Non-destructive culture medium replacement device and method for microalgae

Abstract

The invention discloses a non-destructive culture medium replacement device and method for microalgae, and relates to the technical field of microbiological treatment. Comprising a frame body, a water storage assembly used for storing pure water and calcium-free seawater is arranged above the frame body, a replacement box is arranged in the middle of the frame body and used for containing a dialysis bag containing algae liquid, and the pure water or the calcium-free seawater can flow in and out at the same flow speed through a pipeline and a flow control valve; a collecting assembly used for receiving water discharged from the replacement box is arranged below the frame body. And two groups of symmetrically arranged transverse frames are fixedly mounted in the middle of the frame body. According to the device, a traditional centrifugal method is replaced by physical isolation and micro-vibration auxiliary replacement of the dialysis bag, a knocking assembly driven by a cam is matched with a spring buffer structure, the movement of liquid in the bag is accelerated, meanwhile, mechanical impact force is controlled within a certain amplitude range, it is ensured that the structure of a microalgae cell membrane is complete, and the survival rate is greatly increased; and a high-activity sample is provided for subsequent calcium signal research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial processing, and particularly relates to a non-injury medium replacement device and method for microalgae. BACKGROUND

[0002] In the research of molecular biology and physiology, especially in the research of ion signal transduction mechanism of microalgae model organisms (such as Chlorella, Chlamydomonas, etc.), it is often necessary to construct an experimental system under a specific ion environment. Among them, the "calcium removal-calcium-free supplement" operation is one of the key steps: that is, first remove Ca²⁺ in the original calcium-containing culture medium, and then introduce calcium-free culture solution to establish a "zero calcium starting point". Traditional methods mostly rely on centrifugal washing or static dialysis, but this way has the following problems: Although the centrifugal method is fast, but the high shear force can easily cause damage to the cell membrane of microalgae, affecting the subsequent activity; Although the static dialysis method is gentle, but the efficiency is very low, and it takes several hours or even overnight, and it is difficult to control the dynamic balance of the concentration gradient of the internal and external solutions; More importantly, both methods cannot complete the continuous automatic process of "salt removal-calcium removal-liquid supplement" in the same system, resulting in complicated operation, high pollution risk and poor repeatability.

[0003] In view of the problems in the related art, no effective solution has been proposed so far.

[0004] Therefore, in order to solve the above problems, the present application provides a non-injury medium replacement device and method for microalgae. SUMMARY

[0005] In order to overcome the above technical problems, the purpose of the present application is to provide a non-injury medium replacement device and method for microalgae.

[0006] The purpose of the present application can be realized by the following technical solutions: A non-injury medium replacement device for microalgae, comprising a frame body, a water storage assembly for storing pure water and calcium-free seawater is arranged above the frame body, a replacement box is arranged in the middle of the frame body, the replacement box is used to place a dialysis bag containing algal liquid, and the pure water or calcium-free seawater can enter or exit at the same flow rate through a pipeline and a flow control valve, and a collection assembly for receiving water discharged from the replacement box is arranged below the frame body; Two groups of symmetrically arranged cross frames are fixedly installed in the middle of the frame body, the replacement box is arranged above the cross frames, and a moving assembly is further arranged in the middle of the frame body to enable the replacement box to move horizontally along the cross frames, so as to facilitate the workers to take and place the dialysis bag through the movement of the replacement box; A placing rack for placing the dialysis bag is installed in the inside of the replacement box; The replacement box is provided with a knocking assembly for intermittently and gently knocking the middle region of the dialysis bag, so as to accelerate the liquid movement in the dialysis bag and improve the replacement efficiency; and the replacement box is further provided with a buffer assembly matched with the knocking assembly, so that the force receiving area of the dialysis bag when being knocked can be increased, the knocking force can be dispersed, the local force of the dialysis bag can be prevented from being too large to cause damage, and the disturbance effect of the knocking on the liquid in the bag can be enhanced.

[0007] As a preferred technical scheme of the present application, the water storage assembly comprises symmetrically distributed pure water tanks and calcium-free water tanks, the pure water tanks and the calcium-free water tanks are installed on the frame body through fixing frames, and the top ends of the pure water tanks and the calcium-free water tanks are connected with water inlet pipes connected with external pure water sources and calcium-free seawater sources respectively.

[0008] As a preferred technical scheme of the present application, the bottom end of the pure water tank is installed with a first water pipe, the first water pipe is installed with a first valve, and the bottom end of the first water pipe is installed with a first corrugated expansion pipe; the bottom end of the calcium-free water tank is installed with a second water pipe, the second water pipe is installed with a second valve, and the bottom end of the second water pipe is installed with a second corrugated pipe.

[0009] As a preferred technical scheme of the present application, the collection assembly comprises first and second collection tanks arranged on the frame body, the first collection tank is used for receiving water in the pure water tank, and the second collection tank is used for receiving water in the calcium-free water tank.

