Hardware

1.1.1 Overview

To achieve in-situ dyeing of bacteria, the first question we needed to address was:How can bacteria be fixed at specific locations on fabric without spreading freely?

If we simply apply the bacterial solution directly onto the fabric, it is evident that due to the lack of nutrients and prolonged environmental exposure, the bacteria would struggle to express genes normally or even survive. Furthermore, conventional bacterial culture methods, such as inoculating bacteria onto solid agar plates or cultivating E.coli in liquid media, cannot fulfill the project's requirement for "in-situ dyeing".

Inspired by medical applications of hydrogels, such as creating wound dressings, we designed a hydrogel capable of immobilizing our engineered bacteria. This hydrogel allows the bacteria to receive light signals and stably express pigments and halogenase enzymes. It offers the advantages of being biocompatible with the bacteria and easy to remove.

1.1.2 Previous Results

Hydrogel is an ideal material for immobilizing live engineered bacteria in this project because it provides a hydrous environment that can be infused with nutrients, tryptophan, and antibiotics. It also generally has good light transmittance, which minimally impacts the engineered bacteria's ability to receive light signals. At the project's outset, we experimented with various hydrogel materials, such as gelatin methacryloyl (GelMA) and sodium carboxymethyl cellulose (CMC-Na). However, after numerous preliminary tests, we found that these hydrogels either had excessively long gelation times, involved complicated preparation processes, or were costly. Consequently, we selected and modified a gelation method for a hydrogel material based primarily on sodium alginate.

Sodium alginate-based hydrogels are commonly used as carriers for microbial biosensors. We were attracted by this material's ability to effectively encapsulate microorganisms and its permeability to small molecules. Our method involves thoroughly saturating silk fabric with a sodium alginate solution (allowing the solution to fill the pores within the protein fibers), followed by evenly spraying a calcium chloride solution over the fabric using a spray head. Upon application, calcium ions rapidly cross-link with the sodium alginate to form a gel that completely envelops the fabric.

Figure 1:​​ Fabric coated with hydrogel (dye was added to the sodium alginate solution for visualization purposes)

After successfully forming the hydrogel multiple times, we used it to culture wild-type E.coli BL21(DE3) (not genetically engineered). In these culture experiments, distinct bacterial colonies were visible within the hydrogel after 16 hours of incubation. We collected liquid exuded from the hydrogel and conducted four parallel replicate experiments. The average optical density (OD) value measured was 0.25425, compared to an OD value of 2.147 for the blank control group (culturing E.coli on an LB agar plate). Interpreting this data suggests that the vast majority of bacteria were successfully immobilized and retained within the hydrogel matrix, without significant leakage or loss of viability. This indicates that our hydrogel system can provide a stable microenvironment supporting bacterial growth and possesses the core capability necessary for sustaining bacterial culture.

1.1.3 User Interviews and Expert Consultations​​ ​​

1.1.3.1Mr. Jia Yongguang

Mr. Jia Yongguang is a Distinguished Research Fellow at Beijing Normal University, a Ph.D. and Master's Supervisor. His research focuses on the research and development of polymer materials based on natural molecules, including natural molecule-based "antimicrobial peptide mimics" and biomedical elastomers. He has led two National Natural Science Foundation of China projects and three provincial/ministerial-level projects in this field, and participated in over ten other major key projects. ​

Implementation

We presented our hydrogel experimental procedures and results to Mr. Jia.

Feedback

• Mr. Jia expressed approval of our idea to use hydrogel.

• He raised concerns that bacteria might still escape from the selected hydrogel material during the later stages of cultivation. Although bacterial growth within the sodium alginate hydrogel is slower than in standard liquid culture, escape remains possible under sufficient nutrient conditions.

• He was concerned that the hydrogel might lose a significant amount of water and shrink during the later stages of illumination.

• He pointed out that the gelation method involving spraying a Ca²⁺-containing solution might lead to non-uniform pore sizes within the sodium alginate hydrogel. Furthermore, the very short gelation time could cause inconvenience and difficulties in operations such as mixing with other reagents and shaping.

