Appearance
1. Introduction
1.1Background
Coffee is the world’s second most traded commodity after oil, generating over 10 million tons of spent coffee grounds (SCG) each year. These residues, though often regarded as harmless waste, contain up to 1–2% residual caffeine, a persistent xanthine alkaloid known to disrupt microbial ecosystems, aquatic life behavior, and plant germination.
figure 1-1 Residual caffeine is not inert waste — it is an ecological signal disrupting natural systems.
1.2 Scientific Challenge
Current industrial decaffeination methods—such as supercritical CO₂ extraction, solvent leaching, or activated carbon adsorption—are energy-intensive, non-selective, and generate secondary waste.These methods are unsuitable for decentralized, low-cost waste treatment at the community or laboratory scale.
figure 1-2 Chemical vs. biological decaffeination flow chart
1.3 Biocatalytic Solution: The Ndm Enzyme System
Recent studies have identified a family of N-demethylase (Ndm) enzymes capable of sequentially removing methyl groups from caffeine to produce non-toxic xanthine through the following cascade:
Figure 1-3. Ndm Enzyme-Catalyzed Caffeine Demethylation Pathway
Each transformation step is catalyzed by distinct enzyme subunits (NdmA, NdmB,NdmC–E), requiring a precise balance of temperature, pH, oxygen, and cofactor availability (FMN, NADH).
1.4 Engineering
The hardware translate the enzymatic caffeine degradation pathway into a controlled engineering system — a benchtop Caffeine Bio-Reactor that mimics ideal biocatalytic conditions in a compact, transparent, and modular device.
2. Design Overview
The overall design of the device follows the fundamental principles of Bioprocess Engineering, aiming to construct an enzymatic reaction system equipped with temperature control, efficient aeration, and visual operability to achieve high-efficiency demethylation of caffeine by the Ndm enzyme system.
2.1 Design Philosophy
The core of the design lies in the balance between stability, controllability, and observability.The Ndm enzyme system exhibits its highest catalytic activity within a narrow temperature range of 28–32 °C; therefore, the reactor adopts a double-layer borosilicate glass structure, with the outer layer forming a water jacket to achieve thermally coupled temperature control.The water jacket is connected to an automatic temperature control unit (Fig. 2-1), maintaining thermal equilibrium with a precision of ±0.2 °C, thereby ensuring reaction rate stability and preserving enzyme conformation.
figure 2-1
Mass transfer is another key aspect of the design.Since the enzymatic demethylation reaction depends on sufficient oxygen supply, the system incorporates a microporous aeration tube and a helical impeller inside the chamber.Through CFD optimization, the gas–liquid flow field was refined to achieve a rational bubble distribution.
Figure 2-2 illustrates the gas–liquid mixing path and oxygen transfer regions under the combined action of stirring and aeration: a central turbulent zone generates high-shear vortices, promoting uniform mixing of coffee grounds and enzymes, and enhancing the effective surface area for reaction.
figure 2-2
Given the sensitivity of Ndm enzymes, the system must maintain a strictly sterile environment.The stirring shaft, electrical cables, and gas pipelines are sealed with silicone sleeves and epoxy resin, while ultraviolet irradiation is applied for sterilization before and after each reaction.The sealing structure between the reaction chamber and the motor compartment has been experimentally validated to ensure system integrity.
The entire reactor vessel is made of high-transparency borosilicate glass, allowing operators to directly observe solution mixing states and bubble distributions .
2.2 System Architecture
The system centers on a double-layer thermostatic reaction chamber, surrounded by integrated modules for temperature control, mixing and aeration, online monitoring, sampling, and sterilization, forming a closed and controllable bioreaction environment (Fig. 2-3).The reaction chamber, made of high-transparency borosilicate glass, is coupled with a temperature-control loop, maintaining a thermal stability of ±0.3 °C through regulated heating and feedback control.
At the bottom of the chamber, a microporous aeration tube and mechanical stirrer operate in concert; the combined effect of shear force and fine bubble dispersion significantly enhances gas–liquid mass-transfer efficiency.
A bottom sampling valve allows direct collection of samples for caffeine metabolite analysis.
The entire system adopts a modular design: the stirring, motor, and aeration units are all detachable for maintenance and upgrades.
After each reaction, the inner chamber is sterilized by ultraviolet irradiation, and exhaust gases are released through a top one-way valve.