[0010] As a preferred technical scheme of the present application, the bottom end of the replacement tank is installed with two groups of third water pipes, one group of the third water pipes is used for discharging used pure water in the replacement tank to the first collection tank, the other group of the third water pipes is used for discharging used calcium-free seawater in the replacement tank to the second collection tank, and the two groups of the third water pipes correspond to the first and second collection tanks respectively, the third water pipes are installed with third valves respectively, and the bottom ends of the third water pipes are installed with third corrugated pipes respectively.

[0011] As a preferred technical scheme of the present application, the moving assembly comprises a mounting seat fixedly installed at the middle part of the frame body, an electric push rod is fixedly installed on one side of the mounting seat, and the output end of the electric push rod is connected with the outer wall of the replacement tank; the moving assembly further comprises four groups of first rollers installed at the four corners of the bottom end of the replacement tank, and a supporting leg fixedly installed on one side of the bottom of the replacement tank, and the bottom end of the supporting leg is installed with a second roller; the moving assembly further comprises a guide groove opened on the horizontal frame, and the first rollers are arranged in the guide groove and can roll in the guide groove.

[0012] As a preferred technical scheme of the present application, the bottom end of the stand is fixedly provided with a support at each corner, the bottom end of the support is connected to the inner bottom surface of the replacement box, the top surface of the stand is provided with a placement slot for placing the dialysis bag, the bottom of the placement slot is provided with a through slot, the through slot provides a passage for the acting component of the subsequent knocking assembly, both ends of the placement slot are movably connected with a clamping plate for fixing both ends of the dialysis bag through a spring hinge, and a handle is installed on the clamping plate.

[0013] As a preferred technical scheme of the present application, the knocking assembly comprises a motor seat fixedly installed on the replacement box, a motor is fixedly installed on the motor seat, and a first chain wheel is installed on the output end of the motor; the knocking assembly further comprises a rotating rod rotatably connected to the inside of the replacement box, a second chain wheel is installed on one end of the rotating rod, the first chain wheel and the second chain wheel are transmissionally connected through a chain, and a cam is fixedly installed on the middle part of the rotating rod, the position of the cam corresponds to the position of the through slot.

[0014] As a preferred technical scheme of the present application, the buffer assembly comprises a connecting seat distributed at each corner of the through slot, a telescopic support is installed on the connecting seat, a spring is connected to the telescopic support in a sleeved manner, a movable plate is installed on the top end of the telescopic support, the movable plate matches the through slot, and can move up and down along the height direction of the through slot.

[0015] A non-invasive medium replacement method for microalgae, comprising the following steps: S1, cut a dialysis bag with a length of 10-15 cm and a molecular weight cut-off of 14000 Da, wash the dialysis bag in flowing tap water for 3-4 hours to remove glycerol, then tie one end of the dialysis bag and seal it, turn over the dialysis bag so that the knot is inside, and sterilize the dialysis bag at 121℃ for 20min, and then cool it to room temperature after sterilization; S2, take 30mL of algal liquid in the logarithmic growth phase and put it into the dialysis bag, then place the dialysis bag on the placement stand inside the replacement box, and fix the dialysis bag through the clamping plates at both ends; S3, first close the third valve on the third water pipe above the first collection box, open the first valve, and the pure water in the pure water tank enters the inside of the replacement box through the first water pipe and the first corrugated expansion pipe until the dialysis bag is completely immersed in water, then open the third valve to make the water in the replacement box flow into the first collection box through the third water pipe and the third corrugated pipe, at this time, by adjusting the first valve and the corresponding third valve, the water inflow rate of the first water pipe and the water outflow rate of the third water pipe are made the same, so that the volume of water in the replacement box is maintained stable and continuously updated, thereby maintaining the salinity in the replacement box close to 0PSU; S4, start the motor, the motor drives the first sprocket to rotate, the first sprocket cooperates with the chain to drive the second sprocket to rotate, the second sprocket drives the rotating rod to rotate, the rotating rod drives the cam to rotate, the rotation of the cam can intermittently contact with the movable plate, when the cam lifts the movable plate, the dialysis bag can slightly shake, thereby the movement of the liquid in the dialysis bag can be accelerated, and the replacement efficiency is improved; S5, taking the salinity of the algae liquid in the dialysis bag as an index, the salt ions in the bag are basically removed; S6, after desalination is completed, the first valve is closed, and the water in the replacement tank is completely discharged, then the third valve is closed, then the second valve is opened at the same flow rate as the first valve, so that the calcium-free water in the calcium-free water tank enters the inside of the replacement tank, until the dialysis bag containing the algae liquid needing to be replaced by calcium-free ions is completely immersed in water, then the third valve on the third water pipe above the second collection tank is opened, so that the water in the replacement tank can flow into the second collection tank through the third water pipe and the third corrugated pipe, at this time, the flow rates of the second water pipe and the third water pipe are the same, so that the volume of the water in the replacement tank is maintained stable and continuously updated, and the culture liquid containing calcium ions in the dialysis bag is replaced by calcium-free ion culture liquid, in this process, the operation in S4 is repeated; S7, harvesting mother liquor: carefully open the dialysis bag in the super-clean bench, and use a sterile pipette or a pipette gun to suck out the algae liquid after replacement and balance, as the mother liquid of algal cells for inoculation, this process should be as complete as possible to reduce the residual calcium ions and old culture medium metabolites on the surface and around the algal cells.

