Background and Design

Effective bioremediation of heavy metal pollution using engineered cyanobacteria requires not only advanced synthetic biology tools, but also a reliable, safe, and practical hardware platform. In our project, we recognized early on that the success of our biological circuits would be limited without a bioreactor system designed to maximize both biosafety and operational efficiency. Our hardware development focused on enabling safe interaction between engineered Synechocystis and contaminated water, while preventing environmental escape and supporting easy maintenance and scalability. Therefore, the key direction of our hardware design has been confirmed.

• Maximize contact between engineered Synechocystis sp. PCC 6803 and contaminated water,
• Ensure biosafety and containment to prevent environmental release,
• Facilitate easy maintenance, monitoring, and reuse,
• Support scalability for diverse application scenarios, from laboratory testing to industrial wastewater treatment.

Evolution of Our Bioreactor Design

The inspiration for our hardware design originated from the continuous directed evolution system designed by our team in 2024 (SZ-SHD2024 Hardware). The system utilizes a series of bottles, peristaltic pumps, and a magnetic stirrer to create a controlled environment where Escherichia coli can be subjected to gradually increasing concentrations of a selected stressor—such as chlorite—thereby promoting rapid adaptation and evolution of tolerant strains.

Our hardware journey began with a simple proof-of-concept system consisting of a gas pump and a glass reagent bottle, equipped with basic check valves and filters for input and output. While this system allowed us to verify experimental results and demonstrate the viability of our engineered cyanobacteria in heavy metal uptake, it was clear that this setup was not suitable for repeated use or larger-scale applications. The first prototype’s lack of reusability, limited control over environmental conditions, and minimal biosafety features drove us to develop a more sophisticated solution.

Second Generation: Integrated Bioreactor System

To address these limitations, we engineered a second-generation bioreactor, incorporating feedback from our experiments and best practices in biocontainment.

The reactor is composed of modular chambers that allow for stepwise water treatment, easy cleaning, and straightforward scaling. Water contaminated with heavy metals is introduced via a precision liquid pump, which ensures that the flow rate matches the processing capacity of the cyanobacterial culture inside. This is crucial because it allows for optimal contact time between the bacteria and pollutants, maximizing the efficiency of heavy metal removal.

Key Features and Workflow

1. Multi-Chamber, Modular System

• The reactor is divided into integrated chambers for pre-filtration, bioremediation, and post-treatment, enabling stepwise processing and easy module replacement or cleaning.

2. Flow Control and Homogeneous Mixing

• A precision liquid pump controls the inflow of contaminated water, ensuring the rate matches the bioremediation capacity of the cyanobacteria.
• An internal rotor stirs the culture, preventing sedimentation and ensuring even distribution of bacteria and heavy metals.

3. Aeration and Light Supply

• A dedicated air pump supplies filtered oxygen, supporting optimal photosynthesis and cyanobacterial health.
• External mounting points for LED light panels (sealed with rubber lips when unused) provide customizable light intensity, simulating natural conditions and maximizing biomass productivity.

4. Multi-Stage Filtration and Biosafety

• First Filter (Coarse): Removes large particulates to protect downstream components.
• Second Filter (TFF Membrane): Utilizes tangential flow filtration (TFF), a gold standard in bioprocessing, to:
  oRetain cyanobacterial cells and prevent their release into the environment,
  oAllow clean, treated water to exit safely.
• This dual filtration system is critical for environmental biosafety and regulatory compliance.

5. Sampling and Monitoring Ports

• Designed for easy sampling of water and biomass for real-time monitoring of metal removal efficiency, cell viability, and system parameters.

6. Scalability and Modularity

• Our 3D-printed prototype holds ~5L of culture, suitable for lab and small-scale field tests.
• The modular design allows straightforward scaling—by increasing chamber size, pump capacity, and membrane area, the system can be adapted for industrial or municipal applications.

7. Maintenance and Reusability

• Quick-connect fittings and user-friendly chamber design enable rapid cleaning, sterilization, and reloading of cyanobacterial cultures, supporting continuous operation and cost-effectiveness.

Third Generation: Advanced Bioreactor System for Water Treatment Plants and Natural Water Sources

Building on the successes and lessons learned from our previous bioreactor prototypes, we have designed the third-generation (Gen3) A.L.G.E.A. bioreactor to address the challenges of large-scale deployment in real-world environments, such as municipal water treatment plants and natural water bodies like lakes and rivers. Therefore, in our design, the main algae reaction pool will have a capacity of 10,000,000L, similar to one single pool capacity in a middle scale water treatment plant. Cyanobacteria will grow in different pools to it’s platform stage and pumped into the pool for reaction. The pool will be filled and drained twice a day. This generation emphasizes not only increased capacity and automation, but also enhanced robustness, environmental integration, and long-term biosafety.

Key Features and Workflow

Key Features and Workflow

1. High-Capacity, Modular Infrastructure

• Scalable Processing Units:
  The Gen3 bioreactor is designed as a modular array of interconnected units, each with a processing capacity of up to 20,000,000L per 24 hours. Multiple modules can be deployed in parallel or series, allowing seamless adaptation to the volume and contamination level of the water source.
• Mobile & Stationary Options:
  The system can be configured as a stationary installation for water treatment plants or as transportable modules for temporary or emergency response near lakes, rivers, or mining sites.

2. Adaptive Flow Control and Mixing

• Smart Inflow Regulation:
  Advanced electronic flow meters and automated valves enable real-time adjustment of water inflow, matching the bioremediation capacity of the cyanobacterial culture and responding dynamically to fluctuations in pollution levels.
• Turbine-Based Mixing:
  Robust, energy-efficient turbine mixers ensure complete homogenization of large water volumes, preventing dead zones and maximizing contact between pollutants and engineered cyanobacteria.

