Project Introduction
Hydrogen (H2) is an often-underutilized energy source that can uniquely decarbonize long-distance transport and be used in high-heat industrial applications [1]. In addition to being water and land intensive, 96% of current H2 fuel production is reliant on fossil fuels [1]. In contrast, one method of sustainable H2 production is dark fermentation (DF), which uses organic waste to produce H2 fuel in waste-to-energy (WTE) streams. However, DF suffers from low yields due to its usage in high oxidative-reductive potential (ORP) environments [2]. High ORP environments result in excessive oxidation of hydrogen-producing bacteria (HPB) which inhibits H2 production [3]. Needed is a biological device that can decrease the ORP to –379 mV in environments where DF occurs and thereby increases H2 yields [4].
To fulfill this need, we engineered a biological device (the Cysteinator) which reduces the ORP of dark fermentation media. The Cysteinator supplements DF feedstocks with L-cysteine, an amino acid capable of reducing ORP, increasing H2 production. DF feedstocks can range anywhere from solid bean curd waste to rice winery wastewater, and so the Cysteinator was built to function despite varied environments [5]. We have done so by building in device modularity, accompanied by a comprehensive multi-scale model to predict device behavior in various feedstock compositions.
Included in our project are analyses to determine how the Cysteinator can be implemented in WTE streams and its utility to those streams. Our multi-faceted modeling approach is employed in diverse ways that help optimize testing and future implementation. Accompanied by a Life-Cycle Assessment, Techno-Economic Analysis, and a review of the impact on the UN Sustainable Development Goals, our project characterizes the benefit that the Cysteinator can bring to the global community.
We also deliver a novel composite part, an L-cysteine activated bi-directional switch built based on circuitry native to P. ananatis. This bi-directional switch can be used in a variety of applications and introduces new circuitry for future biological devices. Our multi-faceted modeling approach uniquely characterizes device behavior while abating common assumptions that plague models of metabolically engineered devices. This approach is an example of how modeling can be applied to integrating biological devices with biotechnology. These novel approaches can be used to enhance the utility of future biological devices.
Project Description
Large-scale commercial exploitation of fossil fuels began during the Industrial Revolution, triggering an extreme rise in the release of harmful carbon dioxide (CO2) [6]. CO2 emissions have risen by ~40% in the past 60 years; contributing to ocean acidification, more extreme weather events, and higher temperatures and sea levels [6]. This existential threat underlines the immediate need to address our climate challenges with sustainable energy technologies (Figure 1).
Figure 1. Diagram depicting the effects of CO2 emissions caused by burning fossil fuels.
Hydrogen (H2) is an underutilized energy source with incredible promise to decarbonize long-distance transport and replace fossil fuels in industrial heat generation. With three times the energy density (J/g) than traditional fossil fuel sources, sustainable H2 energy is uniquely situated to address the energy storage and density challenges of current renewable energy [5].
By 2050, global H2 demand is projected to reach 20% of total global energy demand, approximately 585 million tons per year [7]. However, 96% of current hydrogen production is powered by fossil fuel combustion, undermining its potential to truly become a clean energy source [1]. Dark Fermentation (DF), a light-independent process in which anaerobic bacteria convert glucose into H2, is the most promising sustainable H2-production alternative [8].
DF can use a wide variety of feedstocks to prepare bacterial inoculum, and diverse substrates to use as glucose sources in a process independent of external energy [10]. These characteristics enable DF systems to transform the landscape of waste-to-energy (WTE) streams. WTE streams recycle biomass, agricultural residues, industrial wastewater, and municipal solid waste to produce energy, and DF enables this process to efficiently and sustainably produce H2. Along with H2, DF produces volatile fatty acids which can be used in a variety of downstream applications [10]. DF is designed to exploit existing bioreactor designs and can be easily integrated into existing infrastructure. This collection of reasons is why DF is the forefront of sustainable H2 production.
Industrial DF is reliant on Hydrogen-Producing Bacteria (HPB) converting glucose to acetic or butyric acid to produce H2 (see figure 3) [5]. This process is often inhibited by Hydrogen-Consuming Bacteria (HCB) and metabolic competitors, which result in low H2 yields [11]. To limit the proliferation of HCBs and metabolic competitors a variety of pretreatments (pH, chemical, heat, loading shock, etc.) and bioreactor conditions are used (pH, temperature, hydraulic-retention time, etc.) [12]. Yet these alterations to the DF process flow do not sufficiently yield H2 for commercial implementation (see figure 2). The Cysteinator and advancements in DF will contribute to reducing sustainable H2 production costs from $105–$270/MWh to $58-150/MWh, in line with onshore wind energy and utility-scale solar energy production costs at $27-$92/MWh [13-14].
Figure 2. Figure 2 shows the four stages in the DF process flow. Stage 1: Inoculum Preparation consists of the preparation of feedstocks with various pretreatments. This process converts the HPBs in feedstocks into bacterial inoculum where HPBs propagate and are ready to be introduced into the bioreactor. Stage 2: Substrate Preparation consists of the hydrolysis of substrates to convert organic wastes to H2 precursors (eg. glucose) and mechanical shredding to increase surface area. Stage 3: Dark Fermentation consists of the combination of the inoculum and prepared substrate to initiate DF. At this stage the HPBs in the inoculum convert glucose and other sugars to H2 and VFAs. Stage 4: Downstream Processing consists of VFAs being used as precursors in photo-fermentation, microbial electrolysis cells, and other systems to produce more H2 among other useful byproducts.