[0016] Compared with the prior art, the present application has the following beneficial effects: 1. In the present application, the dialysis bag is physically isolated and the auxiliary replacement is assisted by micro-vibration, instead of the traditional centrifugal method, the knocking assembly driven by the cam cooperates with the spring buffer structure, the mechanical impact force is controlled within a certain amplitude range while accelerating the movement of the liquid in the bag, the structure of the microalgae cell membrane is ensured to be complete, and the survival rate is greatly improved, thereby providing high-activity samples for subsequent calcium signal research.

[0017] 2. In the present application, each functional unit is arranged in layers, has high integration and small floor area, and is suitable for routine use in small and medium-sized laboratories. The corrugated pipes, rollers and guide rails are designed to consider the flexibility and sealing safety of movement.

[0018] 3. In the present application, the method obtains the algal cells without old culture medium and metabolites in a gentle way, and the damage to the algal cells is small, the method adopts continuous replacement, can utilize the concentration gradient, and has the effect of efficiently removing a large amount of calcium. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description only need to be some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0020] Figure 1 It is a schematic view of the overall structure of the present application. Figure 2 It is a front view of the present application. Figure 3 It is a schematic view of the moving assembly structure of the present application. Figure 4 It is a schematic view of the knocking assembly structure of the present application. Figure 5 It is a schematic view of the knocking assembly and the placing rack structure of the present application. Figure 6 It is a schematic view of the buffering assembly structure of the present application.

[0021] Reference signs: 1, rack body; 2, water storage assembly; 21, pure water tank; 22, first water pipe; 23, first valve; 24, first corrugated expansion pipe; 25, calcium-free water tank; 26, second water pipe; 27, second valve; 28, second corrugated pipe; 29, fixing frame; 3, cross frame; 4, replacement tank; 41, third water pipe; 42, third valve; 43, third corrugated pipe; 5, moving assembly; 51, mounting seat; 52, electric push rod; 53, first roller; 54, supporting leg; 55, second roller; 56, guide rail groove; 6, placing rack; 61, support column; 62, through groove; 63, clamping plate; 64, handle; 7, knocking assembly; 71, motor seat; 72, motor; 73, first sprocket; 74, rotating rod; 75, second sprocket; 76, chain; 77, cam; 8, buffering assembly; 81, connecting seat; 82, telescopic supporting column; 83, spring; 84, movable plate; 9, collecting assembly; 91, first collecting tank; 92, second collecting tank. DETAILED DESCRIPTION

[0022] The application will be further described below in combination with the accompanying drawings and specific embodiments: Please refer to Figure 1According to the embodiment of the application, a non-invasive medium replacement device for microalgae includes a frame body 1, a water storage assembly 2 arranged above the frame body 1 for storing pure water and calcium-free seawater, a replacement box 4 arranged in the middle of the frame body 1 for placing a dialysis bag containing algal liquid and allowing pure water or calcium-free seawater to flow in and out at the same speed through pipelines and flow control valves, and a collection assembly 9 arranged below the frame body 1 for receiving water discharged from the replacement box 4. The frame body 1 provides a mounting base and a stable working platform for all components such as the water storage assembly 2, the replacement box 4, and the collection assembly 9, integrates all functional modules into one, saves space, and facilitates operation and maintenance. The replacement box 4 is used for placing a dialysis bag containing algal liquid and containing pure water or calcium-free seawater for replacement, thereby realizing the dialysis process. Please refer to Figure 1 and Figure 3 Two groups of symmetrical transverse frames 3 are fixedly installed in the middle of the frame body 1, the replacement box 4 is arranged above the transverse frames 3, and a moving assembly 5 is further arranged in the middle of the frame body 1 to allow the replacement box 4 to horizontally move along the transverse frames 3. Through the movement of the replacement box 4, the staff can conveniently take and place the dialysis bag. The transverse frames 3 are used for installing and supporting the replacement box 4 and providing a track base for the moving assembly 5. Please refer to Figure 4 and Figure 5 A placing rack 6 is installed in the replacement box 4 to place the dialysis bag. The placing rack 6 provides a platform for placing the dialysis bag. Please refer to Figure 4 , Figure 5 and Figure 6 A knocking assembly 7 is arranged on the replacement box 4 to intermittently and gently knock the middle region of the dialysis bag, so as to accelerate the movement of the liquid in the dialysis bag and improve the replacement efficiency. A buffer assembly 8 is further arranged on the replacement box 4 and cooperates with the knocking assembly 7. Through the buffer assembly 8, the stress area of the dialysis bag when being knocked can be increased, so as to disperse the knocking force, avoid damage to the dialysis bag caused by excessive local stress, enhance the disturbance effect of the knocking on the liquid in the dialysis bag, replace manual shaking, save manpower, and control the knocking frequency and intensity. The knocking assembly 7 acts on the dialysis bag through the buffer assembly, thereby avoiding damage to the microalgae caused by direct rigid knocking.