1.1.3.2Ms.Zhang Chengsu

Ms. Zhang Chengsu is an inheritor of the Intangible Cultural Heritage of Wax Dyeing of the Gelao people,at the Haokou Wax Dyeing Workshop in Wulong District, Chongqing.

Figure 2:​​Interview with Ms. Zhang Chengsu

Implementation

• We explained our bacterial dyeing strategy using hydrogel to Ms. Zhang Chengsu and showed her the physical state of the hydrogel.

Feedback

• Ms. Zhang Chengsu found our hydrogel strategy very interesting. She noted a similarity to the wax used in batik (wax dyeing) – whereas wax is used in batik to resistdye in specific areas, our hydrogel is intended to applycolor to specific areas.

• Ms. Zhang considered our hydrogel method convenient and rapid for gel formation, and easy to operate.

​​​​1.2.1 Hydrogel Gelation Strategy​​

In the original system, the rapid ionic cross-linking reaction between Ca²⁺ and sodium alginate is prone to cause a diffusion-limited effect. This often results in a hydrogel with a gradient pore structure, being denser at the surface where cross-linking occurs first, and more porous internally due to limited Ca²⁺ diffusion. This structural heterogeneity can affect the hydrogel's mass transfer efficiency and mechanical properties. To address this, we adopted a slow calcium ion release strategy: Lightweight CaCO₃ powder was added to the sodium alginate solution and uniformly dispersed using an ultrasonic disruptor. Glucono-δ-lactone (GDL) was then added and mixed thoroughly. After standing for 3-4 minutes, the system completely solidifies into a hydrogel. In this process, GDL slowly hydrolyzes in water to generate gluconic acid, gradually lowering the system's pH. The H⁺ ions progressively react with the pre-dispersed CaCO₃, promoting its slow dissolution and the uniform release of Ca²⁺ ions. This avoids the sudden release and uneven diffusion of Ca²⁺ seen in the traditional method. The uniformly released Ca²⁺ ions synchronously cross-link with the sodium alginate polymer chains, eliminating the gradient pore structure and forming a hydrogel with more homogeneous structure and properties, thereby solving the diffusion limitation problem of the original system.

The mass fraction of sodium alginate is a critical parameter governing its gelation potential and solution rheology. At low mass fractions, the sodium alginate chains are too discrete with insufficient cross-linking sites to form a stable gel network. Conversely, excessively high mass fractions lead to a significant increase in solution viscosity and poor fluidity, which not only hinders uniform mixing with other components but may also pose technical challenges for subsequent operations like material molding or spraying.

To resolve this, we systematically characterized the relationship between sodium alginate solution viscosity and its mass fraction to determine the optimal concentration for our project. Using a rotational rheometer maintained at 30°C, we measured the apparent viscosity of sodium alginate solutions at mass fractions of 0.5%, 0.8%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, and 3.5% across different shear rates.

Table 1:​​ Apparent viscosity of sodium alginate solutions as a function of shear rate

Analysis of the experimental data shows that the dynamic viscosity of the sodium alginate solution generally increases with its mass fraction. When the mass fraction reaches 3.5%, the solution becomes very viscous, and insoluble small lumps remain even after prolonged stirring. This change exhibits distinct stages – a significant jump in dynamic viscosity occurs around the 2.0% mass fraction, indicating a critical transition in the internal structure of the system. At this critical mass fraction, the sodium alginate solution maintains appropriate fluidity, and sufficient interaction exists between polymer chains to facilitate the formation of a gel with adequate strength. Considering solution rheology, intermolecular interaction strength, and gelation performance comprehensively, we determined 2.0% to be the optimal sodium alginate mass fraction for this project and subsequent applications.