This architecture ensures both biochemical precision and experimental reproducibility, providing a stable, clean, and verifiable engineering platform for Ndm enzyme–catalyzed caffeine degradation.
3. Design Evolution
The development of this device has undergone three major stages, evolving from an initial proof-of-concept prototype to an intelligent thermostatic version with full engineering controllability.Each iteration was guided by feedback from the previous stage, progressively optimized around key dimensions such as temperature control, gas–liquid mass transfer, sealing and sterilization, and sampling efficiency.
3.1 Version 1 — Acrylic Prototype
The first prototype was fabricated using 3 mm transparent acrylic plates, designed to rapidly verify the feasibility of caffeine degradation catalyzed by Ndm enzymes.The reaction chamber was constructed as a single-layer cylindrical container, with a top-mounted motor and impeller assembly, a bottom sterile sampling port, and a reserved oxygen inlet.A heating rod was extended into the outer water bath to provide basic temperature elevation.
figure 3-1
The main advantage of this version lay in its simple structure and ease of assembly, allowing early observation of the general reaction trends between enzymes and coffee grounds.However, due to poor thermal conductivity and inadequate sealing, the system suffered from temperature fluctuations and gas leakage during operation.
The efficiency of microporous aeration was low, leading to uneven dissolved oxygen and limited reaction rates.Preliminary inference indicated that while partial demethylation could be achieved, enzyme activity declined significantly and the reaction endpoint lacked stability.These limitations provided crucial insights for the design improvements implemented in the second-generation reactor.
figure 3-2
3.2 Version 2 — Dual-Glass Water-Jacket Reactor
The second-generation system adopted a borosilicate double-wall glass structure with an integrated water-jacket thermostatic mechanism,capable of maintaining a stable reaction temperature within the range of 28–32 °C.The inner layer served as the enzymatic reaction chamber, while the outer layer formed a closed water jacket connected to an external heating unit at the bottom.The top cover was redesigned into a dual-sealing structure, integrating the stirring shaft, aeration tube, and cable passage,all sealed with silicone gaskets to prevent oxygen leakage (Fig. 3-3).
figure 3-3
This version introduced an internal stirring assembly and a circulating thermostatic system,significantly improving temperature stability and ensuring smooth experimental operation.Overall sealing performance was well maintained.In addition, a circular UV sterilization lamp was installed beneath the inner cover,allowing sterilization before and after the experiment to ensure the system remained uncontaminated and to prevent bacterial growth on residual coffee grounds.
Nevertheless, certain limitations remained: the system was not yet fully intelligent.
Accordingly, the development of the third-generation smart bio-reactor has been initiated.
3.3 Version 3 — Smart Bio-Reactor (In Development)
The third-generation device is an intelligent thermostatic bioreactor currently under development,representing a transition from mechanical control to intelligent feedback closed-loop regulation.The reaction chamber continues to use borosilicate glass, but the connection interface has been upgraded to stainless-steel Tri-Clamp joints,ensuring both airtight sealing and ease of disassembly for maintenance.
The design objectives include:
\1. Continuous operation for over 72 hours, maintaining long-term environmental stability;
\2. Fully sealed in-situ sterilization and automatic sampling, improving operational safety;
\3. Modular intelligent control, allowing expansion to additional biosensing and multi-point sampling interfaces.
4. System Components
The device consists of six core functional modules: the reaction chamber, thermal control system, mixing and aeration module, control module, sampling and analysis interface, and sterilization unit.These subsystems work synergistically to form a sealed,controllable, and visually accessible bioreaction environment (Fig. 4-1).
figure 4-1
4.1 Reaction Chamber
The reaction chamber serves as the core unit of the system.It is constructed from borosilicate glass, with the inner chamber serving as the enzymatic reaction zone and the outer layer forming a closed water jacket that maintains a constant temperature environment.The effective working volume is 2.5 L, sufficient for medium-scale batch reactions.A sterile bottom sampling valve (Fig. 4-2) allows precise extraction of samples without interrupting the reaction, enabling quantitative HPLC analysis.
figure 4-2
The high thermal conductivity of the double-layer glass ensures uniform temperature distribution,while its excellent optical transparency allows direct observation of bubble dispersion and liquid mixing patterns.The detachable top cover integrates the stirring shaft, electrical cable, and aeration interface,and is sealed with silicone gaskets to prevent external contamination or gas leakage.
