Human Practices | TUDarmstadt

Introduction

Water is essential for sustaining life. However, on Mars, water is limited and not safe to drink. In this era of space exploration, we recognize that access to safe water is a major challenge that we cannot ignore.

Mars, one of the next destinations for human exploration by NASA [1, 2], has water in the form of ice layers [3]. Unfortunately, this water is contaminated with perchlorates, which are highly toxic compounds that make it unsafe for consumption [4, 5]. The focus of our project is to tackle this problem by engineering Bacillus subtilis 168 to break down perchlorates. This approach will give astronauts safer access to Martian water supplies and reduce reliance on expensive transportation from Earth.

At the same time, our project has significant applications here on Earth. Perchlorate contamination is not just found in water sources; it is often detected in soil and crops, which poses risks to human health and the environment [6]. By modifying our system for use on land, our project could help create safer water and food supplies around the world.

While our project offers exciting opportunities for sustainable water purification on Mars, it is equally important to consider potential risks and their potential effects. On Mars, the failure of our system could lead to exposition of astronauts to perchlorate contamination. Thus, technical challenges, reliability, and quality checks must be strictly controlled to operate the biological system in space.

Biosafety is another important concern. Accidental release of genetically modified bacteria on Mars could possibly interfere with future astrobiology research , while increased resistance to the disinfectant chlorite could complicate sterilization. This highlights the importance of strictly closed systems and containment strategies.

Even though it remains a challenge to directly contact any astronaut, the group of persons who would later use our system, we gained various advice and opinions from different experts that helped us shape our project into a more concise and in-depth version, while also acknowledging these risks and the responsibility our project is holding.

Wetlab

Ph.D. Matthew Youngblut

Ph.D. Matthew Youngblut is a specialized scientist in biochemistry, molecular biology, and microbiology. He worked as postdoctoral researcher at the University of California, Berkeley on designing and carrying out expression and purification of multiple enzymes, such as the perchlorate reductase (PcrAB) [7]. In the early stages of our project, we reached out to gain a deeper understanding of PcrAB and insights into our project proposal.

Practical expertise: We stayed in contact since the first meeting. Ph.D. Youngblut provided us with practical guidance on handling PcrAB.

Perchlorate reductase from Azospira oryzae is recommended due to its good system for characterization.
Core perchlorate reductase genes require chaperones for proper folding and maturation.
Methyl viologen is highly oxygen-sensitive, it should be reduced with zinc powder prior to use for enzymatic assays.
Phenazine methosulfate offers a suitable alternative electron mediator, being less oxygen-sensitive than methyl viologen.
Perchlorate reductase shows substrate inhibition at high substrate concentrations. Therefore, we might need to rely on nitrate reductase at high perchlorate concentraions.

Assoc. Prof. Stefan Hofbauer

Stefan Hofbauer is an Assoc. Prof. at BOKU University, Vienna, whose research focusses on procaryotic heme biosynthesis. As he worked intensively with chlorite dismutase during his Ph.D [8, 9, 10]. , we were very happy he was ready to talk to us.

He evaluated our project idea and encouraged us to use chlorite dismutase from Azospira oryzae as it is fast and well-studied [11, 12]. He also approved our plan to use Gram-positive Bacillus subtilis as chassis, as it is able to synthesize heme b and thus assemble active chlorite dismutase [13]. In addition, he provided us with many practical insights regarding protein purification, enzyme characterization and safe handling of chlorite.

Use of chlorite dismutase from A. oryzae as this chlorite dismutase variant is highly active and well studied.
B. subtilis is able to produce active holo-chlorite dismutase.
Purification using a non-cleavable his-tag, as this method is well established and the his-tag does not interfere with enzymatic activity.
UV spectroscopic characterization is possible, though Clark-oxygen-sensing-electrode is more reliable. As we cannot access a Clark-electrode, we were happy to hear about UV-spectroscopic assays. Hofbauer advised us to stir the cuvette and to bear in mind that measuring low chlorite concentrations is not precise.
Use low enzyme concentrations to be able to monitor the very fast reaction rather than lowering temperature, as this might increase the enzyme’s activity.
Check UV-absorbance of chlorite stock solution before use, as chlorite could spontaneously decompose.

Prof. Dr. Viktor Stein

A valuable contribution to our chlorite dismutase protein engineering project came from Prof. Dr. Viktor Stein, an expert in the field at our university, TU Darmstadt. Initially, our concept was to design a new enzyme using the method of constrained hallucination, which, in theory, would allow us to preserve enzymatic activity while partially altering the protein structure toward a predicted, and potentially more stable, configuration.

After consulting Professor Stein, we received critical insights that helped reshape our approach:

Feasibility concerns: He pointed out that developing such a pipeline, and successfully transferring it to the lab, would be challenging within the available timeframe. He also emphasized that our project lacked a clear and useful goal.
Recommendations: He encouraged us to continue working on the protein but advised keeping the structure close to the wild-type and focusing on specific, well-informed mutations.
Promising direction: He suggested that the most promising aspect of our work might lie in the possibility of producing a functional monomeric enzyme.
Additional suggestions: He proposed an alternative strategy: fusing our enzyme with perchlorate reductase (PcrAB) to enhance perchlorate degradation effectiveness.