The bacterial strains were able to survive stably and maintain normal metabolic activity within the hydrogel, successfully achieving functional expression of the indigo synthesis genes. Furthermore, after 16 hours of continuous culture, observation of the sodium alginate hydrogel morphology confirmed its overall structural integrity, with no significant swelling, shrinkage, rupture, or degradation, demonstrating stable physicochemical properties of the hydrogel during the experimental period. Notably, significant indigo pigment production was observed in three out of five gradient control groups, with visible coloration effects, further validating the suitability of this culture system for target product synthesis by the engineered bacteria.

Figure 3:​​ Hydrogel after 16 hours of bacterial culture (The three experimental group hydrogel culture plates all exhibited a significant deepening of indigo-blue hue, with both color uniformity and intensity markedly higher than the external control groups. The control group hydrogels remained in their initial light or colorless state throughout, showing no indigo-blue change)

Afterwards, we simulated fabric dyeing using the hydrogel. Indigo-producing E.coli were encapsulated within the hydrogel, which was then applied to cover the fabric. After 16 hours of cultivation, the fabric was removed, the hydrogel was washed off, and observable color indicated the feasibility of using hydrogel-based bacterial encapsulation for fabric dyeing.

Figure 4:​​ Hydrogel Dyeing Experiment (In the third image, the left four fabric swatches had hydrogel containing indigo-producing E.coli added, showing distinct blue dyeing. The second swatch from the right had hydrogel added but no E.coli. The rightmost swatch had no hydrogel added)

1.2.2 Bacterial Encapsulation Strategy​​

Addressing the issue of bacterial escape in microbial immobilization systems, the experimental team initially employed auxotrophic bacterial strains as a primary control strategy. However, this single measure proved to have significant limitations in effectively containing bacterial escape and was insufficient for long-term stable culture requirements. To further enhance the barrier properties of the system, we adapted a tough polyacrylamide-alginate hydrogel composite coating applied over the core sodium alginate hydrogel carrier. This functional hydrogel shell layer serves to contain the E.coli within the sodium alginate hydrogel, preventing their escape, while also acting as a barrier against contamination from external microbes. The specific operational steps and a schematic diagram of this encapsulation structure are shown in Figure 5.

Figure 5:​​ Schematic diagram of the bacterial encapsulation strategy

We encapsulated E.coli in two groups using hydrogels: one group was coated with the polyacrylamide-sodium alginate hydrogel composite, while the other group consisted only of the bacteria-loaded sodium alginate hydrogel without the outer coating. Both encapsulated hydrogel groups were placed in liquid culture medium. After a period of cultivation, the OD values were measured. The group with the composite coating exhibited an OD value of 0.034, whereas the uncoated group had an OD value of 2.046.

Figure 5 illustrates the strategy we ultimately adopted for hydrogel spraying, which involves first spraying the sodium alginate hydrogel followed by overlaying a polyacrylamide-sodium alginate hydrogel composite layer on its surface. However, when validating its effectiveness, we employed a different approach: we first added bacterial suspension to the sodium alginate pre-gel solution, then utilized a syringe injection method to obtain sodium alginate hydrogel microspheres. These sodium alginate microspheres were subsequently immersed in the polyacrylamide-sodium alginate pre-gel system. Due to the high viscosity of this solution, it forms an encapsulating layer around the microspheres. The microspheres were then placed in a cross-linking agent to solidify the outer encapsulation. This operation can be repeated several times.

Figure 6:​​ Physical diagram of the results (In the image, A represents unwrapped sodium alginate microspheres, while B represents microspheres with multiple layers of encapsulation).

1.2.3 Hardware Experiments Notebook

2.1.1 Incubation Chamber

Figure 8:Incubation Chamber

As illustrated in the figure above, the structure of the designed incubation chamber is presented. Its core function is focused on maintaining a constant temperature and providing light protection during the bacterial cultivation process, thereby ensuring the stability of the microbial growth environment. The outer wall is fabricated from black polylactic acid (PLA) material, which is not only a commonly used raw material in 3D printing but also effectively blocks external light interference throughout the cultivation period. Additionally, owing to its inherent thermal insulation properties, it helps maintain a stable internal temperature, fulfilling the fundamental requirements of light avoidance and temperature consistency for microbial cultivation.

