Overview

In the current fields of molecular biology, synthetic biology, proteomics, and biopharmaceuticals, microbial cultivation and the extraction of intracellular active substances constitute fundamental and critical experimental steps. However, existing technical workflows face significant bottlenecks:

1. High equipment cost Our field research indicates that mainstream micro-fermentation tank equipment on the market is expensive, creating a significant barrier to entry.

2. Complex operation and challenging real-time monitoring During experiments, we found that real-time monitoring remains difficult with such equipment. Researchers must still invest significant time in manual operations and prolonged monitoring, especially requiring continuous oversight during nighttime hours. This substantially impacts experimental efficiency and increase researcher workload. Furthermore, existing workflows rely on multiple disparate devices working in tandem, resulting in cumbersome procedures with poor reproducibility.

3. High contamination risk Both existing shake flask and micro-fermentation methods require manual sampling, resulting in elevated overall contamination risks that severely compromise research stability and efficiency.

Figure1.1 Overall Presentation Diagram
Figure1.1 Overall Presentation Diagram
Figure1.1 Overall Presentation Diagram

To systematically address these challenges, our project team innovatively proposed the design of an “Integrated Fermentation & Processing System (iFPS)” based on real-world research needs. This system deeply integrates mechatronics and intelligent control technologies, seamlessly combining three core functional modules—micro-fermentation culture, high-speed centrifugal collection, and mechanical agitation disruption—into a single compact benchtop device. This achieves true end-to-end automation from “post-inoculation” to “crude lysate collection.” The system not only significantly enhances experimental consistency and operational safety but also keeps equipment costs around $100-just 1% of the cost of existing micro-fermentation devices. fundamentally solving problems points in synthetic biology practice—namely high costs, low efficiency, and heavy reliance on manual labor.

Device Development Process

Throughout development, we strictly adhered to standard hardware development procedures and the iGEM-advocated Design-Test-Learn-Build cycle, structured into five phases.

Figure 1.2 R&D Process Diagram
Figure 1.2 R&D Process Diagram
Figure 1.2 R&D Process Diagram

The primary task in this phase is to precisely identify core issues within the current laboratory workflow. Through researcher interviews and observation of experimental procedures, we pinpointed the project’s pain points Based on this, we defined the core design requirements for the device and established the ultimate objectives to be achieved. All design decisions stem from these requirements.

This phase involved a comprehensive review of existing technical solutions. We analyzed the strengths and weaknesses of commercially available stand alone equipment (shakers, centrifuges, homogenizers) and conducted in-depth research on hardware solutions related to automation and miniaturization from previous iGEM projects. This approach avoided redundant work while providing inspiration and technical feasibility validation for our innovative single-motor-driven multi-mode switching solution.

In this phase, we translated concepts into concrete engineering designs. Using SolidWorks software for 3D modeling, we meticulously considered the dimensions, materials, and compatibility of all components. This iterative process involved multiple design revisions and improvements based on team discussions and feedback.

This phase marks the critical transition from virtual to physical reality. We manufacture all custom parts via methods like 3D printing, procure standard components, and complete the assembly and debugging of the first prototype. Subsequently, rigorous system integration testing is conducted in the wet lab. This testing phase is crucial for identifying any design flaws, performance limitations, or unexpected behaviors. The validation results from the wet lab provide insights for further design iterations and refinements.

During this phase, we invited target users (other iGEM teammates, lab peers, faculty members) to experience the device operation and gathered feedback on human- machine interaction, operational safety, and structural stability. This feedback directly informed the final design iterations. Ultimately, all design files, code, and bill of materials were finalized, and comprehensive assembly guides and user manuals were developed to ensure project replicability.

Requirement and Cost Analysis

Through interviews with multiple laboratory researchers, we discovered that the process of extracting substances in laboratories consumes significant time.Simultaneously, the need to move between different instruments significantly increases the error rate. Based on this, we conducted research into traditional laboratory processes and the problems inherent within them.