[0023] Please refer to Figure 1 The water storage assembly 2 includes symmetrical pure water tanks 21 and calcium-free water tanks 25. The pure water tanks 21 and the calcium-free water tanks 25 are installed on the frame body 1 through fixing frames 29. The fixing frames 29 rigidly connect the water tanks and the frame body 1, offset the vibration during device operation, make the water tanks not shake, ensure stable water supply pressure, avoid unstable liquid inflow caused by liquid level fluctuation, and connect the top ends of the pure water tanks 21 and the calcium-free water tanks 25 with water inflow pipes respectively connected with external pure water sources and calcium-free seawater sources.

[0024] Please refer to Figure 1 and Figure 2 The bottom end of the pure water tank 21 is provided with a first water pipe 22, the first water pipe 22 is provided with a first valve 23, the bottom end of the first water pipe 22 is provided with a first corrugated expansion pipe 24, the bottom end of the calcium-free water tank 25 is provided with a second water pipe 26, the second water pipe 26 is provided with a second valve 27, the bottom end of the second water pipe 26 is provided with a second corrugated pipe 28, the valve controls the on-off and flow rate of the replacement liquid, and the liquid inlet requirement of the subsequent replacement tank can be accurately matched by adjusting the valve opening, so as to avoid liquid overflow of the replacement tank 4 due to too fast liquid inlet or low replacement efficiency due to too slow liquid inlet; the corrugated expansion pipe has the structural characteristics of being stretchable and bendable, and can adapt to the displacement of the replacement tank 4; when the replacement tank 4 enters and exits the rack body, the corrugated expansion pipe can be retracted, so that it does not hinder the replacement tank 4 and does not limit the movement range of the replacement tank.

[0025] Please refer to Figure 1 The collection assembly 9 includes a first collection tank 91 and a second collection tank 92 arranged on the rack body 1, the first collection tank 91 is used for receiving water in the pure water tank 21, the second collection tank 92 is used for receiving water in the calcium-free water tank 25, the first collection tank 91 and the second collection tank 92 realize classified recovery of different waste liquids (desalination waste liquid, calcium-free replacement waste liquid), avoid mixing of the two kinds of waste liquids, and do not need to additionally separate salt-containing and calcium-containing waste liquids during subsequent treatment, thereby reducing environmental protection treatment cost.

[0026] Please refer to Figure 1 and Figure 2 The bottom end of the replacement tank 4 is provided with two groups of third water pipes 41, one group of the two groups of third water pipes 41 is used for discharging used pure water in the replacement tank 4 to the first collection tank 91, the other group of the two groups of third water pipes 41 is used for discharging used calcium-free seawater in the replacement tank 4 to the second collection tank 92, and the two groups of third water pipes 41 correspond to the first collection tank 91 and the second collection tank 92 respectively, the third water pipes 41 are provided with third valves 42, and the bottom ends of the third water pipes 41 are provided with third corrugated pipes 43; the corresponding structure of the double-pipe line and the double collection tank realizes directional discharge of the waste liquid, when the device is in the desalination stage, the pipe line corresponding to the first collection tank 91 is opened, when the device is in the calcium-free replacement stage, the pipe line corresponding to the second collection tank 92 is opened, so that the waste liquid does not flow in the wrong direction, and the accuracy of classified recovery is ensured; the third valve 42 controls the water discharge flow rate, and the water inlet flow rate is equal to the water discharge flow rate through linkage adjustment of the valve and the water storage pipe line, so that the liquid level in the replacement tank 4 is always stable, and the dialysis bag is prevented from being exposed to air due to liquid level fluctuation.

[0027] Please refer to Figure 1 and Figure 3The moving assembly 5 comprises a mounting seat 51 fixedly installed in the middle of the frame body 1, one side of the mounting seat 51 is fixedly installed with an electric push rod 52, the output end of the electric push rod 52 is connected with the outer wall of the replacement tank 4; the moving assembly 5 further comprises four groups of first rollers 53 installed at the four corners of the bottom end of the replacement tank 4, and a supporting leg 54 fixedly installed at one side of the bottom of the replacement tank 4, the bottom end of the supporting leg 54 is installed with a second roller 55; the moving assembly 5 further comprises a guide rail groove 56 opened on the cross frame 3, the first rollers 53 are arranged inside the guide rail groove 56 and can roll in the guide rail groove 56, the electric push rod 52 provides automatic power to replace manual pushing and pulling, reduces the operation strength, and the pushing force is uniform, avoiding the replacement tank 4 from tilting due to uneven manual force; the matching structure of the first rollers 53 and the guide rail groove 56 limits the moving track of the replacement tank 4, ensures that it enters and exits the frame body 1 along a straight line, avoids jamming or deviation during displacement, and protects the pipeline and device components; the structure of the supporting leg 54 and the second roller 55 disperses the weight of the replacement tank, avoids the abrasion of the first rollers 53 caused by excessive force, prolongs the service life of the rollers and the guide rail groove, and makes the replacement tank 4 more stable during movement; compared with the traditional manual pulling design, the moving structure realizes automatic and stable displacement, which improves the operation efficiency and reduces the risk of device failure.