Figure 9:Incubation Chamber Lid

Furthermore, to achieve effective sealing, We designed two grooves on the incubation chamber lid to accommodate elastic sealing rings. These rings form tightly fitting sealed surfaces, improving the contact at the interfaces and thereby enhancing both gas isolation and thermal retention performance.

The portable bioculture chamber incorporates a multi-chamber internal design, divided into three independent cultivation compartments. These compartments are physically separated by removable thermal insulation partitions, providing thermal environment isolation. Based on this structure, cultivation experiments can be conducted in two modes: Firstly, precise control of temperature and gas atmosphere can be independently applied to each compartment, enabling parallel experiments under multiple different cultivation conditions. Secondly, upon removal of the thermal insulation partitions, the three compartments can be integrated into a unified cultivation space to meet bacterial culture needs under large-scale or uniform conditions, significantly enhancing the experimental applicability and operational flexibility of the device.

2.1.2 Fermentation Tank

To investigate the impact of shear stress on microbial growth, this study utilized E.coli BL21(DE3) as the experimental strain. Under identical initial conditions—including bacterial suspension concentration, cultivation temperature, medium composition, and cultivation time—three parallel bacterial liquid samples were subjected to 8 hours of cultivation using two methods: shaker oscillation and stirring with an impeller. After the cultivation period, the growth status of the bacterial strains was characterized by measuring the OD₆₀₀ values of the bacterial suspensions. The specific detection results are presented in the table below.

Table 2:OD Values of E.coli BL21(DE3) after Cultivation under Different Shear Stress Conditions

As evidenced by the data presented in the table, discernible differences remain in the OD₆₀₀ values of the bacterial cultures subjected to the two shear stress application methods. This indicates that within the experimental system, shear stress continues to exert an influence on the growth status of E.coli BL21(DE3). To mitigate the impact of shear forces during bacterial cultivation, we have designed a novel fermentation distribution structure that reduces stirring-induced shear stress. By replacing the traditional impeller with an air sparger system, homogeneous mixing of the fermentation broth, air, and supplemental components is achieved. Compared to conventional fermentation tanks, this design offers the dual advantages of reducing energy consumption associated with mechanical stirring while simultaneously avoiding mechanical disruption of microbial cellular structures caused by impeller-induced shear forces.

Figure 10: Air Sparger-type Mixing Device

As illustrated, the air sparger comprises upper and lower annular pipes. The lower ends of these pipes are equipped with multiple vertical conduits for connection and aeration purposes. Both the upper and lower annular manifolds are fitted with gas intake ports. The upper and lower annular pipes are equipped with spray heads oriented at a 30° angle to the radial direction. When all spray heads are activated, the inclined nozzles generate rotational airflow, thereby creating a spiral gas stream within the fermentation tank that induces liquid agitation. This configuration simultaneously increases the gas-liquid contact area, consequently enhancing oxygen dissolution efficiency.

Additionally, when the air sparger is not required, an air pump can be directly connected for aeration purposes.

Figure 11: Fermentation Tank without Air Sparger (arrows indicate aeration direction)

2.2.1 Hybrid Mixing Structure

Given that our liquid system primarily uses hydrogel as the base matrix, resulting in high viscosity, we have developed a composite mixing structure that integrates an anchor impeller with a helical ribbon impeller. The helical ribbon impeller induces axial upward movement of the viscous fluid during rotation, facilitating the mixing of thick solutions. Meanwhile, the anchor impeller effectively addresses issues such as sediment accumulation at the bottom, dead zone residues, and wall adhesion of high-viscosity materials.