Figure 3. Chemical reactions that DF utilizes to produce H2. R1 depicts the conversion of glucose to acetic acid. R2 depicts the conversion of glucose to butyric acid. A balance between these two pathways is approximated to determine theoretical H2 yield in DF bioreactor systems.
Virginia iGEM has concluded that adding L-cysteine will increase H2 production through DF by enhancing the proliferation of HPBs and increase their hydrogenase activity. L-cysteine is a reducing agent that lowers oxidation-reduction potential (ORP) of DF feedstocks, and its addition to feedstocks has shown to increase H2 yields by 60% [15]. Therefore, we assert that an additional pretreatment step of adding L-cysteine to DF feedstocks be implemented into a modified DF process flow.
When L-cysteine concentrations in the feedstocks exceed 300 mg/L HPB biomass growth is inhibited thereby reducing total H2 production [16]. To ensure that excess L-cysteine supplementation does not inhibit HPB biomass growth the Cysteinator contains a novel L-cysteine activated kill-switch. When L-cysteine concentrations in the feedstock reach 300 mg/L the kill-switch arrests cell growth leading to cell death. Kill-switch activated cell death also serves as biocontainment of the Cysteinator and ensures that it does not propagate outside of the DF bioreactor.
As both feedstocks vary wildly, the best way to seamlessly integrate L-cysteine supplementation into the DF process flow is through a modular biological device such as the Cysteinator (see Figure 4). The Cysteinator is an E. coli bacterium that contains a modular L-cysteine overexpression circuit, and a novel modular L-cysteine activated kill-switch circuit. A modular device has the benefit of being optimized for use in a variety of feedstock environments. For this reason, the Cysteinator is accompanied by a multi-scale model capable of optimizing device behavior in varied feedstock environments.
Figure 4. The Cysteinator will be integrated into Stage 1 of the DF process flow to reduce feedstock ORPs allowing for HPBs to propagate, producing an inoculum
Figure 5. Figure 5 is a visualization of what the Cysteinator does most simply, converts organic wastes into L-cysteine.
In order to make sustainable H2 a commercially viable energy alternative, we built the Cysteinator. The Cysteinator is a modular device adaptable to diverse feedstocks while secreting L-cysteine to precisely 300 mg/L. .
Project Goals
To ascertain the full potential of the Cysteinator we divided our project into three goals.
- Goal 1: Validate Device Functionality
- Goal 2: Understand Device Implementation
- Goal 3: Evaluate Device Utility
Goal 1: Validate Device Functionality
The Cysteinator is a two-plasmid device. The first plasmid confers overexpression & export of L-cysteine. When extracellular L-cysteine concentrations reach 300 mg/L, the second plasmid—a novel L-cysteine activated kill switch—uses an L-cysteine induced transcription factor to trigger transcription of a toxin that arrests chassis growth and results in cell death.
Testing the Cysteinator is occurring in three steps: (1) validate that the overexpression plasmid results in extracellular L-cysteine accumulation, (2) validate that the L-cysteine kill-switch plasmid is effective and is regulated by L-cysteine accumulation, (3) validate that the plasmids work together. To predict device behavior and inform our testing scheme we built a comprehensive multi-scale model amenable to our device's modularity.
Look at our Engineering Page for more information about the plasmids.
Look at our Experiments Page for more information about the testing & assembly.
Look at our Modeling Page for more information about predicting device behavior.
Look at our Results Page for more information about our progress.
Goal 2: Understand Device Implementation
Implementing a biological device into a complex bioreactor process flow involves: (1) understanding the current technology, (2) understanding how device will be integrated into the current technology, and (3) understanding what future testing needs to be done and how to streamline it.
To understand the current technology, we conducted a comprehensive literature review of DF systems and engaged with relevant stakeholders. Building on this work, we also engaged with stakeholders to understand how the Cysteinator could be integrated into bioreactors. This activity was further supplemented with continuing to design, build, and test our model, predicting device behavior in commercial environments. Our work concluded with evaluating the total body of our work, consulting with experts, and planning the next steps in implementing the Cysteinator.
Look at our Sustainability Page for more information about current technology & plans for device integration.
Look at our Modeling Page for more information about predicting device behavior.
Look at our Contribution Page for more information about what we’ve done & our next steps.
Goal 3: Evaluate Device Utility
Our device was built with the goal to enact positive change in our local, national, and global communities by furthering sustainable technologies. To understand the scope of the impact our device has on this goal we reviewed how our device aligns with the UN’s Sustainable Development Goals. Engaging with stakeholders emphasized the importance of understanding market viability of our device and for this reason we also conducted a Life-Cycle Analysis and Techno-Economic Analysis of our devices and its resultant modified DF process flow.
Look at our Sustainability Pages for more information about how our device impacts our selected SDGs.
In summary, Virginia iGEM delivers the Cysteinator, a multi-scale model, and multiple comprehensive analyses that demonstrate how DF becomes market viable and advances sustainable energy development.