Figure 1.3 Flowchart for Laboratory Shaker Culture and Miniature Fermentation Methods
Figure 1.3 Flowchart for Laboratory Shaker Culture and Miniature Fermentation Methods
Figure 1.3 Flowchart for Laboratory Shaker Culture and Miniature Fermentation Methods
Evaluation Criteria Existing Traditional Solution (Micro-fermentation tank + Centrifuge + Grinder)
Time Cost Extensive manual operations (aliquoting, transferring, resuspending, balancing, setting up equipment) require approximately 60–75 minutes of active handling time.
The experiment requires continuous operation: Monitoring fermentation conditions necessitates 24-hour uninterrupted sampling and experimentation. The prolonged continuous work and disrupted sleep patterns are difficult for researchers to endure.
Total process duration: Approximately 1.5–2.5 hours from culture completion to obtaining lysate.
Equipment Cost High initial procurement cost: The price of a miniature fermenter is at least $10,000. The combined cost of three separate pieces of equipment—a rocking incubator, centrifuge, and shaker—is approximately $5,000.
Laboratory Space Occupancy Significant space requirements: Three large devices occupy at least 2–3 square meters of valuable laboratory space.
Labor and Training Costs High demands: Personnel must be trained to proficiently operate three instruments and master aseptic techniques.
Value wastage: PhD students and researchers spend time on repetitive tasks.
Result Consistency and Risk Poor consistency: Manual operations inevitably introduce errors, resulting in high coefficient of variation.
High Risk: Multiple sample transfers increase cross- contamination hazards. Moreover, manual sampling from conical flasks and miniature fermenters carries a high risk of contamination.

Thus, we identified three major pain points: cumbersome and time-consuming operations, high equipment costs and space requirements, and poor result reproducibility. Based on this, we defined the core design requirements for the equipment: achieving full automation and integration of the three key functions: fermentation, centrifugation, and agitation/disruption. The goal is to reduce manual operation time by over 90% and significantly enhance data consistency. All design decisions stem from these requirements.

Project Philosophy and Design

Project Overview

Our hardware team has consistently focused on addressing practical challenges encountered in synthetic biology micro-fermentation, aiming to provide solutions and fresh perspectives for tackling specific problems. Our design philosophy centers on “integration, automation, and miniaturization, Modular.” The system appears as a compact desktop unit internally divided into two modules: an upper fermentation module and a lower integrated centrifugal agitation module. Controlled by a single STM32 chip, the system utilizes multiple control chips to drive motor rotation. An OLED display shows culture temperature, pH, and motor parameters for real-time monitoring of cultivation conditions. It also comes equipped with numerous components and modules for users to choose from, catering to diverse needs and practical situations.We have backed up all the models and codes for others to refer to and reproduce.

Core Module Function Design and Testing

a. Fermentation Culture and Fluid Control: Enables strain expansion culture and employs flow switches to control two liquid release stages for microbial enrichment and transfer of concentrated culture.

Figure 3.2.1 Schematic Diagram of Fermentation Condition Control Module
Figure 3.2.1 Schematic Diagram of Fermentation Condition Control Module
Figure 3.2.1 Schematic Diagram of Fermentation Condition Control Module
Figure 3.2.2 Schematic of Liquid Control and Microorganism Enrichment Module (a:Leakage Control Device, b:Mortise-and-Tenon Structure)
Figure 3.2.2 Schematic of Liquid Control and Microorganism Enrichment Module (a:Leakage Control Device, b:Mortise-and-Tenon Structure)
Figure 3.2.2 Schematic of Liquid Control and Microorganism Enrichment Module (a:Leakage Control Device, b:Mortise-and-Tenon Structure)
Figure 3.2.3 Turn clockwise to open the flow switch
Figure 3.2.3 Turn clockwise to open the flow switch
 Figure 3.2.3 Reverse continuous stirring
Figure 3.2.3 Reverse continuous stirring
Figure 3.2.3 Turn clockwise to open the flow switch
Figure 3.2.3 Reverse continuous stirring

b. Centrifugation and Agitation-Induced Lysis: Primarily separates cells from the culture medium. Adding glass beads followed by agitation lyses cells to release intracellular squalene.