Thanks to his feedback, we were able to refine our plans and set more realistic and achievable goals.

Ph.D. Garrett Roberts Kingman

Dr. Roberts Kingman is a postdoctoral researcher in astrobiology at NASA Ames Research Center. He specialized on evolutionary genetics, synthetic biology, and microbial adaptation in extreme environments. His current work focuses on engineering E. coli and B. subtilis to reduce perchlorate [14, 15] .

We reached out to Dr. Roberts Kingman to learn more about implementing perchlorate reduction in a Gram-positive chassis and to identify a suitable assay for detecting perchlorate degradation. Surprisingly, the main limitation is not the difference between Gram-positive and Gram-negative hosts. B. subtilis works quite well, the problem lies in optimization of the molybdenum cofactor biosynthesis, which is essential for enzymatic function. This is an important optimization we need to tackle after establishing B. subtilis as perchlorate reducer in our lab.

Assay recommendation: Use a methylene blue assay under alkaline conditions. Centrifugation and vigorous vortexing are key steps to properly form the methylene blue–perchlorate complex.
Material compatibility: The organic phase with dichloroethane (DCE) is incompatible with polystyrene but compatible with polyethylene, so we switched to using polyethylene labware to improve measurement reliability. Alternatively, quartz cuvettes may also be employed.
Chassis insight: B. subtilis produces a nitrate reductase, which belongs to the DMSO reductase family of molybdopterin-dependent enzymes, just like perchlorate reductase. This suggests it produces the necessary molybdenum cofactors, making it a suitable starting chassis.
Space readiness: Media composition and avoiding chloride buildup are important challenges we should address.

Bioreactor

Rik Volger

Rik Volger is a PhD candidate in Bioprocess Engineering at Delft University of Technology (TU Delft). His research focusses on hydrodynamics as well as the use of microorganisms in bioreactors for biomining in space [16]. We discussed our bioreactor project with Rik and showed him our initial plans. He gave us valuable advice on the optimal design for reliable operation and recommended a bioreactor type suitable for our lab. Apart from that he connected us with Zachary Hale and his colleague Leon Williams.

First plan of the Bioreactor: Rik approved our overall concept, but we were advised that the use of solid media to grow bacteria on could lead to problems by the formation of biofilms.
Batch-system: He suggested a batch-system bioreactor as it is easier to work with in the lab on a small scale.
Anaerobicity: Rik pointed out that achieving anoxic conditions might not be as difficult as expected, since in most bioreactors the bigger challenge is ensuring an even distribution of oxygen.
Stirred tank bioreactor: Since we are working under anaerobic conditions, Rik suggested using a stirring device to ensure an even distribution of nutrients.
Pure culture instead of mixed culture: Because our approach aims to use these bioreactors in space, Rik recommended using a pure culture of one organism rather than a co-culture, as mixed cultures can cause unwanted side effects and byproducts.

Prof. Dr. Miriam Agler-Rosenbaum

Prof. Dr. Miriam Agler-Rosenbaum is head of department of the Bio Pilot Plant at the Leibniz HKI. Her main research areas are bioelectrochemical systems, defined microbial mixed cultures and droplet microfluidics for microbial applications. Prof. Dr. Christian Hertweck encouraged us to contact her.

Our discussion about designing and building a bioreactor changed the way we viewed the project. She pointed out that we were approaching it mainly from a practical and mechanical perspective. Instead, we should also consider the biosynthetic pathway of the reaction and the metabolism of our chassis when designing the bioreactor.

Importance of metabolism: Prof. Dr. Miriam Agler-Rosenbaum emphasized that when designing a bioreactor, it is important to consider not only the environmental conditions of the microorganism but also its metabolism, whether native or altered through genetic engineering. For example, using genetically modified B. subtilis to degrade perchlorate does not alter the growing conditions of the organism but the efficiency of the reaction can be dependent on the substrates provided in the media or disrupted by by-products.
Simple bioreactor schemes: She explained that when the metabolism is taken into consideration, bioreactors can be as simple as bags, shaken on a rotation plate, particularly in the case of batch systems.
Disposable solutions: Prof. Dr. Miriam Agler-Rosenbaum noted that disposable solutions, such as plastic bags, may be less suitable for space-related projects, since resources in space scenarios are often very limited.
Filtration type: Prof. Dr. Miriam Agler-Rosenbaum pointed out that our originally planned dead-end filtration would likely not be the best solution in the later stages of the project or when considering practical applications. She suggested considering alternative filtration methods, such as crossflow filtration.

Zachary Hale and Leon Williams

Zachary Hale (right) and Leon Williams (left) are members of the Advanced Concepts Team at the European Space Agency (ESA). The team serves as ESA’s internal think tank, exploring bold and long-term ideas in science and technology to support future space missions.

We were introduced to them by Rik Volger. Zachary and Leon focus on computational modelling of specific reactions, such as perchlorate degradation.