Figure 13: Hybrid Mixing Structure

2.2.2 Tank Design

Furthermore, the bottom of our portable raw material mixing tank features a slightly curved design to optimize fluid dynamics and mass transfer efficiency, thereby enhancing fermentation performance. For high-viscosity liquids, the arched base reduces material sedimentation at the tank bottom while facilitating easier cleaning and eliminating dead zones. Additionally, during high-temperature sterilization, the smooth contour of the curved base prevents incomplete sterilization that may occur in flat-bottomed corners due to uneven heat conduction.

The portable raw material mixing tank incorporates a movable lid design. The lid is configured with dedicated inlet and outlet ports to facilitate convenient sampling and material loading/unloading operations. Furthermore, we have integrated a drive shaft connection on the lid for interfacing with the power transmission system.

Figure 14: Tank Body and Lidd

2.3.1 Overview

Building upon our research group's optogenetic control system, we have developed the following hardware setup. Viewed from right to left, the apparatus comprises three core workstations: the power station, printing nozzle station, and illumination station. Vertically, we have constructed a three-tier aluminum profile structure to strategically position the motors, pneumatic cylinders, and portable mixing tank, ensuring optimal space utilization. During the patterning process, the textile substrate is first positioned on the placement platform. The bacterial culture is premixed with hydrogel and other components, then delivered to the printing nozzle via a perfusion pump. The motor-driven lead screw mechanism precisely positions the nozzle to deposit the pre-gel solution onto the substrate. After 1-2 minutes for gelation, the pneumatic system transfers the substrate to the illumination station where customized LED arrays with optical filters activate the bacterial gene circuits through specific wavelength exposure, inducing targeted pigment production.

Figure 15: Overall Printer Model and Engineering Drawing

The main framework utilizes aluminum profiles, which offer lightweight construction, ease of assembly, and excellent durability. The hollow channel design of the profiles conveniently accommodates the integration of linear guides, motor systems, and pneumatic components. This aluminum extrusion-based structure provides an optimal combination of lightweight properties, assembly convenience, and structural robustness, while its hollow channels offer inherent advantages for embedding motion systems and actuators.

Figure 16: Aluminum Profile Support Structure

Furthermore, we have designed an enclosing shell for the entire system to provide light-blocking functionality. The shell is divided into separable left and right sections that detach from the internal functional components, facilitating convenient cleaning and maintenance of our hardware system.

Figure 17: Enclosing Shell

This photoactivation printing equipment adopts a highly modular design architecture, where each functional module possesses independent operational capabilities. When multiple modules operate under coordinated control mechanisms, they collectively fulfill the requirements for bacteria-based biological dyeing, enabling the generation of specific patterns and integration of biological functions.

2.3.2 Motor-Lead Screw Drive Module

To achieve complete coverage of the textile area by the printing nozzle, this study designed a motor-lead screw drive module. This module employs a lead screw as the core transmission component, with a nut seat assembled on the screw carrying and securing the motion platform equipped with the nozzle. The motor drives the rotation of the lead screw, thereby propelling the nut seat and nozzle platform along the screw's axial direction in linear motion. Additionally, pulley assemblies are installed at both ends of the nozzle platform to prevent reversal or deflection during movement while reducing frictional resistance between the platform and the support structure, thus providing auxiliary guiding and sliding functions.

Figure 18: Motor and Lead Screw

The designed ball screw has a total length of 306 mm, with fixed and support ends configured at each terminal to achieve axial positioning. To meet the movement requirements of the nozzle platform, the actual effective travel of the intermediate lead section is 172 mm. We have equipped this ball screw with a coupling that interfaces with the aforementioned drive motor, ensuring coaxial alignment and stability in power transmission. Furthermore, to address emergency operational needs during power outages or in non-electrified scenarios, we have additionally configured a hand crank device. This manual drive mechanism enables operational control of the lead screw movement.

