Serial Bioreactor System (SeBiS)
Overview
The implementation of the R-DetoX 2.0 engineered E. coli must be considered to ensure its adequate containment. In the context of this project, bacteria cannot be directly released into the contaminated water source for degradation to occur. Therefore, a strategy must be considered to effectively employ the prototype. SeRIAL BiOREACTOR SYSTEM (SeBiS) is a theoretical process designed to leverage bioreactors to deliver the recombinant bacteria safely. As illustrated in Figure 1, the process follows a defined workflow: bacterial growth, RDX detection, RDX degradation, byproduct mitigation, and water testing prior to release.
Figure 1. SeBiS Conceptual Workflow.
Why a Bioreactor System
A bioreactor is a controlled vessel used to support biological processes such as bacterial growth, biodegradation, and biochemical reactions. It provides an optimal environment by regulating factors like temperature, pH, and oxygen levels, allowing microorganisms or cells to function efficiently for a specific purpose and maintaining that environment by the use of sensors. In this project, the bioreactor is central as it allows determination of scale-up considerations to both cultivating recombinant E. coli and later using it to mediate the degradation cycle of RDX from the contaminated water. There are parameters that do not depend on scale; that is, they are constants independent of the process size [1]. The purpose of the testing system being proposed with bioreactors instead of test tubes is to effectively simulate all the conditions and factors that could influence our bioremediation system when implemented on a larger scale. Therefore, the proposed Serial Bioreactor System (SeBiS) design helps us visualize a testing process that can provide more information for process scale-up and the vision of future integration into treatment plants.This project specifically considers a batch bioreactor system, where all nutrients and bacterial cultures are added at the beginning, and no additional input or output occurs during the process. In the first stage, a process of scaling up the recombinant bacteria takes place. A culture from a frozen stock will be transferred to a flask, then to a seed bioreactor, and eventually to a batch bioreactor, where ideal conditions for growth will be simulated until the desired bacterial biomass is reached. This is done since gradual scale-up ensures healthy, active inoculum and stable, distributed growth in the production reactor [2]. Once sufficient biomass is obtained, the next stage focuses on promoting effective interaction between the engineered bacteria and the RDX-contaminated water to enable biodegradation of RDX. The resulting byproducts, formaldehyde and nitrite, are still potential contaminants and will be further treated downstream using activated carbon for formaldehyde and an electrolytic reactor for Nitrite. Finally, a validation and water quality assessment step aims to confirm the removal of all contaminants before environmental discharge.
Figure 2. Process Flow Diagram (PFD) of the Proposed Bioprocess for E. coli-Mediated RDX Degradation.
All Phases
THE BIOREACTOR GROWTH SYSTEM
The first phase aims to cultivate the engineered E. coli until the desired biomass production is obtained. The scale up process consists of these mayor steps: the bacteria is inoculated from a frozen stock into a test tube, then to a culture shake flask, and eventually into a batch bioreactor, where the environment is carefully controlled to support optimal growth.
Figure 3. Scale-up of growth of recombinant E. coli from frozen stock to shaken flask to bioreactor
Parameters such as pH, temperature, dissolved oxygen, glucose feed concentration, agitation speed, and air flow rate need to be monitored throughout the process. When it comes to the medium, M9 salts minimal medium with glucose will be considered as the basal medium [3], because its colorless nature facilitates the visualization of the reporter gene’s expression, which produces a red pigment. These types of mediums are preferred for the testing of the R-DetoX 2.0 prototype due to its chromogenic nature, which can indicate the performance of the system and can be monitored analytically in-line without compromising the sterility of the process. Alternatively, RPMI1640 or rich LB medium can be used for the growth of the prototype [4]. Once this upstream process is done, the culture generated during Phase 1 will be then transferred to the enzyme reaction bioreactor, where degradation occurs.
Operation parameters:Some growth parameters for recombinant E. coli in a bioreactor are independent of the vessel's working volume, while others depend on it and must be adjusted accordingly, such as agitation (rpm) and aeration rate (vol/time).
Table 1. Operation Parameters Independent of Scale.
| Parameter | Value | Monitoring | Adjustment | Source |
|---|---|---|---|---|
| Temperature | 37°C | Temperature Sensor | Heating Jacket | [5] Murungan et al. (2024) |
| pH | 7 | pH Sensor | Buffer tank | |
| Dissolved Oxygen | 30–35% | Oxygen Flowmeter | Oxygen Inlet |
Enzymatic Degradation of RDX Bioreactor
In the Second Phase, the objective is to expose the recombinant E. coli containing our prototype to the RDX-contaminated water, allowing the process to continue until both RDX degradation and bacterial lysis are complete. Prior to feeding, the water is passed through a filter that contains large rocks and fine sand to remove large particles. The culture from the first batch bioreactor will be transferred into the second batch bioreactor, and the contaminated water will enter from a second input until the desired volume of water is obtained. In this bioreactor, RDX will interact with the riboswitch component of the prototype, triggering the production of the enzymes flavodoxin reductase XplB and fused flavodoxin-cytochrome P450 XplA [6]. These enzymes will be the biological scissors that will degrade the RDX molecule and consequently, produce the byproducts: formaldehyde and nitrite (see Figure 4). The KillSwitch will be activated after byproduct formation, and bacterial lysis will take place. Before passing on to the Third Phase, the bioreactor will have a momentary shut-down with the purpose of letting the bacterial components deposit themselves at the bottom of the bioreactor. Subsequently, the final solution will be transferred to the next Phase, leaving behind sludge containing dead bacteria—which will be passed down to a biowaste container. Ideally, the remaining components inside the second bioreactor should be mainly water, formaldehyde and nitrite.