[0028] Please refer to Figure 4 and Figure 5 The bottom corners of the placement rack 6 are fixedly installed with supporting columns 61, the bottom ends of the supporting columns 61 are connected with the inner bottom surface of the replacement tank 4, the top surface of the placement rack 6 is provided with a placement groove for placing the dialysis bag, the bottom of the placement groove is provided with a through groove 62, the through groove 62 provides a channel for the acting components of the knocking assembly 7, both ends of the placement groove are movably connected with clamping plates 63 for fixing both ends of the dialysis bag through spring hinges, handles 64 are installed on the clamping plates 63, the supporting columns 61 lift the placement rack 6, so that the dialysis bag can be completely immersed in the liquid in the replacement tank 4, ensuring sufficient contact between the dialysis bag and the replacement fluid, and avoiding local non-replacement; the placement groove positions the dialysis bag to prevent it from shifting during knocking, ensuring that the knocking assembly 7 can act on the dialysis bag accurately, and avoiding uneven replacement caused by deviation; the through groove 62 provides a space for the cam and the movable plate of the buffer assembly of the knocking assembly 7 to pass through, so that the knocking force can directly act on the bottom of the dialysis bag, avoiding disturbance failure caused by the placement rack 6 blocking; the movable clamping plates 63 fix both ends of the dialysis bag to prevent the algae liquid in the bag from spilling due to shaking, avoiding loss and pollution of the algae liquid, and the handles 64 are designed to facilitate the staff to quickly fix or disassemble the dialysis bag.

[0029] Please refer to Figure 4 and Figure 5, the knocking assembly 7 includes a motor seat 71 fixedly installed on the replacement box 4, a motor 72 fixedly installed on the motor seat 71, and a first sprocket 73 installed at the output end of the motor 72; the knocking assembly 7 further includes a rotating rod 74 rotatably connected inside the replacement box 4, a second sprocket 75 installed at one end of the rotating rod 74, and a transmission structure of a chain 76 connecting between the first sprocket 73 and the second sprocket 75; a cam 77 is fixedly installed at the middle part of the rotating rod 74, and the position of the cam 77 corresponds to the position of the through slot 62; the motor 72 provides a stable power source, and the transmission structure of the sprocket and the chain transmits the rotary motion of the motor 72 to the rotating rod 74; compared with gear transmission, chain transmission is more suitable for the space inside the replacement box 4, and is more convenient to install and maintain; the structure of the rotating rod 74 and the cam 77 converts the rotary motion into reciprocating lifting action, and the eccentric design of the cam 77 allows it to intermittently contact the movable plate of the buffer assembly when rotating, thereby realizing intermittent knocking of the dialysis bag, accelerating the convection of the liquid in the bag, and compared with traditional manual shaking or magnetic stirring, the knocking structure realizes automatic and controllable disturbance, improves the replacement efficiency, avoids inconsistency of manual operation, and has stable power transmission and low fault risk.

[0030] Please refer to Figure 6 , the buffer assembly 8 includes a connecting seat 81 distributed at four corners of the through slot 62, an extension strut 82 installed on the connecting seat 81, a spring 83 connected to the extension strut 82 in a sleeved manner, and a movable plate 84 installed at the top end of the extension strut 82; the movable plate 84 is matched with the through slot 62 and can move up and down along the height direction of the through slot 62; the combination of the extension strut 82 and the spring 83 provides elastic buffering, avoids damage to the dialysis bag or rupture of the microalgae cells caused by rigid contact; the movable plate converts the “point contact” of the cam into “surface contact”, increases the stress area of the dialysis bag, and makes the knocking force uniformly act on the bottom of the dialysis bag, thereby avoiding cell damage caused by excessive local stress; compared with the rigid knocking design without buffering, the structure solves the problem of microalgae damage caused by mechanical disturbance through elastic buffering and surface contact, while not affecting the replacement efficiency.

