Hardware

Introduction

We attempted to design a petri dish for our experiments on solid and liquid cultures of C. elegans to facilitate C. elegans experiments in the laboratory. During the design study, we found that the current 24-well plate used for C. elegans culture has a mixture of C. elegans, bacterial liquid, and microplastic samples, which is not convenient for molecular detection of each component. At the same time, since the spiked samples are all in the same well, it is not possible to make flexible contact and separation between the bacterial fluid and C. elegans. In addition to this, solid and liquid cultures need to be carried out on different 24-well plates, which does not allow the conversion between solid and liquid. Therefore, we designed the C. elegans solid-liquid culture model, which can meet multiple types of C. elegans experiments in the laboratory.

In addition, in order to link the laboratory scenario with industrial treatment, we came up with the idea of designing a C. elegans-E. coli co-culture fermenter to degrade microplastic wastewater through E. coli secretion of enzymes and primary enrichment, as well as secondary enrichment of E. coli by C. elegans to ultimately achieve biological remediation.

The hardware we designed is intended for applications in laboratory nematode cultures and industrial microplastic wastewater treatment processes.

Section 1: The design of C. elegans culture model

1.1 Overview

In order to solve a series of problems encountered in our C. elegans experiments in both liquid and solid cultures, as well as to provide relevant assistance for the subsequent drug sieving experiments and to further realize our goals, we designed such an integrated device for C. elegans culture and drug sieving. We use filter membranes and dividers to separate C. elegans, E. coli, water samples and pharmaceuticals to make them suitable for our C. elegans culture experiments, as well as easy to disassemble and assemble. Externally, the device is small, square and has excellent portability, optimized based on commercially available 24/96-well plates of common specifications. We have designed a double decker bayonet so that the C. elegans culture plate can be converted between liquid and solid cultures with multiple usage scenarios (Figure 1.1). This culture device can help to further improve and complete experiments.

1.2 Structure

The innovative design of the culture device includes "semi-retention", "solid-liquid conversion" and "partitioned detection" petri dish structure. The modular design links C. elegans culture with molecular testing, and customizes solutions for different culture scenarios. The bottom tray of the device can be sterilized for multiple use, while the top embedded 24/96 well plate facilitates the transfer and detection of C. elegans or samples, and can be discarded and replaced with a new plate after completion of the experiment through special treatment, preventing C. elegans from leaking out and causing biological contamination, and avoiding biosafety issues. The overall structure is basically in line with the general specification of 24/96 well plate, which is suitable for the application of 24/96 well plate related testing instruments (e.g. enzyme labelling instrument). At the same time, the interchangeable design greatly increases the possibility of industrial mass production.

1.2.1 Bottom Bracket Section

This 24-well plate bottom tray is designed to optimize the culture of C. elegans, focusing primarily on separable cultures of bacterial and enzymatic fluids with nematodes. The total height of the bottom tray is 4cm, the vertical and horizontal dimensions are 14.3 * 10.8cm², and the 24 holes at the bottom can be used for independent or repetitive spiking of different experimental variables, such as E. coli (OP50/BL21(detection)/BL21(secretion)), secreted proteins, water samples, PET/TPA suspensions, etc., according to the requirements of the specific experiments. The inner wall of the bottom tray is designed with two catches, the upper catch a can be used for solid culture of nematodes after inserting the spacer and placing a 24-well plate; the lower catch b can be used for liquid culture of nematodes after inserting the spacer and placing a 24-well plate (Figure 1.2). We use transparent polystyrene material for easy observation and excellent biocompatibility for bacterial growth.