Figure 3.2.4 Experimental Design Table
Figure 3.2.4 Experimental Design Table
Figure 3.2.4 Experimental Design Table
$$ A = \varepsilon \cdot c \cdot I $$
Figure 3.2.5 Contour Plot of Absorbance
Figure 3.2.5 Contour Plot of Absorbance
Figure 3.2.5 Contour Plot of Absorbance
Figure 3.2.6 Schematic of the Mode Switching Module
Figure 3.2.6 Schematic of the Mode Switching Module
Figure 3.2.6 Schematic of the Mode Switching Module
Figure 3.2.7 CommunicatingCentrifuge Tube Kit
Figure 3.2.7 CommunicatingCentrifuge Tube Kit
Figure 3.2.7 CommunicatingCentrifuge Tube Kit

c. Intelligent Control System: Primarily used to automate the entire device, reducing manual operation time

To integrate control of the entire apparatus and monitor cell culture conditions in real time, we developed the following circuit control system based on the STM32F103C8T6 microcontroller:

Figure 3.2.8 Wiring Diagram
Figure 3.2.8 Wiring Diagram
Figure 3.2.8 Wiring Diagram

Equipment

Condition Monitoring Module Drawing inspiration from HUST-China 2023’s fermentation condition monitoring design, we employed a DS18B20 + temperature probe as the temperature monitoring module and a pH composite electrode + pH sensor as the pH detection module. (Hardware | Huazhong University of Science and Technology - China - iGEM 2023)

Figure 3.2.9 pH Calibration Curve
Figure 3.2.9 pH Calibration Curve
Figure 3.2.9 pH Calibration Curve

Motion Control Module We selected distinct motors for the three key motion control points based on thorough research and analysis of each motor’s characteristics, implementing optimal designs:

Top: The top motor drives the stirring rod and activates the flow switch, requiring high precision. We selected the 11HS2810S stepper motor, which enables precise speed and angle control via PWM modulation and delivers substantial torque.

Bottom: The bottom motor controls centrifugation and oscillation, demanding strict speed regulation. We selected the 36-3530 brushless DC motor. Its brushless design enables high rotational speeds at low power consumption.

External: The external motor controls timed nutrient supplementation to regulate nitrogen levels in the culture medium. We fabricated a simple peristaltic pump that achieves timed supplementation (of ammonium sulfate) by adjusting the motor’s speed and rotation status. For the peristaltic pump’s control motor, we selected the MG996R servo motor. It is easy to program, allows precise specification of rotation angles, and consumes minimal power.

Figure 3.2.10 Peristaltic Pump
Figure 3.2.10 Peristaltic Pump
Figure 3.2.10 Peristaltic Pump

Software

We developed the program using Keil5. The primary functional code is as follows:

Figure 3.2.11 Main Code
Figure 3.2.11 Main Code
Figure 3.2.11 Main Code

Product Iteration

This section documents the challenges encountered during hardware development and their corresponding solutions.

Version 1.0

Initially, our device design comprised only an upper chamber for fermentation and a lower chamber for enrichment and lysis. In the upper chamber, we incorporated a stirring rod. Two openings were added to the chamber’s side, each equipped with a slide valve for controlled liquid addition and removal. At the tank bottom, a drain pipe allowed the concentrated bacterial solution to be sequentially transferred into centrifuge tubes below via a plug-screw mechanism upon fermentation completion, followed by removal for weighing. After centrifugation in the lower tank, the shaft was manually switched from concentric to eccentric mode, and grinding beads with buffer solution were added to initiate agitation and cell disruption.

However, this version still required significant manual labor, leading us to develop V2.0.

Figure 3.3.1 Schematic of the First-Generation Model
Figure 3.3.1 Schematic of the First-Generation Model
Figure 3.3.1 Schematic of the First-Generation Model

Version 2.0

Figure 3.3.2 Schematic of the second-generation model
Figure 3.3.2 Schematic of the second-generation model
Figure 3.3.2 Schematic of the second-generation model

Compared to the first generation, we introduced a mortise-and-tenon structure to mechanically link the flow switch and stirrer rod. The top knob can drive either the stirrer rod or the flow switch depending on its rotation direction. Simultaneously, we designed an automatic switching bearing that alters the motor’s rotation direction to toggle between centrifugal and agitation modes.