[0031] A non-damage medium replacement method for microalgae includes the following steps: S1, cut a dialysis bag with a length of 10-15 cm and a molecular weight cut-off of 14000 Da, wash the dialysis bag in flowing tap water for 3-4 hours to remove glycerol, then tie one end of the dialysis bag and seal it, turn over the dialysis bag so that the knot is inside, and sterilize the dialysis bag at 121℃ for 20min, and then cool it to room temperature after sterilization; S2, take 30mL of algal liquid in the logarithmic growth phase and put it into the dialysis bag, then place the dialysis bag on the placing rack 6 inside the replacement box 4, and fix the dialysis bag through the clamping plates 63 at both ends; S3, first close the third valve 42 on the third water pipe 41 above the first collection tank 91, open the first valve 23, the pure water in the pure water tank 21 enters the inside of the displacement tank 4 through the first water pipe 22 and the first corrugated expansion pipe 24, until the dialysis bag is completely immersed in water, then open the third valve 42, so that the water in the displacement tank 4 can flow into the first collection tank 91 through the third water pipe 41 and the third corrugated pipe 43, at this time, by adjusting the first valve 23 and the corresponding third valve 42, the water inflow rate of the first water pipe 22 and the water outflow rate of the third water pipe 41 are the same, so that the volume of water in the displacement tank 4 is maintained stable and continuously updated, thereby maintaining the salinity in the displacement tank 4 close to 0 PSU; S4, start the motor 72, the motor 72 drives the first sprocket 73 to rotate, the first sprocket 73 cooperates with the chain 76 to drive the second sprocket 75 to rotate, the second sprocket 75 drives the rotating rod 74 to rotate, the rotating rod 74 drives the cam 77 to rotate, the rotation of the cam 77 can intermittently contact with the movable plate 84, when the cam 77 lifts the movable plate 84, the dialysis bag can slightly shake, thereby accelerating the movement of the liquid in the dialysis bag and improving the displacement efficiency; S5, take the salinity of the algal liquid in the dialysis bag as an index, judge whether the salt ions in the bag have been basically removed; S6, after desalination is completed, close the first valve 23, and make the water in the displacement tank 4 flow out completely, then close the third valve 42, and then open the second valve 27 at the same flow rate as the first valve 23, so that the calcium-free water in the calcium-free water tank 25 enters the inside of the displacement tank 4, until the dialysis bag containing the algal liquid needing to be replaced by calcium-free ions is completely immersed in water, then open the third valve 42 on the third water pipe 41 above the second collection tank 92, so that the water in the displacement tank 4 can flow into the second collection tank 92 through the third water pipe 41 and the third corrugated pipe 43, at this time, the flow rates of the second water pipe 26 and the third water pipe 41 are the same, so that the volume of water in the displacement tank 4 is maintained stable and continuously updated, and the dialysis bag is replaced by calcium-free ion culture solution, in this process, the operation in S4 is repeated; S7, harvest the mother liquor: carefully open the dialysis bag in the super clean bench, use a sterile pipette or a pipette gun to suck out the algal liquid after displacement and balance, as the algal cell mother liquor for inoculation, this process should be as complete as possible to reduce the calcium ions and old culture medium metabolites remaining on the surface and around the algal cells.

[0032] The working principle of the non-invasive medium replacement device and method for microalgae is as follows: the dialysis bag containing algal liquid is placed on the placing rack 6, the two ends of the dialysis bag are fixed by the clamping plate 63, then the electric push rod 52 is started to be retracted, thereby driving the replacement box 4 to move to the inside of the rack body 1 until the replacement box 4 is moved to a position corresponding to the water storage assembly 2 and the collection assembly 9, then the first water pipe 22 is opened (at this time, the third valve 42 is in a closed state), the pure water in the pure water tank 21 flows into the inside of the replacement box 4 through the first water pipe 22 and the first corrugated expansion pipe 24 until the dialysis bag is completely immersed in water, then the third valve 42 above the first collection tank 91 is opened, so that the water in the replacement box 4 can flow into the first collection tank 91 through the third water pipe 41 and the third corrugated pipe 43, at this time, by adjusting the first valve 23 and the corresponding third valve 42, the water inflow rate of the first water pipe 22 and the water outflow rate of the third water pipe 41 are made to be the same, so that the volume of the water in the replacement box 4 is maintained stable and continuously updated, thereby maintaining the salinity in the replacement box 4 close to 0 PSU, in the process, the motor 72 is started, the motor 72 drives the first sprocket 73 to rotate, the first sprocket 73 cooperates with the chain 76 to drive the second sprocket 75 to rotate, the second sprocket 75 drives the rotating rod 74 to rotate, the rotating rod 74 drives the cam 77 to rotate, the rotation of the cam 77 can intermittently contact the movable plate 84, when the cam 77 lifts the movable plate 84, the dialysis bag can slightly shake, thereby accelerating the movement of the liquid in the dialysis bag and improving the replacement efficiency, after the desalination operation is completed, the first valve 23 is closed, and the water in the replacement box 4 is completely discharged, then the third valve 42 is closed, then the second valve 27 is opened, so that the calcium-free water in the calcium-free water tank 25 enters the inside of the replacement box 4 until the dialysis bag is completely immersed in water, then the third valve 42 on the third water pipe 41 above the second collection tank 92 is opened, so that the water in the replacement box 4 can flow into the second collection tank 92 through the third water pipe 41 and the third corrugated pipe 43, at this time, the flow rates of the second water pipe 26 and the third water pipe 41 are the same, so that the volume of the water in the replacement box 4 is maintained stable and continuously updated, and the culture solution containing calcium ions in the dialysis bag is replaced by the culture solution without calcium ions.