1.2.2 Culture Plate Section

The specification of this 24-well culture plate is 12.7*8.4*2.1cm³, against the common 24-well plate on the market, the main difference between the two is the ring-shaped opening at the bottom of each well. For this part of the gap, we attached a layer of cellular filters with a pore size of 5 μm to it as a way to limit the C. elegans activity to the inside of the well plate. When we choose the bayonet a of the bottom tray of the device, the annular opening portion does not come into contact with the liquid in the lower wells, and nematode growth medium can be prepared in 24-well plates for solid culture of C. elegans. When we choose bayonet b of the bottom bracket of the device, the annular opening part contacts the liquid in the lower well, and liquid exchange occurs. At the same time, E. coli in the lower wells can enter the upper wells through the filter, and molecules such as proteins can be exchanged, allowing for liquid culture of the C. elegans. Since the device is removable, the lower wells can be replenished at any time to meet the food requirements of the C. elegans. In addition, the upper and lower devices can be separated for detection, splitting the upper layer to detect the fluorescence status of C. elegans, splitting the lower layer to detect the TPA(phthalic acid) or PETase(polyethylene terephthalate) content or enzyme activity, the fluorescence status of E. coli, and so on. Through different assembling and sampling methods, a variety of solid, liquid, solid-liquid, and liquid-liquid cultivation and detection methods of C. elegans can be realized.

1.2.3 Baffle Section

The specification of the baffle is 13.3*8.8*0.2cm³, which can be adapted to different heights of 24-well plates with different bayonets. One end is designed with a curved handle, which is convenient for the experimenter to extract and move.

1.2.4 Cover Section

The specification of the top cover is 14.3*10.8*1.5cm³, which is made of transparent polystyrene material for easy observation and preventing contamination of stray bacteria for storage and cultivation.

1.3 Workflow

The contents below show the basic usage of our 24/96-well C. elegans culture model.

1.3.1 Preparation

Prepare the required experimental materials, including E. coli, bacterial solution, enzyme solution, C. elegans samples, water samples, and PET/TPA suspension. Ensure that all parts of the 24-well culture device, including the bottom tray, culture plate, partition and top cover, have been sterilized to prevent contamination by stray bacteria.

1.3.2 Assembly

Place the bottom tray portion of the transparent polystyrene material flat on the bench, selecting either bayonet a or b according to the experimental requirements, and use the bayonet to secure the divider to ensure that it is securely bonded to the culture plate.
--If performing a solid culture, select bayonet a and place the 24-well culture plate on top.
--If performing a liquid culture, select bayonet b and place the 24-well culture plate on top.

1.3.3 Sample Addition

(1) Bottom well:
--The bottom tray section allows for independent or duplicate addition of E. coli (e.g. OP50, BL21, etc.), water, PET/TPA suspensions, or other secreted proteins as required for specific experiments in each well.
--If liquid culture is performed, ensure that the bottom pore liquid is able to exchange material with the top pore through the cell strainer.

(2) Upper well:
--If solid culture is performed, prepare NGM medium in the upper 24-well plate and place C. elegans samples.
--If performing a liquid culture, add the liquid medium and the C. elegans sample directly, ensuring that the cell strainer limits the C. elegans' range of motion.

1.3.4 Culture

The samples are cultured either solid or liquid, depending on the experimental requirements. When liquid culture is performed, the E. coli solution in the bottom tray portion can be regularly replenished according to the food requirements of the C. elegans. During the incubation process, the transparent top cover prevents contamination by stray bacteria and facilitates observation of the sample status without opening the unit.

1.3.5 Detection

(1) Solid culture detection: Observe the activity and growth status of C. elegans on NGM medium through microscope.
(2) Liquid culture detection: E. coli activity, enzyme activity in bottom wells can be detected, or TPA enrichment status can be observed by fluorescent labelling.
(3) Partitioning: the upper 24-well plate detects the fluorescence status and growth of C. elegans; the bottom layer detects the fluorescence status of E. coli, the content of TPA/PETase or the concentration of other secreted proteins.

1.3.6 Disassembly and Cleaning

After the experiment, the bottom tray can be sterilized and reused, and the 24-well plate can be discarded or specially treated according to requirements to prevent biological contamination caused by C. elegans leakage.