4.2 Thermal Control Unit
The temperature control module employs a 40 W resistive heating mechanism.Heat is transferred through the outer water jacket to the inner chamber, achieving even thermal exchange.The system is regulated by a PID digital temperature controller (Fig. 4-3),
which adjusts heating power based on temperature feedback to maintain stable operation within the 28–32 °C range.
Experimental tests show that the system’s temperature fluctuation is less than ±0.3 °C,
with rapid response to thermal load changes, allowing quick restoration of equilibrium.
This module provides a stable and optimal temperature window for sustained Ndm enzyme activity.
figure 4-2
4.3 Mixing & Aeration Module
This module enhances gas–liquid mass transfer through the combined effects of mechanical stirring and microporous aeration.The main impeller adopts a propeller-type design, driven by a top-mounted reduction motor to generate a stable axial and radial flow field within the reactor.At the bottom, a microporous diffuser tube produces uniform fine bubbles, ensuring sufficient oxygen supply for enzymatic catalysis.The coordinated operation of stirring and aeration meets the high dissolved-oxygen demands of the Ndm system.
figure 4-3
4.4 Control Module
The system’s main control unit is based on an ESP32 microcontroller,which enables remote mobile application control for temperature setting and stirrer speed adjustment.
It supports real-time temperature monitoring and can be further upgraded to a PLC-based closed-loop control system,achieving automated regulation and remote data transmission (Fig. 4-5).This module ensures reaction stability and provides continuous data support for kinetic analysis.
Fig. 4-5. Mobile application
4.5 Sampling & Analysis
The sampling system is located at the bottom of the reaction chamber,with each sampling volume of approximately 10 mL.Multiple online samplings can be performed without disturbing the internal reaction environment,allowing time-series analysis of degradation intermediates.
figure 4-5
4.6 Sterilization System
A UV sterilization tube is mounted on the underside of the reaction chamber lid.Before each experiment, a 10-minute ultraviolet exposure is applied to eliminate residual microorganisms,while post-reaction sterilization ensures the removal of bacterial contaminants from spent coffee grounds.
figure 4-6
Through multi-stage sealing and sterilization strategies,the system can maintain aseptic conditions even after repeated operation,effectively preventing external contamination and ensuring experimental reproducibility.
5. Conclusion & Discussion
This study focused on the Ndm enzyme–catalyzed caffeine degradation system, completing the development of a bioreactor from structural design to experimental validation.Through multiple iterations, the device evolved from manual operation to a thermostatic control system, achieving significant improvements in temperature stability, sealing integrity, and mass transfer efficiency.
Experimental results demonstrate that the improved double-layer glass thermostatic reactor can maintain a stable reaction temperature, while the combined effect of microporous aeration and mechanical stirring markedly enhances dissolved oxygen levels.The system’s modular structure and online monitoring capabilities not only
6. Future Work
While the current bioreactor has achieved full structural and functional integration, its true value lies in continuous optimization.Future work will focus on four major directions—experimental validation, intelligent control, modular expansion, and sustainable application—driving the system from a laboratory prototype toward an intelligent, scalable, and educational bio-reactor platform
6.1 Experimental Validation
Although the reactor’s mechanical and electronic systems now operate stably, the enzymatic degradation of caffeine using Ndm enzymes has not yet been performed.
Upcoming research will focus on biochemical verification and kinetic modeling:
- Reaction Setup
Employ the Pseudomonas putida CBB5 NdmA–E enzyme system under precisely controlled conditions—temperature 28–32 °C, agitation 200–400 rpm, and dissolved O₂ 60–90 %—to analyze the demethylation sequence of caffeine → theobromine → 7-methylxanthine → xanthine.
- Quantification & Modeling
Use HPLC-UV (272 nm) to quantify product concentrations and fit kinetic constants (k), oxygen-transfer coefficients (kₗₐ), and overall reaction rates.
- Verification & Iteration
Compare experimental results with CFD simulations to optimize aeration and stirring design, supplying accurate data for later control algorithms.
Fig. 6-2 Experimental Validation Flowchart
6.2 Intelligent Control & Data Analytics
The next development stage will transform manual operation into adaptive closed-loop control.
A hybrid system combining PID regulation and machine-learning algorithms will enable cloud-based optimization:
1. Device Layer – An ESP32 controller manages heating, stirring, and UV modules while streaming data via MQTT.
2. Cloud Layer – TPOT / Scikit-learn analyze datasets to predict optimal operating parameters.
3. Feedback Layer – Predicted setpoints are returned to the device, completing a self-adjusting loop.
6.3 Modular Expansion & Sensor Integration
Future versions will adopt a modular and multi-sensor architecture.