[0033] In the description of the present application, it should also be noted that, unless otherwise explicitly specified and limited, the terms "connected", "connected" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium. For those skilled in the art, the specific meaning of the above-mentioned terms in the present application can be understood according to the specific circumstances.

[0034] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that the technical solutions described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalent ones. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A device for non-invasive medium replacement of microalgae comprising a frame (1), characterized in that: The upper part of the frame body (1) is provided with a water storage assembly (2) for storing pure water and calcium-free seawater, the middle part of the frame body (1) is provided with a replacement box (4), the replacement box (4) is used for placing a dialysis bag containing algal liquid, and pure water or calcium-free seawater can be made to flow in and out at the same flow rate through pipelines and flow control valves, and the lower part of the frame body (1) is provided with a collection assembly (9) for receiving water discharged from the replacement box (4). The middle part of the frame body (1) is fixedly provided with two groups of symmetrically arranged cross frames (3), the replacement box (4) is arranged above the cross frames (3), and the middle part of the frame body (1) is further provided with a moving assembly (5) enabling the replacement box (4) to horizontally displace along the cross frames (3), and through the movement of the replacement box (4), the staff can conveniently take and place the dialysis bag. The inside of the replacement box (4) is provided with a placing rack (6) for placing the dialysis bag. The replacement box (4) is provided with a knocking assembly (7) for intermittently and gently knocking the middle part of the dialysis bag, so as to accelerate the liquid movement in the dialysis bag and improve the replacement efficiency, and the replacement box (4) is further provided with a buffer assembly (8) matched with the knocking assembly (7), and through the buffer assembly (8), the stress area of the dialysis bag when being knocked can be increased, so that the knocking force is dispersed, the local damage of the dialysis bag caused by excessive stress is avoided, and the disturbance effect of the knocking on the liquid in the bag is enhanced.

2. A device for non-injurious medium replacement of microalgae according to claim 1, characterized in that: The water storage assembly (2) comprises symmetrically distributed pure water tanks (21) and calcium-free water tanks (25), the pure water tanks (21) and the calcium-free water tanks (25) are installed on the frame body (1) through fixing frames (29), and the top ends of the pure water tanks (21) and the calcium-free water tanks (25) are connected with water inlet pipes connected with external pure water sources and calcium-free seawater sources respectively.

3. A device for non-injurious medium replacement of microalgae according to claim 1, characterized in that: The bottom end of the pure water tank (21) is provided with a first water pipe (22), the first water pipe (22) is provided with a first valve (23), and the bottom end of the first water pipe (22) is provided with a first corrugated expansion pipe (24). The bottom end of the calcium-free water tank (25) is provided with a second water pipe (26), the second water pipe (26) is provided with a second valve (27), and the bottom end of the second water pipe (26) is provided with a second corrugated pipe (28).

4. A device for non-injurious medium replacement of microalgae according to claim 1, characterized in that: The collection assembly (9) comprises first and second collection boxes (91) and (92) arranged on the frame body (1), the first collection box (91) is used for receiving water in the pure water tank (21), and the second collection box (92) is used for receiving water in the calcium-free water tank (25).

5. A device for non-injurious medium replacement of microalgae according to claim 4, characterized in that: The bottom end of the replacement box (4) is provided with two groups of third water pipes (41), one group of the two groups of third water pipes (41) is used for discharging used pure water in the replacement box (4) to the first collection box (91), the other group of the two groups of third water pipes (41) is used for discharging used calcium-free seawater in the replacement box (4) to the second collection box (92), and the two groups of third water pipes (41) correspond to the first and second collection boxes (91) and (92) respectively, the third water pipes (41) are provided with third valves (42), and the bottom ends of the third water pipes (41) are provided with third corrugated pipes (43).

6. A device for non-injurious medium replacement of microalgae according to claim 1, characterized in that: The moving assembly (5) comprises a mounting seat (51) fixedly installed in the middle of the frame body (1), one side of the mounting seat (51) is fixedly installed with an electric push rod (52), and the output end of the electric push rod (52) is connected with the outer wall of the replacement box (4); The moving assembly (5) further comprises four groups of first rollers (53) installed at the four corners of the bottom end of the replacement box (4), and a supporting leg (54) fixedly installed at one side of the bottom of the replacement box (4), and the bottom end of the supporting leg (54) is installed with a second roller (55); The moving assembly (5) further comprises a guide rail groove (56) opened on the cross frame (3), and the first roller (53) is arranged in the guide rail groove (56) and can roll in the guide rail groove (56).