Figure 19: Schematic Diagram of Lead Screw and Hand Crank

2.3.3 Nozzle System

In bacterial dyeing experiments based on the hydrogel strategy, sodium alginate hydrogel and polyacrylamide-sodium alginate composite hydrogel need to be applied sequentially. To achieve orderly delivery and precise compounding of the two hydrogels during spraying, we designed a multi-nozzle series delivery system. By configuring independent drive pumps on both sides of the system, programmed control of pump output can be implemented according to process requirements. This enables simultaneous input of sodium alginate pre-gel and polyacrylamide-sodium alginate pre-gel system, which are mixed in the nozzle convergence zone before being sprayed. Furthermore, by individually activating a single-side pump while closing the other side's pathway, an operational mode where only the corresponding nozzle outputs a single pre-gel system can be achieved.

Figure 20: Nozzle Design

We reserved space at the lowest level of the entire hardware setup for placing two portable raw material mixing tanks: one for loading sodium alginate pre-gel mixed with bacterial suspension and culture medium, and the other for polyacrylamide pre-gel. The ends of the nozzle pipelines can be directly inserted into the portable mixing tanks to draw solutions via the pumps.

Figure 21: Relative Position of Portable Mixing Tank and Pipelines

2.3.4 Cylinder-Slide Rail Power System

Figure 22: Slide Rail and Slider

We adopted MGN series ball-type linear guides and constructed the platform using aluminum profiles. Two parallel guide rails are embedded to facilitate the sliding of the placement plate driven by the sliders.

Figure 23: Cylinder and Connection Components

The cylinder is installed at the bottom of the entire hardware setup. Through connecting components, it drives the placement plate to perform linear back-and-forth movement.

2.3.5 Placement Plate Module

Figure 24: Upper and Lower Placement Plates

The placement plate serves as the support structure for the hydrogel. We have designed it as two separable plates. The upper plate primarily functions to receive the sodium alginate hydrogel and textile substrate, while the lower plate connects to the cylinder and serves as the carrier for linear transfer. We have incorporated specific features on the lower plate to facilitate easy removal of the upper plate.

Figure 25: Lower Placement Plate (arrows indicate the structure designed for manual removal of the upper plate)

To maintain temperature during illumination and enable high-temperature sterilization after printing, we have integrated a polyimide (PI) heating film and a sheet-type thermocouple into the upper placement plate. These components are connected to our development board via a relay. Utilizing a control principle similar to PID, we regulate the heating film to maintain a stable temperature around 37°C for extended periods, while also allowing rapid heating to approximately 100°C for E.coli sterilization. For details, please refer to our programming section.

Figure 26: Polyimide (PI) Heating Film

2.3.6 Photoactivation Module

After hydrogel application is completed at the right-side printing station, the placement plate slides along the rails to the left-side photoactivation station. We have designed observation windows on the enclosure of the photoactivation module to monitor mechanical operation and hydrogel status during printing. When light blocking is required, we use a light-proof cloth for shielding. This design minimally impacts the illumination effect while providing operational convenience.

Figure 27: Schematic Diagram of Light-proof Cloth Position

As shown in the diagram, we have reserved space on both sides of the light source and optical filters to accommodate cooling devices such as fans for the light source.

Our illumination strategy utilizes the property of light filters to transmit specific wavelengths of light, enabling bacteria within the hydrogel at designated locations to receive specific wavelengths and activate their genetic pathways. Our experimental project involves three types of bacteria engineered with different plasmids. Two of these convert L-tryptophan into 6-bromotryptophan and 4-nitrotryptophan, respectively, while the third strain converts the modified tryptophans and unmodified L-tryptophan into 6,6'-dibromoindigo (purple), 4,4'-dinitroindigo (green), and indigo, respectively. Therefore, if bacteria in a specific area only receive green light illumination, they cannot produce the green pigment; the same applies to the purple pigment. To address this, we have designed an illumination strategy. We separate the target pattern into three colors – purple, green, and blue – and split it into two light filters. First, we retain only the green and purple channels. Then, using the grayscale image of the purple channel, we convert purple to red. Finally, we merge the green and red images to create our first light filter, designed to activate E.coli genes responsive to red and green light, promoting the bromination or nitration of tryptophan and its release extracellularly. Our second light filter is a blue-only filter, designed to activate the genetic pathways of bacteria responsive to blue light.