However, during practical testing, we encountered the following issues:

  1. Leakage at the bottom requires liquid to be evenly distributed into the centrifuge tubes below, making it impossible to ensure proper balancing;
  2. Subsequent processing of microbial cells required multiple rounds of liquid removal and addition within the centrifuge tubes, making the operation cumbersome;
  3. The mode-switching bearing only altered the radius of circular motion during oscillation mode, failing to achieve true centrifugation.

To address these issues, we continuously optimized the design and launched V3.0.

Version 3.0

Figure 3.3.3 Schematic Diagram of Third-Generation Model
Figure 3.3.3 Schematic Diagram of Third-Generation Model
Figure 3.3.3 Schematic Diagram of Third-Generation Model
Figure 3.3.4 Third-Generation Circuit Wiring Diagram
Figure 3.3.4 Third-Generation Circuit Wiring Diagram
Figure 3.3.4 Third-Generation Circuit Wiring Diagram

In the third-generation design, we introduced an automated control system. A DC motor-driven stirring rod was added to the top of the upper tank, along with two small openings for pH and temperature sensors to enable real-time monitoring of fermentation conditions.

To address the challenges of unbalanced centrifuge tubes and cumbersome liquid removal, we introduced a communicating centrifuge tube kit.

To ensure the mode-switching bearing achieves true oscillation functionality, we installed a one-way bearing on the drive shaft to transmit unidirectional motion, enabling selection between centrifugal and oscillation modes.

Although we successfully resolved the issues encountered in the third version, we now face a new challenge:

  1. The brushed DC motor proved difficult to control for precise angular rotation, causing the inner plate to frequently stop at undesirable positions and preventing successful single-pass liquid discharge.
  2. The 3D-printed servo gears proved too flexible to effectively rotate the outer tank for secondary drainage.

Version 4.0

Ultimately, we iterated to V4.0 as the final hardware product for this project. Through research, we discovered that stepper motors can simultaneously achieve continuous rotation and precise angle control. By positioning both discharge outlets on the base plate and utilizing the stepper motor’s fixed-angle rotation, we resolved the issues present in the third version.

Modularity

Our device continues last year’s modular and “building block” design philosophy, enhancing tank interchangeability and portability. Different modules can be easily swapped based on varying scenarios and fermentation requirements to maximize environmental benefits and operational efficiency while reducing costs. This year, we added several new designs, including: Stirring Module, Fermentation Tank Module, Flow Control Module, Temperature Control Module, and Aeration Module.

Agitator Module

During fermentation, stirring rods are commonly used within fermentation tanks to mix reactants and ensure uniform reactions, facilitating efficient processes and thorough nutrient absorption. Different stirring rods are required based on the physicochemical properties of the fermentation broth, such as viscosity and dissolved oxygen levels.

Figure 3.4.1 Agitator Module
Figure 3.4.1 Agitator Module
Figure 3.4.1 Agitator Module

Figure 3.4.1(a) shows a fan-blade agitator designed for vigorous liquid agitation. Its curved profile reduces resistance during mixing, making it suitable for environments requiring large-scale culture medium agitation.Figure 3.4.1(b) shows a spiral-rising agitator that uniformly mixes microbial cultures with nutrients. Its thin blades easily divide the liquid, making it suitable for high-viscosity culture media.

Flow Control Module

During fermentation, scenarios often arise where controlling the flow rate or volume of fermentation broth is necessary to optimize subsequent processes.

Figure 3.4.2 Flow Control Module
Figure 3.4.2 Flow Control Module
Figure 3.4.2 Flow Control Module

Figure 3.4.2(a) depicts a flow control module featuring porous construction for uniform leakage, enabling seamless integration into our equipment. The triple-hole design enhances operational flexibility. Figure 3.4.2(b) shows a flow control valve that regulates flow from the upper to lower tubes via a rotary handle, offering simple operation suitable for infrequently used flow control valves.

Speed Control Module

During cell centrifugation, the required rotational speed is influenced by cell size. To facilitate switching the centrifuge’s rotational speed, we designed a speed adjustment module.