1.4 Summary

Based on the original 24/96-well plate, this C. elegans culture and drug sieve detection integrated device combines solid-liquid conversion, partition detection and other innovative designs, which significantly improves the flexibility of the experiment and the convenience of operation. Through the modular design, the device is not only suitable for solid and liquid C. elegans culture, but also capable of detecting the interaction between various substances, which is helpful for the in-depth study of the physiological state of C. elegans. Through the partition design, the fluorescence state and biological activity of C. elegans and E. coli can be detected separately, which improves the experimental efficiency. The use of cell filters effectively restricts the activity range of C. elegans, prevents biological contamination and enhances the biosafety of the device.

Section 2: The design of our fermenter

2.1 Overview

In order to help construct a co-culture system for E. coli and C. elegans, and to further our goal of treating microplastic-containing wastewater, we designed this fermenter for bacteria and C. elegans co-culture. The fermenter has multiple components that can adapt to complex co-culture environments to ensure effective growth and interaction between E. coli and C. elegans. The transparent structure of the fermenter facilitates real-time monitoring of the biological reactions inside, as well as the observation of C. elegans activity.

Externally, the fermenter has a simple design, symbolizing the harmony between biotechnology and environmental remediation. The bottom of the unit is piped with a multi-layer filter membrane to ensure ecological safety by ensuring that treated water samples are discharged and preventing any modified microorganisms from being released into the external environment.

The design of this fermenter takes into account the ease of disassembly and assembly, and the modular design makes it easier to clean and maintain, making it suitable for different types of experiments. With this fermenter, we expect to further promote the development of co-culture technology and contribute to the research of microplastic pollution control.

2.2 Structure

The entire fermenter system contains three tanks. The first tank is responsible for the culture of E. coli (secreted) and the secreted pETase is pressed into the second tank by a piston. In the second tank, the initially filtered aqueous humor is discharged into the tank, where it is degraded by the pETase into small molecules TPA and EG. The aqueous humor containing TPA is pressed by a piston into the third tank. The third tank is responsible for the culture of E. coli (enriched) and C. elegans. Inside the tank E. coli collects TPA while C. elegans are transferred to liquid culture after hatching and maturing on a C. elegans culture platform and then enriched for E. coli. The purified water is discharged from the bottom of the third tank, and the outlet has a multi-layer filter membrane to prevent leakage of E. coli and C. elegans.

The fermenter adopts a modular design concept to enhance the adaptability and ease of operation of the fermentation system through flexible structural combinations, providing a customized solution for the co-culture of bacteria and C. elegans, and improving the efficiency of industrial processing. The fermenter is provided with a variety of loadable modules, such as a stirring module to enhance the mixing of the culture medium, a temperature control module to ensure constant culture conditions, and a dissolved oxygen platform to regulate the supply of oxygen in the culture environment.

The modular design of the fermenter significantly reduces costs at the same time. Each module can be produced by ordinary 3D printing, and the manufacturer can replace the components at will according to the industrial processing needs, achieving a high degree of industrial simulation at low cost and providing an ideal research platform for large-scale co-culture experiments.

2.2.1 Piston Section

The piston is located in the upper part of the tank on the left side and allows the internal pressure and volume of the fermenter to be adjusted by vertical movement. The black rubber at the bottom of the piston has the sealing ability to avoid the interaction between the external air and the internal fermentation liquid, ensuring the sterility of the system and preventing contamination by stray bacteria. The transparent structure of the tank facilitates the experimenter to monitor the growth status of the strain and enzyme secretion activities in real time.

2.2.2 Pipe Connection Section

The pipework connection section consists of two sides of butt-jointed transparent pipework, with the left pipework being tightly connected to the right tank by means of swivel joints. The sealing of the joints is well designed to prevent leakage of culture medium or outside air, ensuring the sterility of the system. The insertion port of the membrane at the connection of the tubes allows the experimenter to insert membranes of different pore sizes to filter specific strains or molecules to ensure the purity and safety of the culture.

2.2.3 Tank Section

The tank is composed of a cylinder at the body and a cone at the bottom of the tank, which facilitates the enrichment of some microorganisms, molecules and other sediments at the discharge port. Cylindrical piping on the upper level to facilitate multiple refilling of the fermenter.