Figure 28: Physical Image of Our Light Filters and the Filter Patterns Processed in Photoshop (The image with the more prominent red channel is the processed one)

Due to the addition of nutritional components and buffer reagents, the hydrogel system tends to exhibit a characteristic light yellow color. This results from the selective absorption of blue-violet light by culture substances, leading to an imbalance in the transmitted light spectrum. To correct this spectral distortion, our solution involves installing a blue-violet light compensation filter on the lens assembly near the hydrogel sample. By specifically supplementing the absorbed blue-violet light wavelengths, we achieve precise balance in the transmitted light spectrum, ensuring the reliability of optical detection data.

Due to the incorporation of nutritional components and buffer reagents, the hydrogel system tends to exhibit a characteristic light yellow tint. This phenomenon arises from the selective absorption of the blue-violet spectrum within visible light by culture substances, leading to an imbalance in the transmitted light spectrum distribution. To correct this spectral distortion, our solution proposes the installation of a blue-violet light compensation filter on the lens assembly side adjacent to the hydrogel sample. By directionally supplementing the absorbed blue-violet spectral bands, we achieve precise balancing of the transmitted light spectrum, thereby ensuring the reliability of optical detection data.

Figure 29: Schematic Diagram of the Lens Assembly

The light source we selected is an LED lamp, which achieves full-spectrum illumination control of the sample by adjusting the PWM signal duty cycles of the R, G, and B pins to regulate the luminous intensity of the three primary color LEDs.

2.4.1 Engineer Li

Engineer Li is an engineer at an electrical manufacturing plant with over ten years of work experience.

Implementation

• We presented our physical prototype to Engineer Li and provided a detailed explanation and demonstration of the overall hardware operation.

• We gained valuable insights for design improvements.

Feedback

• Engineer Li commended our modular design and power system architecture.

• Engineer Li pointed out that the current nozzle design, where fluids from both sides share a single pipeline, is structurally unreasonable.

• Engineer Li noted that the current nozzle requires disassembly for cleaning after each hydrogel spraying cycle. He recommended exploring optimized solutions for easier handling, such as preliminary cleaning when the placement plate moves to the next station.

• Engineer Li expressed concern that the current cylinder-slide rail system lacks safety mechanisms to prevent disengagement, posing a risk of the slider detaching from the rail.

2.4.2 Ms. Chen Hong

Ms. Chen Hong is an inheritor of the intangible cultural heritage of Gelao batik at the Haokou Gelao Batik Workshop in Wulong District, Chongqing. She has prior experience using automated batik equipment.

Figure 30: Interview with Ms. Chen Hong

Implementation

• We demonstrated the operational procedures required for end-users of our hardware product to Ms. Chen Hong.

• We obtained inspiration for future enhancements.

Feedback

• Ms. Chen Hong acknowledged the high automation level and user-friendly operation of our hardware.

• Ms. Chen Hong noted the similarity between our hardware and automated batik equipment previously used in heritage workshops. She expressed confidence that future modifications based on our hardware could lead to the development of machines capable of automated batik production, thereby contributing to the preservation of intangible cultural heritage.

2.5.1 Nozzle Iterative Design

Figure 31: Schematic Diagram of the Nozzle

As illustrated, this nozzle system is specifically designed for the orderly and precise spraying of sodium alginate and polyacrylamide-sodium alginate composite hydrogels. The system consists of a fluid delivery pipeline network, multiple nozzle sets, and two independent valves. The pipeline network adopts a dual-branch structure, allowing independent transportation of the two hydrogel materials. At the nozzle, we have incorporated a three-way-valve-like structure. During the spraying operation, the valve on one side is first opened to allow the sodium alginate pre-gel system to be sprayed through the nozzle. Subsequently, the pump output pressure is reduced, while the valve on the other side is opened, enabling the polyacrylamide-sodium alginate pre-gel system to mix with the sodium alginate pre-gel system at the nozzle outlet, and the mixture is then sprayed through the nozzle. The use of two valves allows precise control over the flow of both solutions, enabling switching and on/off control of the supply channels for the two hydrogels, thereby ensuring that the two hydrogels are sprayed strictly in the preset sequence.