Figure 4. Biocatalyzed routes proposed for the degradation of RDX. Pathway A corresponds to the route proposed under anaerobic conditions; pathway B is that postulated to occur under aerobic conditions [7]
The purpose of having a model of two serial bioreactors has to do with the working volume of the two different vessels and the growing phases of the bacterial culture. The first bioreactor will cultivate the recombinant E. coli to its maximum volume capacity. This culture will pass on to a bigger bioreactor that can handle both the volume of the culture and the volume of the water without overstepping the maximum capacity of work volume. Consequently, the parameters for the second batch bioreactor will be numerically different to those of the first vessel. The efficacy of the recombinant E. coli requires a sufficient number of healthy bacteria before reacting with RDX, a xenobiotic contaminant. Therefore, once the culture in the Growth Bioreactor reaches a high biomass, it will be transferred to the second batch bioreactor.
Electrochemical Reactor Byproduct Mitigation
After the biological degradation stage, the effluent still contains formaldehyde and nitrite, which must be removed before discharge. First, formaldehyde is captured by passing the effluent through an activated carbon adsorption unit. Activated carbon (AC) has been demonstrated as an effective and low-cost adsorbent for formaldehyde removal in aqueous solutions, achieving removal efficiencies exceeding 90% using commercial and biomass-derived AC adsorbents—for example, a study reported that commercial AC reached 99.2% removal [8]. Once formaldehyde is largely removed, the remaining solution, primarily water and nitrite, is directed to an electrochemical reactor.
An electrochemical reactor is a device that uses an electric current to drive chemical reactions. It typically has two electrodes (anode and cathode) in contact with an electrolyte. When a voltage is applied, electrons move through an external circuit and ions move through the electrolyte, causing chemical changes. In this system, the electrochemical reactor is designed to target nitrite (NO₂⁻) reduction, converting it into dinitrogen gas (N₂).
During electrochemical nitrogen reduction, several products such as ammonia (NH₃), nitrous oxide (N₂O), and dinitrogen (N₂) can form depending on the electrode material and applied potential. Achieving selectivity toward N₂ is particularly desirable because it is inert, environmentally safe, and naturally abundant in the atmosphere. Furthermore, nitrogen gas has extensive industrial applications, making its formation not only environmentally benign but also potentially valuable from a resource recovery perspective. A study by Miao Li et al. demonstrated that by optimizing electrode composition and working conditions, nitrate reduction could reach 87% removal with 100% selectivity to N₂ [9]. This highlights how electrochemical parameters can steer product distribution toward nitrogen gas.
Reactions and Mechanisms
In the SeBiS system, the electrochemical reactor aims to promote the reduction of nitrite (NO₂⁻) into N₂ through a stepwise proton–electron transfer mechanism.At the anode, water oxidation occurs:
\( 2\mathrm{H}_2\mathrm{O} \rightarrow \mathrm{O}_2 + 4\mathrm{H}^+ + 4e^- \)
At the cathode, nitrite ions accept electrons (reduction) and react with protons from water to form nitrogen gas:
\( 2\mathrm{NO}_2^- + 8\mathrm{H}^+ + 6e^- \rightarrow \mathrm{N}_2 + 4\mathrm{H}_2\mathrm{O} \)
Finally, balancing electrons to obtain the net reaction allows mitigation of Nitrite into Nitrogen gas and Oxygen:
Anode equation x 3:
\( 6\mathrm{H}_2\mathrm{O} \rightarrow 3\mathrm{O}_2 + 12\mathrm{H}^+ + 12e^- \)
Cathode equation x 2:
\( 4\mathrm{NO}_2^- + 16\mathrm{H}^+ + 12e^- \rightarrow 2\mathrm{N}_2 + 8\mathrm{H}_2\mathrm{O} \)
Net equation:
\( 4\mathrm{NO}_2^- + 4\mathrm{H}^+ \rightarrow 2\mathrm{N}_2 + 3\mathrm{O}_2 + 2\mathrm{H}_2\mathrm{O} \)
* Note: The net equation suggests that the reaction must occur under acidic conditions.
Reactor Design Considerations
The effectiveness of an electrolytic reaction is influenced by variables such as electrode surface area, applied voltage, current density, electrode material, electrolyte composition, temperature, and reaction time. The SeBiS Electrochemical Reactor, shown in (Figure 5), provides precise energy control, optimized electrode surfaces, and effective mixing, creating a stable environment for continuous and efficient oxidation–reduction reactions.
Figure 5. SeBiS Electrochemical Reactor Design Featuring Semicircular Electrodes.
