standard, and the culture is completed. If, in a certain detection stage, there are test tube samples with turbidity detection values ​​that meet the standard, the corresponding test tube is removed after the detection, and the other sample liquids are cultured for the next round. The separation stage: For culture tubes with turbidity detection values ​​that meet the standards, the target environmental functional microorganisms and non-target environmental microorganisms are separated under the influence of gravity.

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