Furthermore, we have reserved a semi-enclosed space below the pump where cleaning agents for flushing the pipelines and nozzles can be stored in advance for ready use.

2.5.2 Placement Plate Iterative Design

Figure 32: Iterative Design of the Placement Plate

We have replaced the original single upper placement plate with two separate plates and elevated the rear plate to function as a waste collection box. Each time the nozzle completes the spraying operation, the cylinder moves the placement plate to the illumination station. At this point, the waste box is precisely positioned below the nozzle. By switching the pipeline connection of the nozzle to the cleaning solution, the pipeline can be flushed, and the waste liquid generated can be sprayed directly into the waste box via the nozzle.

Figure 33: Schematic Diagram of the Relative Position of the Waste Box

2.5.3 Cylinder Iterative Design

We have readjusted the position of the piston in the cylinder and shortened its stroke. This ensures that when the piston reaches its extreme position, the waste box is correctly located below the nozzle and the front placement plate is accurately positioned within the illumination station, thereby eliminating the risk of the slider disengaging from the rail.

Figure 34: Relative Position of the Placement Plate When the Cylinder Is at the Leftmost Position

We will present the operational workflow of our entire hardware system in the form of an animation.

Figure 35: Product Rendering

3.1.1 Main Controller: Turing ESP32-S3​​

This project utilizes the Turing ESP32-S3 as the main control board. This board features abundant GPIO resources, powerful dual-core processing capability, and good support for MicroPython, making it suitable as the control core for complex peripherals. The development board we used is shown below. ​​

Figure 37:​​ CAD diagram and physical image of the development board

3.1.2 RGB LED Light Source​​

The system uses a common anode RGB LED as the light source. Signals are output via the PWM (Pulse Width Modulation) pins of the ESP32-S3 to precisely control the brightness of the red, green, and blue color channels, thereby producing the desired light color and intensity. ​​

Figure 38:​​ Regulation of light color through control

3.1.3 28BYJ-48 Stepper Motor and ULN2003 Driver Board​​

During the testing phase, we used a 28BYJ-48 stepper motor to drive the printhead movement. This motor is connected to the ESP32-S3 via a ULN2003 driver board. This motor type offers high torque and precise step angles, making it suitable for applications requiring precise positioning. During the system verification phase, we employed a 60-series servo motor as the power source, interfacing it to the development board using an external RS-485 conversion chip for programmatic control.

Figure 39: Motor Operation Controlled by a Development Board

3.1.4 DS18B20 Temperature Sensor​​

A DS18B20 digital temperature sensor is used to monitor the culture plate module. This sensor uses the 1-Wire protocol, offers high accuracy, and has a simple connection interface. ​​

3.1.5 Relay Module​​

The system uses a CZ057-type single-channel 12V relay module to control the heating pad. This relay module features optocoupler isolation, supports high/low level triggering, and can safely control the operation of the 12V heating pad. The relay is controlled via the GPIO10 pin of the ESP32-S3. When the pin outputs a high level, the relay engages, completing the circuit to power the heating pad. ​​

3.1.6 Polyimide (PI) Heating Film and Power Supply​​

A polyimide (PI) heating film is used as the heating element, operating at 12V. This heating film is thin, flexible, has fast thermal response, and provides even heat distribution, making it suitable for precise temperature control applications. A 12V power supply provides the operating voltage for the heating pad, ensuring its stable operation. The heating pad's power is switched on and off via the relay module, enabling controlled heating of the culture plate module.

3.1.7 Connection Cables​​

M2M (Male-to-Male) DuPont wires are used for connections between the various components and the development board. ​​

3.2 Software Development​​ ​​