7. A device for non-injurious medium replacement of microalgae according to claim 1, characterized in that: The bottom end of the placing rack (6) is fixedly installed with a support column (61) at four corners, the bottom end of the support column (61) is connected with the inner bottom surface of the replacement box (4), the top surface of the placing rack (6) is provided with a placing groove for placing the dialysis bag, the bottom of the placing groove is provided with a through groove (62), the through groove (62) provides a channel for the acting component of the subsequent knocking assembly (7), and the two ends of the placing groove are both movably connected with a clamping plate (63) for fixing the two ends of the dialysis bag through a spring hinge, and the clamping plate (63) is installed with a handle (64).

8. A device for non-injurious media replacement of microalgae according to claim 7, characterized in that: The knocking assembly (7) comprises a motor seat (71) fixedly installed on the replacement box (4), the motor seat (71) is fixedly installed with a motor (72), and the output end of the motor (72) is installed with a first sprocket (73); The knocking assembly (7) further comprises a rotating shaft (74) rotatably connected in the replacement box (4), one end of the rotating shaft (74) is installed with a second sprocket (75), the first sprocket (73) and the second sprocket (75) are transmissionally connected through a chain (76), the middle part of the rotating shaft (74) is fixedly installed with a cam (77), and the position of the cam (77) corresponds to the position of the through groove (62).

9. A device for non-injurious media replacement of microalgae according to claim 7, characterized in that: The buffer assembly (8) comprises a connecting seat (81) distributed at four corners of the through groove (62), the connecting seat (81) is installed with a telescopic support column (82), the telescopic support column (82) is connected with a spring (83) in a sleeved mode, the top end of the telescopic support column (82) is installed with a movable plate (84), the movable plate (84) is matched with the through groove (62) and can move up and down along the height direction of the through groove (62).

10. A method for non-injurious media replacement of microalgae, characterized in that: The method comprises the following steps: S1, cut 10-15 cm long, and cut the dialysis bag with a molecular weight cut-off of 14000 Da, wash the dialysis bag in flowing tap water for 3-4 hours to remove glycerol, then tie one end of the dialysis bag and seal it, turn over the dialysis bag so that the knot is inside, and sterilize the dialysis bag at 121℃ for 20min, and then cool to room temperature after sterilization; S2, take 30mL of algal liquid in the logarithmic growth phase and put it into the dialysis bag, then place the dialysis bag on the placing rack (6) in the replacement box (4), and fix the dialysis bag through the clamping plates (63) at both ends; S3, first close the third valve (42) on the third water pipe (41) above the first collection tank (91), open the first valve (23), the pure water in the pure water tank (21) enters the inside of the displacement tank (4) through the first water pipe (22) and the first corrugated expansion pipe (24), until the dialysis bag is completely immersed in water, then open the third valve (42), so that the water in the displacement tank (4) can flow into the first collection tank (91) through the third water pipe (41) and the third corrugated pipe (43), at this time, by adjusting the first valve (23) and the corresponding third valve (42), the water inflow rate of the first water pipe (22) and the water outflow rate of the third water pipe (41) are the same, so that the volume of water in the displacement tank (4) is maintained stable and continuously updated, thereby maintaining the salinity in the displacement tank (4) close to 0 PSU; S4, start the motor (72), the motor (72) drives the first sprocket (73) to rotate, the first sprocket (73) drives the second sprocket (75) to rotate through the chain (76), the second sprocket (75) drives the rotating rod (74) to rotate, the rotating rod (74) drives the cam (77) to rotate, the rotation of the cam (77) can intermittently contact with the movable plate (84), when the cam (77) lifts the movable plate (84), the dialysis bag can slightly shake, thereby accelerating the movement of the liquid in the dialysis bag and improving the displacement efficiency; S5, take the salinity of the algae liquid in the dialysis bag as an index, judge whether the salt ions in the bag have been basically removed; S6, after desalination is completed, close the first valve (23), and make the water in the displacement tank (4) flow out completely, then close the third valve (42), then open the second valve (27) at the same flow rate as the first valve (23), so that the calcium-free water in the calcium-free water tank (25) enters the inside of the displacement tank (4), until the dialysis bag containing the algae liquid needing to be replaced with calcium-free ions is completely immersed in water, then open the third valve (42) on the third water pipe (41) above the second collection tank (92), so that the water in the displacement tank (4) can flow into the second collection tank (92) through the third water pipe (41) and the third corrugated pipe (43), at this time, the flow rates of the second water pipe (26) and the third water pipe (41) are the same, so that the volume of water in the displacement tank (4) is maintained stable and continuously updated, and the culture solution containing calcium ions in the dialysis bag is replaced with calcium-free ion culture solution, in this process, the operation in S4 is repeated; S7, harvest the mother liquor: carefully open the dialysis bag in the super clean bench, use a sterile pipette or pipette gun to suck out the algae liquid after displacement and balance, as the inoculum of algal cells, this process should be as complete as possible to reduce the calcium ions and old culture medium metabolites remaining on the surface and around the algal cells.

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