3. Optimized Aeration and Illumination

• High-Throughput Aeration:
  Industrial-grade air compressors deliver filtered oxygen to support dense cyanobacterial populations, while redundant systems guarantee uninterrupted aeration.
• Solar-Assisted LED Lighting:
  The system integrates both high-efficiency LED panels and solar collectors, reducing energy costs and enabling 24/7 operation—even in remote locations. Light intensity and spectrum can be customized to maximize photosynthetic performance and productivity.

4. Multi-Tiered Filtration and Environmental Biosafety

• Triple-Stage Filtration:
  oPrimary Coarse Filter: Removes large debris and sediments commonly found in natural water sources.
  oSecondary TFF Membrane: Ensures complete retention of engineered cyanobacteria and microalgae, allowing only treated water to pass.
  oFinal UV & Sterile Filtration: Treated water flows through a UV sterilization chamber and fine sterile filter, neutralizing any remaining microorganisms or genetic material before environmental discharge.
• Containment and Leak Detection:
  Integrated sensors continuously monitor for possible leaks or membrane failures, automatically shutting down the system and alerting operators to prevent unauthorized release of GEMs (genetically engineered microorganisms).

5. Advanced Monitoring, Automation, and Data Integration

• Real-Time Sensing Suite:
  Embedded sensors monitor critical parameters such as pH, temperature, dissolved oxygen, heavy metal concentrations, and cyanobacterial density.
• Remote Control and Data Logging:
  The Gen3 system is fully IoT-enabled, allowing remote monitoring, process optimization, and maintenance scheduling via a secure online dashboard. All operational data is logged for compliance and research purposes.

6. Environmental Integration and Energy Efficiency

• Eco-Friendly Materials:
  Construction materials are selected for durability, corrosion resistance, and low environmental impact.
• Energy Recovery and Water Recycling:
  Optional modules can recover heat and recycle treated water for secondary uses, increasing overall sustainability.

7. Ease of Maintenance and Long-Term Operation

• Automated Cleaning and Sterilization:
  The system features automated CIP (clean-in-place) and SIP (sterilize-in-place) protocols that minimize downtime and human intervention.
• Modular Replacement:
  Key components such as filtration membranes, pumps, and sensors are designed for hot-swap replacement, ensuring continuous operation even during repairs or upgrades.

Productivity Estimation of A.L.G.E.A. Bioreactor Systems

To provide a quantitative estimation of the biomass productivity and bioremediation capacity for the three generations of our A.L.G.E.A. bioreactor hardware. We applied experimentally derived biological parameters to the specifications of each hardware design, we can forecast the potential performance at different scales, from laboratory proof-of-concept to full-scale industrial deployment.

Methodology

The estimations are based on foundational parameters derived from our experimental results and on operational models appropriate for the scale of each hardware generation.

Foundational Parameters

he following key performance metrics were refered from our "result" section(https://2025.igem.wiki/sz-shd/results#result%207 )

• Organism: Engineered Synechocystis sp. PCC 6803 expressing the AtPCS and PseMT genes.
• Average Growth Rate: 0.175 OD/day, as determined from the Gompertz model fit of wild-type Synechocystis growth (Section 15).

• Biomass Conversion Factor: 0.0087 g (wet weight) / OD·mL, based on direct measurement of harvested cyanobacterial biomass (Section 16).

• Bioremediation Rate: 1.314 mg of total heavy metals removed per liter of water. This rate is based on the 24-hour performance of our engineered E. coli system (Section 7) and is used as the target performance proxy for the engineered Synechocystis.

Operational Models

Two distinct operational models are applied to reflect the different scales and designs of the hardware:

• Continuous Flow Model (for Gen 1 & Gen 2): This model applies to our smaller-scale systems. Contaminated water flows continuously through the reactor, where a stable, growing culture of cyanobacteria removes heavy metals. Productivity is measured as a daily rate of water treatment and biomass generation.
• Sequential Batch Reactor (SBR) Model (for Gen 3): This model is applied to the industrial-scale Gen 3 system. Algae are grown to a high density (OD 3.0) in separate "nursery" pools. The main treatment pool is filled with a mixture of this dense algae and contaminated water for a fixed 12-hour treatment cycle. The pool is then drained for downstream processing, and the cycle repeats, allowing for two full treatment cycles per day. This model focuses on total daily throughput and the required daily consumption of biomass.

Productivity Estimations by Generation

Generation 1: Proof-of-Concept Bioreactor
Operating under a continuous flow model with an assumed culture volume of 1 L:

• Water Treatment Capacity: 2 L/day
• Estimated Biomass Productivity: 1.53 g/day (wet weight)
• Estimated Bioremediation Productivity: 2.63 mg of metals/day

Generation 2: Integrated Bioreactor System
Operating under a continuous flow model with a 5 L culture volume:

• Water Treatment Capacity: 100 L/day
• Estimated Biomass Productivity: 7.66 g/day (wet weight)
• Estimated Bioremediation Productivity: 131.4 mg of metals/day

Generation 3: Advanced Industrial System
Operating under an SBR model with a 10,000,000 L batch volume and two cycles per day:

• Daily Water Throughput: 20,000,000 L/day
• Estimated Bioremediation Productivity: 26.28 kg of metals/day
• Required Daily Biomass Consumption: 522 tons/day (wet weight at OD 3.0)