Project Description

Living on Mars?

Mars has long fascinated humanity and is a key focus for future space exploration due to its similarities to Earth and its potential to support life, past or present [1]. At the same time, the vision of living on Mars reflects humanity’s broader ambitions — expanding into space, ensuring long-term survival, and inspiring innovation [2]. With growing interest in Mars exploration, the utilization of local resources becomes increasingly important. Among these, access to reliable and sustainable water supplies is crucial for future missions [3]. Mars itself has large amounts of water with more than 21 million km³ of ice on or close to the surface [4]. However, theses natural reserves are contaminated with highly toxic perchlorate salts, rendering them unusable [5].

The World Health Organization (WHO) sets the guideline value for perchlorate in drinking water at 70 µg/L [6]. Yet, perchlorate concentrations in Martian water are suspected to exceed the safe limit by several orders of magnitude [5]. Transporting water from Earth, on the other hand, is not a viable option due to its extremely high costs [7]. While existing recycling systems such as the Water Recovery System provide partial solutions, they cannot fully cover the water demand, making additional sources indispensable [8]. Developing methods to remove perchlorate and generate drinkable water is therefore critical for the success of future crewed Mars missions.

As NASA states, vapor distillation, ion-exchange resins, and reverse osmosis can remove perchlorates, but they demand high power, frequent resupply of bulky consumables, and generate concentrated waste streams [9]. In contrast, synthetic biology offers a self-regenerating, low-mass solution that directly breaks down perchlorates into harmless chloride and useful oxygen, avoiding waste buildup and heavy resource demands. Inspired by a NASA project [9], we are engineering the bacterium Bacillus subtilis to produce enzymes which efficiently degrade perchlorate. This space-tested, well-characterized bacterium grows rapidly and forms spores that survive for years, even in environmental extremes like space [9, 10]. Such durable spores can easily be transported to Mars, making B. subtilis an excellent candidate for robust perchlorate removal on the Red Planet.

Our envisioned water cleaning setup on Mars begins with harvesting ice from the surface or shallow subsurface and transporting it to a base where our bioreactor is located. The ice is melted using solar energy, and—depending on perchlorate concentrations—diluted with previously purified water to avoid levels that are too high for treatment. Inside the bioreactor, spores of our engineered Bacillus subtilis germinate and degrade the perchlorates. We envision, the bacteria using cellulose derived from plant photosynthesis as their carbon source and electron donor [11]. After the degradation process, the bacteria are removed by sterile filtration or kept as probiotic for astronauts [9]. If necessary, excess chloride is balanced by further dilution with recycled water and the cleaned water can then be used for drinking, hygiene, and agriculture, while wastewater is collected, treated with established systems [8], and fed back into the cycle to ensure sustainability.

Synthetic Biology for Clean Martian Water

Certain bacteria, such as some members of the genus Azospira, naturally contain pathways for perchlorate degradation (Figure 1). They use perchlorates as terminal electron acceptors, fully reducing them to harmless chloride, water, and oxygen. The enzyme perchlorate reductase reduces perchlorate (ClO₄^-) to chlorate (ClO₃^-) and chlorite (ClO₂^-) in two steps while oxidizing an electron donor. Subsequently, chlorite is converted into chloride (Cl^-) and oxygen (O₂) by chlorite dismutase, the second enzyme of the pathway. The produced oxygen is further used as an additional electron acceptor by the facultative anaerobic organisms [12].

We introduce the genes encoding perchlorate reductase (pcrABCD) and chlorite dismutase (cld) into Bacillus subtilis 168. In two steps of Gibson Assembly, we aim to construct a plasmid carrying both genes in separate transcriptional units (Figure 2). By using the shuttle vector pMK4, cloning can be done in Escherichia coli before transforming B. subtilis with the fully assembled plasmid (see Cloning).

To understand the underlying biochemistry of the perchlorate degradation pathway, we characterize both perchlorate reductase and chlorite dismutase in vitro. Perchlorate reductase (BBa_254G21JZ) (Figure 3) is of special interest, as it catalyzes the rate limiting step of perchlorate degradation [13]. It requires correctly assembled molybdopterin cofactor and [Fe-S]-clusters for activity, rendering its heterologous expression challenging. Its oxygen-sensitivity further complicates purification [14]. We show here – to our knowledge – first heterologous expression of active perchlorate reductase in E. coli BL21. We establish an activity assay [15] and investigate key mutations that shall diminish substrate-inhibition [14] (see in vitro characterization of perhlorate reductase).
Chlorite dismutase (BBa_25VZ3X3W) (Figure 4), the second enzyme, is also crucial, as it detoxifies the transiently produced chlorite rapidly [16] which would otherwise harm our chassis [17]. We report its first purification by an iGEM team and investigate an alternative activity assay [18, 19] (see Chlorite dismutase activity assay). Additionally, we design variants exhibiting specific mutations to change oligomeric structure and analyze them in the lab (see Chlorite dismutase monomerization).

For monitoring perchlorate concentrations, we apply a solvent extraction method, utilizing methylene blue which complexes specifically with perchlorate (Figure 5) [21]. This complex can then be extracted using dichloroethane and perchlorate concentrations can be derived from the extract’s UV/Vis-spectrum. We optimized the assay to achieve sensitive and reproducible detection of perchlorate in the 0-5 ppm range, enabling its application both in vivo and in water samples. This approach provides a rapid and cost-effective method for perchlorate monitoring (see Detection method).

To evaluate the performance of our engineered B. subtilis in a more realistic environment, we design a DIY bioreactor (Figure 6) in which the bacteria grow anaerobically. Built from readily available materials, the modular, low-cost setup of the bioreactor enables replication and adaptation for different applications (see bioreactor).

Since gravity on Mars is considerably lower than on Earth, and microgravity strongly affects cellular growth, gene expression and biofilm formation [22, 23], we build a random positioning machine (RPM) that can simulate microgravity (Figure 7). An RPM works by continuously reorienting samples in two perpendicular axes, neutralizing gravity’s directional bias when averaged over time [24, 25]. We create an affordable, open-source, and validated RPM with clear documentation, turning theoretical designs into practical, accessible hardware for research, schools and other iGEM teams (see Random Positioning Machine).

In summary, our project combines synthetic biology, innovative engineering, and accessible design to address one of the key challenges of human exploration on Mars: access to clean water. By engineering Bacillus subtilis for perchlorate degradation, developing reliable assays, and creating open-source hardware such as a DIY bioreactor and an RPM, we provide tools that are both practical and scalable for future space missions.

Perchlorates on Earth

At the same time, perchlorate contamination is not only a Martian problem but also a concern on Earth, where natural and human activities have led to detectable levels in water and food supplies [26]. In places like China’s Yangtze River, concentrations exceeding safe limits have been found near fireworks manufacturing facilities [27], and in Europe, fertilizer regulations had to be tightened to reduce perchlorate uptake by crops [28]. By tackling perchlorate degradation for Mars, our project also contributes to addressing a pressing environmental challenge on Earth, showing how space-driven innovation can provide sustainable solutions at home.