2.2.4 Stirring rod section and other components

The stir bar section has a multi-functional integrated design that contains the defoaming unit, temperature control unit and mixing unit, which are linked and controlled by the wheel system above. The sophisticated design of this structure ensures that the key functions of mixing, temperature regulation and defoaming during the fermentation process work in synergy.

The defoaming device is located at the upper end of the stirring bar and is arranged on the crossbar through a plurality of toothed discs. When a large amount of foam is generated during the fermentation process, the defoaming device can pick through the air bubbles by means of the toothed structure, so as to achieve the reduction of the generation of foam, and to avoid the foam from affecting the mixing of the culture liquid and the growth of the bacterial strain and the C. elegans.

The temperature control unit is located in the center of the stirring bar and is used to monitor and regulate the temperature inside the fermenter. The device can precisely control the temperature through built-in sensors to ensure a constant fermentation environment. For temperature-sensitive strains (e.g. modified fluorescent E. coli), the device ensures the right incubation temperature to maximize protease secretion or fluorescent signal expression. The temperature control device is used in conjunction with a stirring device to maintain a homogeneous mixing of the culture broth and to ensure an even distribution of temperature throughout the fermentation broth.

The stirring device is located at the lower end of the stirring rod and consists of a plurality of fan blades, which is capable of continuously stirring the culture medium during the fermentation process, promoting uniform mixing of the fermentation liquid, and maintaining a stable concentration distribution of oxygen, nutrients, and bacterial strains in the culture liquid, thereby enhancing the fermentation efficiency.

2.2.5 Oxygen Dissolving Section

The dissolved oxygen platform consists of two annular pipes with a columnar vent pipe, the annular pipes have several openings on one side, oxygen is passed in by the columnar pipe that extends out of the tank and is discharged through the openings. This design facilitates the delivery of oxygen to the solution in the fermenter and meets the specific oxygen needs of strains such as C. elegans or E. coli.

2.2.6 C. elegans culture platform

The C. elegans culture platform is loaded inside a third fermenter, which is vertically movable through a bayonet inside the fermenter and can be selectively placed in a liquid environment inside the tank. The double bayonet design is inspired by our C. elegans solid-liquid culture model, where we applied the solid-liquid culture concept used in the laboratory to the industrial fermenter to ensure incubation of C. elegans on solid plates and transfer to liquid culture for enrichment of E. coli with TPA.

2.3 Workflow

The contents below show the basic usage of our fermenter model.

2.3.1 Preparation of medium:

Add appropriate amount of medium in the fermenter to ensure uniform distribution of nutrients and provide growth conditions for E. coli and C. elegans.

2.3.2 Loading modules:

Modules such as stirring rods, temperature control devices and dissolved oxygen platforms are installed to ensure precise control of temperature, oxygen and media mixing during the incubation process.

2.3.3 Inoculation of microorganisms:

Inoculate E. coli and Cryptosporidium hidradii C. elegans into the fermenter to ensure aseptic operation and ensure a sterile environment in the fermenter by means of a piston sealing device.

2.3.4 Control and monitoring:

According to the specific situation, transfer the E. coli bacterial liquid from the left tank extrusion to the right tank, activate the stirring device to ensure that the medium is evenly mixed, activate the temperature control module to maintain a constant temperature, and regulate the supply of oxygen through the dissolved oxygen platform, and observe the changes in microbial growth status and reaction.

2.3.5 Treatment and collection:

Filtering and collection of treated microplastic wastewater samples through a funnel and membrane system to avoid any leakage of untreated microorganisms and to ensure ecological safety.

2.3.6 Cleaning and maintenance:

After the experiment, disassemble the modules of the fermenter for cleaning and maintenance to ensure that the fermenter is in the best condition for the next experiment.