The chamber allows the regulation of temperature, via a heating jacket, and solution homogeneity via an agitator. Without the agitation, dead zones or concentration gradients could form, drastically reducing system performance. The environment created by the chamber is essential for maintaining consistent reaction conditions and optimizing electrochemical performance.
The electric distribution is the most critical aspect of our design (Figure 6). The energy is managed by a control system that regulates critical parameters such as voltage, current, and operating time. Electricity travels from a Direct Current (DC) power supply through conductive wires to the electrodes. To achieve high selectivity toward N₂ rather than other nitrogen species (like ammonia), the choice of electrode material is crucial. Miao Li et al. showed that using a Ti/IrO₂–Pt anode combined with a Fe cathode in an undivided cell, and along with the presence of NaCl in the electrolyte, enabled high selectivity for N₂ while minimizing ammonia and nitrite intermediates [9]. At the same time, the anode facilitates complementary oxidation reactions, maintaining charge balance in the system.
Figure 6. Electrodes for Energy Distribution
The electrodes feature a semicircular, large plane area that aims to increase the surface area in contact with solution. This allows more particles to come in contact with the electrodes to increase the probability of reaction.
Through this electrochemical process, the nitrite concentration is reduced, and harmless nitrogen gas is released, completing the byproduct mitigation stage. Combined with prior formaldehyde adsorption, this ensures that the treated effluent meets environmental safety standards before discharge, providing a robust finishing step to the treatment process.
Water Testing & Validation
The final stage ensures that SeBiS achieves the process objectives: effective RDX removal, byproduct mitigation, and safe environmental containment of viable recombinant organisms before discharge. The Water Testing & Validation Phase involves analytical testing to: (1) quantify RDX reduction through the process; (2) quantify byproducts (e.g., formaldehyde, nitrite) and confirm their removal by downstream treatment; (3) demonstrate Kill-Switch efficacy and absence of viable recombinant E. coli in the treated effluent; (4) and establish acceptance criteria that ensures no harm in accordance to the Environmental Protection Agency (EPA) guidelines.
To support these analysis, water will be temporarily stored in a sampling tank, and triplicate samples will be collected to test each target analyte—RDX, formaldehyde, nitrite, and recombinant E. coli—resulting in a total of twelve validation samples. Figure 7 shows the method used for each target.
Figure 7.Bioprocess validation protocol by detecting the presence of formaldehyde, nitrite and RDX after the last stage
The acceptance criteria for each target analyte are based upon exposure limits established by the EPA (Table 2). Results that comply with the acceptance criteria for all the target analytes indicate that the water can safely return to the environment and mark the end of the process for that batch. Failure to comply with any of the acceptance criteria would result in recirculation of the water from the storage tank to the degradation bioreactor for further treatment.
Table 2. Acceptance Criteria Based on Established Guidelines.
| Target | Limit Value | Source |
|---|---|---|
| RDX | 5.8 μg/L | [10] U.S. Environmental Protection Agency, 2017 |
| Formaldehyde | 8 μg/L | [11] New York State Department of Environmental Conservation, 2000 |
| Nitrite | 1.0 mg/L | [12] U.S. Environmental Protection Agency, n.d. |
| E. coli | 100 cfu / 0.1 L | [13] U.S. Environmental Protection Agency, 2021 |
* In cases where different values were given, the worst case was assumed, and the lowest limit was selected.
Moving Forward
The SeBiS model serves as an in-silico bioprocess framework, establishing a foundation for the controlled application of engineered E. coli in the bioremediation of energetic pollutants such as RDX. Future work should focus on the experimental validation of SeBiS under controlled laboratory conditions and its potential integration into treatment systems.
SeBiScould be incorporated into existing wastewater or industrial treatment plants, where streams contaminated with highly energetic compounds—such as RDX—could be directed into dedicated bioreactor modules equipped with the recombinant E. coli. Through this integration, SeBiS could operate as a specialized biodegradation unit, complementing conventional treatment stages to achieve more complete detoxification. Further developments may also include optimizing bioreactor parameters and applying digital monitoring platforms to automate process control and performance validation. Ultimately, SeBiS could redefine how we address energetic waste contamination, paving the way for cleaner water, safer ecosystems, and a future where synthetic biology and engineering work hand in hand.
References
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[10] U.S. Environmental Protection Agency, "RDX (1,3,5-trinitroperhydro-1,3,5-triazine) TFS," Sep. 15, 2017. [Online]. Available: EPA Fact Sheet
[11] New York State Department of Environmental Conservation, "Human health fact sheet: Ambient water quality value for protection of human health and sources of potable water – Formaldehyde (CAS No. 50-00-0)," Apr. 2000. [Online]. Available: NY DEC Fact Sheet
[12] U.S. Environmental Protection Agency, "Consumer factsheet on: Nitrates / Nitrites [Archived fact sheet]," U.S. EPA. [Online]. Available: EPA Nitrates/Nitrites
[13] U.S. Environmental Protection Agency, "E. coli (Escherichia coli): Factsheet on water quality parameters (EPA 841F21007F)," Jul. 2021. [Online]. Available: EPA E. coli Factsheet