Figure 3.4.3 Speed Control Module
Figure 3.4.3 Speed Control Module
Figure 3.4.3 Speed Control Module

Figure 3.4.3(a) shows a 7-tooth gear connected to the drive shaft. Figure 3.4.3(b) depicts a gear set designed for speed adjustment, featuring 14, 21, and 28 teeth respectively. These gears amplify the motor speed by 2x, 3x, and 4x, allowing speed switching through height adjustment for quick and convenient operation.

Aeration Module

Lipase yeast requires substantial oxygen during fermentation, making an efficient aeration system critical for enhancing fermentation efficiency. To improve oxygen utilization, we have designed the following three aeration systems.

Figure 3.4.4 Aeration Module
Figure 3.4.4 Aeration Module
Figure 3.4.4 Aeration Module

Figure 3.4.4(a) shows an aeration unit where gas is introduced radially through the disc. Typically used downward, it features small outlet holes and high gas pressure, suitable for systems with high oxygen demand.Figure 3.4.4(b) depicts another aeration unit connected to a long gas transport pipe. It diverts part of the gas flow into spherical chambers before diffusing outward, increasing gas-liquid contact area. This configuration serves as an ideal transfer station for multiple aeration systems.Figure 3.4.4(c) depicts an aeration line that can be laid along the tank bottom. It disperses bubbles in a fishbone pattern. As the bubbles rise, they agitate the liquid and dissolve, featuring a simple structure suitable for tubular fermenters.

Fermentation Modules

To accommodate diverse fermentation requirements and conditions, we have concurrently designed several variants of fermentation apparatus.

Figure 3.4.5 Fermentation Modules
Figure 3.4.5 Fermentation Modules
Figure 3.4.5 Fermentation Modules

Figure 3.4.5(a) A 200 ml fermentation vessel enables larger-scale fermentation and extraction.Figure 3.4.5(b) shows a fermenter with baffles added to increase oxygen supply during agitation.

Product Usage Procedure:

  1. Install communicating centrifuge tubes and sterile filters; close all flow switches
  2. Open the top lid, add the culture medium, and inoculate the microbial strain.
  3. Connect the power cord and press buttons in sequence to activate corresponding functions.
Figure 3.4.6 Button Layout Diagram
Figure 3.4.6 Button Layout Diagram
Figure 3.4.6 Button Layout Diagram

The red button powers on the device and illuminates the display. The yellow button activates the upper motor for continuous clockwise rotation. The left white button controls the upper motor for 30° counterclockwise rotation. The blue button controls the lower motor rotation. The white button reverses the lower motor rotation direction. The green button activates the nutrient replenishment system.

User Feedback

Throughout the design and development process, we actively engaged with multiple experts and users in the field, extensively gathering feedback to continuously optimize the device. This valuable input provides crucial support for our ongoing product iterations.

Expert Recommendations: Equipment Architecture Design Direction

We first consulted Professor Yang Xuezhi from the National Bio-Manufacturing Industry Innovation Center. He systematically introduced common design approaches for industrial equipment and pointed out: integrated hardware design is suitable for fixed, single-process workflows, while scenarios requiring higher flexibility should adopt industry-standard modular connection methods. Based on this advice, we established an integrated hardware-centric design direction and ultimately successfully developed the iFPS device.

Initial Prototype Testing and Key Improvements

We presented the initial prototype to Professor Wang Jinchun at the Engineering Practice Innovation Center of Huazhong University of Science and Technology. While acknowledging the device’s innovation, he proposed the following critical improvements from a practical application perspective:

To address these issues, we have developed corresponding improvement plans: After structural validation, critical bearings and moving parts will be replaced with metal materials to enhance longevity and operational stability; a removable, independently sterilizable “reaction inner chamber” will be designed to effectively resolve cleaning and sealing challenges; a dedicated PCB board will be developed to integrate circuits, improving system safety and anti-interference capabilities.