2.4 Safety Guarantee

In order to ensure the safety issue of the fermenter, we have made some modifications to its outlet. It ensures that the engineered E. coli and C. elegans are confined inside the fermenter while the treated water samples can be discharged smoothly. On the one hand, it is convenient to reuse the C. elegans and E. coli to enrich the TPA products; on the other hand, it ensures that the C. elegans and the modified engineering bacteria will not cause any harm to the environment to ensure ecological safety.

In the schematic diagram on the left, the fermenter outlet is divided into three sections. The topmost support frame places a 5 μm cell strainer to prevent C. elegans leakage and recycle the C. elegans. The middle support frame holds a 0.22 μm filter membrane to prevent leakage due to C. elegans which not able to enrich all engineered E. coli as the last line of defense. The bottom square platform is a spectrophotometer for detecting the OD value of the solution, which consists of a light source, monochromator, detector, and display system, ensuring that the water samples are free of E. coli.

The schematic diagram on the right shows the exact structure of our spectrophotometer. A light source (e.g. a deuterium lamp) treated with a monochromator provides a stable supply of UV light at a wavelength of 260-280 nm. This light source emits the same two light paths to the detector on the other side of the fermenter outlet, one passing through the sample for the test result and one reaching the detector directly for the reference result. The influence of the environment or fluctuations in the light source on the measurement result is excluded by comparing the intensity difference between the two beams. We use a photomultiplier tube as the detector, which is capable of detecting very weak light signals due to its high sensitivity. In the future, we will try to equip the spectrophotometer with a digital display or computer interface in order to display the results.

2.5 Summary

We have designed a fermenter for the industrial treatment of microplastic-containing wastewater by co-culture of E. coli and C. elegans. The modular design of the fermenter allows for easy adjustment and operation, and can be 3D printed to reduce costs. Its core functions include agitation, temperature control and dissolved oxygen to ensure a stable biological reaction environment during the co-culture process. At the same time, through the filter membrane and funnel system, the fermenter can effectively control the outflow of microorganisms to ensure the biosafety of the research process.

Section 3: Design evolution process

3.1. C. elegans culture model 1.0

Firstly, we tried to achieve the separation of C. elegans detection from culture with the most straightforward idea. Accordingly, we designed a double-layer structure to separate the 24-well plate from the bottom tray to facilitate the addition of different types of samples to the upper and lower layers. At the same time, a 1-cm diameter pipe was designed on one side of the device to facilitate the replenishment of the lower layer. However, the bottom tray of the device was not regionally separated, which made it inconvenient to conduct independent experiments on C. elegans in different wells.

In response to these problems, we made the following adjustments.

3.2. C. elegans culture model 2.0

In the next step, we modified the lower bottom bracket by matching each upper hole with a lower hole cavity, and at the same time endowed the lower cavity with a gradient design to facilitate the rapid replenishment of the 24 wells with liquid when feeding for the first time. And 24 refill ports were designed on both sides of the corresponding position, which is convenient to refill different cavities independently. However, this design is not necessary, which will affect the amount of subsequent replenishment, and adjacent cavities have the potential risk of contaminating each other. At the same time, this design will lead to part of the C. elegans hole depth is too large, not convenient for C. elegans addition and detection. At the same time, the 2*12-well plate design makes the device too narrow and long, which does not match most of the 24-well plate on the market.

In response to the above problems, we made final adjustments and obtained the final hardware design.

Future Plan

For the C. elegans culture model...

In the future, the device has the potential for further development and application in the following aspects: combined with automated operating equipment, such as automatic sampling apparatus and detection equipment, it can further improve the automation of the experiment and reduce the burden of manual operation. At the same time, we can try to make more surface modifications on the polystyrene material to adapt to the cultivation needs of more bacteria, fungi and cells, and expand its application areas. The C. elegans culture model we designed will have some industrial application prospects.

For the fermenter model...

In the future, we will introduce more circuit control elements to assist the stirring system and temperature control system. Meanwhile, we will try to integrate the fluorescence detection system into the fermenter to facilitate our real-time monitoring of TPA enrichment by E. coli and C. elegans. In addition, we will optimize the spectrophotometer at the outlet of the fermenter to improve its detection sensitivity.