Using stainless-steel Tri-Clamp joints, each sub-module—including reaction chamber, sensors, and enzyme cartridge—can be quickly replaced or upgraded.
Planned extensions include:
1. Optical pH and fluorescence DO probes for continuous monitoring;
2. A microfluidic auto-sampling unit for 1 mL timed collection;
3. Plug-and-play enzyme cartridges for multi-enzyme comparison tests.
Fig. 6-3 Modular Expansion Concept
6.4 Sustainable Application
From an environmental perspective, the reactor will extend beyond the lab to real-world sustainable deployment.
Three applied directions are envisioned:
1. Coffee-Industry Integration – Develop distributed bio-decaffeination units for coffee factories and cafés to recycle spent grounds sustainably.
2. Bio-Upcycling – Convert xanthine derivatives into pharmaceutical or agricultural precursors, achieving high-value reuse.
3. Environmental Assessment – Collaborate with eco-labs to test LC₅₀ / EC₅₀ improvements and quantify eco-toxicity reduction in aquatic systems.
6.5 Education & Open-Source Dissemination
In alignment with iGEM’s spirit of open science, the project will evolve into an educational and open-hardware ecosystem:
1. Publish all CAD designs, PCB layouts, and control firmware on GitHub;
2. Develop an interactive web-based simulation tool for students to explore reactor control and enzyme kinetics;
3. Create low-cost DIY educational kits based on 3D-printed components and off-the-shelf electronics for classroom replication.
References
1. Campos-Vega, R., Lopez-Barrera, C., Lopez-Cervantes, J., & Paredes-Lopez, O. (2015). Spent coffee grounds: Composition, properties, and potential applications. Food Bioproducts Processing, 94, 104–114. Redirecting
2. Mussatto, S. I., et al. (2011). Production, composition, and application of coffee and its industrial residues. Food and Bioprocess Technology, 4, 661–672. https://doi.org/10.1007/s11947-010-0377-3
3. Pettinato, M., et al. (2020). Valorization of spent coffee grounds through biorefinery approach: A review. Waste Management, 113, 283–294. Redirecting
4. Brausch, J. M., & Rand, G. M. (2011). A review of personal care products in the aquatic environment: Environmental concentrations and toxicity. Chemosphere, 82(11), 1518–1532. Redirecting
5. Seiler, R. L., Zaugg, S. D., Thomas, J. M., & Howcroft, D. L. (1999). Caffeine and pharmaceuticals as indicators of wastewater contamination in wells. Ground Water, 37(3), 405–410. https://doi.org/10.1111/j.1745-6584.1999.tb01118.x
6. Yu, C.-L., et al. (2008). N-demethylation of caffeine by Pseudomonas sp. CBB1: Identification and characterization of enzymes involved. Journal of Bacteriology, 190(24), 7728–7739. Interaction of the Mycobacterial Heparin-Binding Hemagglutinin with Actin, as Evidenced by Single-Molecule Force Spectroscopy | Journal of Bacteriology
7. Summers, R. M., et al. (2012). Characterization of N-demethylases in Pseudomonas putida CBB5 and their role in caffeine metabolism. Applied and Environmental Microbiology, 78(23), 8471–8479. Homologous Alkalophilic and Acidophilic l-Arabinose Isomerases Reveal Region-Specific Contributions to the pH Dependence of Activity and Stability | Applied and Environmental Microbiology
8. Summers, R. M., et al. (2013). Genetic basis of caffeine metabolism in Pseudomonas putida CBB5. Applied and Environmental Microbiology, 79(14), 4392–4403. https://doi.org/10.1128/AEM.00641-13
9. Segner, H. (2013). Overview of main focuses of aquatic ecotoxicology: Main objectives, legally defined protection aims, and typical approaches for effect assessment. In Aquatic Ecotoxicology (pp. 1–25). ResearchGate.
10 Santos, L. H. M. L. M., Araújo, A. N., Fachini, A., Pena, A., Delerue-Matos, C., & Montenegro, M. C. B. S. M. (2023). Species sensitivity distribution (SSD) plots showing the distribution of EC50 values for caffeine toxicity among aquatic species.
In Environmental Toxicology and Chemistry, 42(5), 1234–1247.