Figure 3.5.1 Discussion with Professor Wang Jinchun
Figure 3.5.1 Discussion with Professor Wang Jinchun
Figure 3.5.1 Discussion with Professor Wang Jinchun

Expanding Application Scenarios and Functional Optimization

We also invited Dr. Chen Yulong from the Institute of Resource Biology and Biotechnology at Huazhong University of Science and Technology to participate in testing. He provided valuable suggestions from the perspectives of synthetic biology and large-scale fermentation:

Based on these recommendations, we redesigned the fermentation module by incorporating baffled fermentation vessels of multiple specifications and integrating an oxygen supply unit. Dr. Chen’s proposed application scenarios also guided us toward new R&D directions. We are currently exploring the use of this equipment for exosome collection, further validating its scalability and practical value in bioprocessing.

Figure 3.5.2 Exchange with Dr. Chen Yulong
Figure 3.5.2 Exchange with Dr. Chen Yulong
Figure 3.5.2 Exchange with Dr. Chen Yulong

Equipment Component List and Cost

Serial No. Name Unit Price Quantity Total Price
1 Consumables 0.2￥/g 1703.74g 340.74￥
2 MG995 996R Metal Standard Servo 19.16￥ 1 19.16￥
3 Micro 28 Stepper Motor 38￥ 1 38￥
4 MUH Unidirectional Bearing 18.37￥ 1 18.37￥
5 DS-430 Self-Resetting Non-Locking Button 0.5￥ 2 1￥
6 One-way Bearing 3.58￥ 1 3.58￥
7 KCD Mini Boat-Shaped Pushbutton 1.75￥ 4 7￥
8 L298N Chip 6.86￥ 1 6.86￥
9 STM32F103C8T6 chip 6￥ 1 6￥
10 830-hole breadboard 4.1￥ 1 4.1￥
11 Temperature + pH Sensor 104￥ 1 104￥
12 Brushless DC Motor 110￥ 1 110￥
13 Metal Components, Wires     5￥
Total       663.81￥≈93.2＄

Discussion

Contributions to Synthetic Biology and the iGEM Community

Significance of the Integrated Automated Cultivation and Lysis Mechanism

This integrated automated cultivation and lysis mechanism will benefit all teams focused on protein expression screening, metabolite engineering, and cell factory optimization. Our device establishes a mechanism that automates the process from cultivation to lysis on a low-cost benchtop platform, enabling easy replication by other teams. In this way, it introduces the option of “desktop-scale automated sample preparation” for subsequent teams and projects beyond iGEM, thereby broadening the application scope of synthetic biology in rapid iteration and standardized characterization.

Significance of the Single-Motor Reversible Multi-Mode Drive Mechanism

As a device capable of automatically switching between centrifugation and shaking modes using a single power source, it will assist all teams in developing compact, cost- controlled automated biological experimental equipment. One advantage of its modular integrated design is the ability to simplify control systems and reduce manufacturing costs, enabling complex functionality even under resource constraints.

Significance of the Device’s Self-Balancing Centrifuge Tubes

Achieving balance during centrifugation is a critical challenge in automated experimental systems. Our self-balancing centrifuge tubes, based on the communicating vessels principle, instantly achieve mass equilibrium upon liquid injection. This innovation eliminates the cumbersome, time-consuming manual weighing and balancing steps required in traditional centrifugation, providing essential support for true “one-button start” fully automated workflows.

Conclusion

Our device is designed to precisely address real-world issues through human-practice feedback loops, meeting diverse needs while continuously optimizing performance. Furthermore, its versatility and replicability unlock broader application potential.

(Overview, Requirements and Cost Analysis, Project Philosophy and Design , Discussion)

Core functions and structure performed well in the prototype, demonstrating the device’s practicality and functionality. This strongly indicates that with further refinement, it can evolve into a commercialized and practical model in the future.

(Project Philosophy and Design, Product Usage Procedure, Discussion)

We conducted multiple user tests and interviews, gathering diverse feedback on the entire device. This feedback was either incorporated into the device’s actual production or integrated into plans for future enhancements and implementation.

(Requirement & Cost Analysis, User Feedback)

All design information, 3D printing data, electronic schematics, parts lists, and assembly instructions for the final prototype have been fully documented and released in a format easily accessible for replication.

In summary, we have developed and demonstrated an automated crude cell extraction device integrating fermentation, centrifugation, and shaking, effectively addressing challenges in synthetic biology development and application.

We are more than willing to provide the source code and model for reference by future iGEM teams. You can contact us via email.