Kinetic data of the perchlorate degradation reaction: Leon Williams and Zacchary Hale noted that the data we collect in kinetic studies could be of interest, particularly in relation to the perchlorate degradation reaction.
Different approaches: We learned from Zacchary and Leon that very similar projects can be approached in vastly different ways. Their perspective through computational modelling and our more practical approach could strongly complement each other in the future.

Conclusion

We sincerely thank all the experts who shared their time and knowledge, helping us shape our project into what it is today. Ph.D. Matthew Youngblut's insights inspired and really helped us to work on perchlorate reductase expression and characterization as well as trying out site directed mutations to reduce substrate inhibition. Assoc. Prof. Stefan Hofbauer confirmed chlorite dismutase should be expressible in B. subtilis and pointed us towards an alternative activity assay, we could use. Ph.D. Garrett Roberts Kingman informed us about a low-cost perchlorate detection method and the potential bottleneck of molybdopterin cofactor synthesis. After consulting Prof. Viktor Stein, we realized we would need to completely change our protein engineering plans and did try out a more feasible approach in the end. In addition, the many expert contacts helped to develop our bioreactor. E.g. we moved away from our initial solid-media idea and were conscious about the importance of media composition to meet the chassis' metabolism in the future.

The advice and support we received from these experts was crucial in shaping our approach. Their perspectives helped us recognize challenges we had not thought about, refine our ideas into more practical solutions, and ensure that our design could address both scientific and real-world needs. By continuously updating our work based on their advice, our project grew from an initial concept into a more solid and feasible design that we are happy to present.

References

[1] Stephen Carney, Mars Exploration. [Online]. Available: https://science.nasa.gov/planetary-science/programs/mars-exploration/

[2] Jessica Taveau, NASA Says Mars Rover Discovered Potential Biosignature Last Year. [Online]. Available: https://www.nasa.gov/news-release/nasa-says-mars-rover-discovered-potential-biosignature-last-year/

[3] Arizona State University, Mars education, water. [Online]. Available: https://marsed.asu.edu/mep/water

[4] Piotr Rzymski, "Perchlorates on Mars: Occurrence and implications for putative life on the Red Planet," 2024. [Online]. Available: https://pmc.ncbi.nlm.nih.gov/articles/PMC4834222/

[5] Leonard David, Toxic Mars: Astronauts Must Deal with Perchlorate on the Red Planet. [Online]. Available: https://www.space.com/21554-mars-toxic-perchlorate-chemicals.html

[6] Craig M Steinmaus, "Perchlorate in Water Supplies: Sources, Exposures, and Health Effects," 2017. [Online]. Available: https://pmc.ncbi.nlm.nih.gov/articles/PMC4834222/

[7] Matthew D. Youngblut, "Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues," 2016. [Online]. Available: https://doi.org/10.1074/jbc.M116.714618

[8] S. Hofbauer et al., "Transiently produced hypochlorite is responsible for the irreversible inhibition of chlorite dismutase," (in eng), Biochemistry, vol. 53, no. 19, pp. 3145–3157, 2014, doi: 10.1021/bi500401k.

[9] S. Hofbauer et al., "Impact of subunit and oligomeric structure on the thermal and conformational stability of chlorite dismutases," (in eng), Biochimica et biophysica acta, vol. 1824, no. 9, pp. 1031–1038, 2012, doi: 10.1016/j.bbapap.2012.05.012.

[10] S. Hofbauer, I. Schaffner, P. G. Furtmüller, and C. Obinger, "Chlorite dismutases - a heme enzyme family for use in bioremediation and generation of molecular oxygen," (in eng), Biotechnology journal, vol. 9, no. 4, pp. 461–473, 2014, doi: 10.1002/biot.201300210.

[11] C. G. van Ginkel, G. B. Rikken, A. G. Kroon, and S. W. Kengen, "Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme," (in eng), Archives of microbiology, vol. 166, no. 5, pp. 321–326, 1996, doi: 10.1007/s002030050390.

[12] I. Schaffner, S. Hofbauer, M. Krutzler, K. F. Pirker, P. G. Furtmüller, and C. Obinger, "Mechanism of chlorite degradation to chloride and dioxygen by the enzyme chlorite dismutase," (in eng), Archives of biochemistry and biophysics, vol. 574, pp. 18–26, 2015, doi: 10.1016/j.abb.2015.02.031.

[13] N. Falb, G. Patil, P. G. Furtmüller, T. Gabler, and S. Hofbauer, "Structural aspects of enzymes involved in prokaryotic Gram-positive heme biosynthesis," (in eng), Computational and structural biotechnology journal, vol. 21, pp. 3933–3945, 2023, doi: 10.1016/j.csbj.2023.07.024.

[14] L. J. Rothschild, G. A. Roberts Kingman, C. R. Stoker, and S.J. Hoffman, DETOXIFYING MARS: THE BIOCATALYTIC ELIMINATION OF OMNIPRESENT PERCHLORATES.

[15] Garrett A Roberts Kingman 1, Justin L Kipness 2, Lynn J Rothschild, "Raiding nature's genetic toolbox for UV-C resistance by functional metagenomics," 2025. [Online]. Available: https://pubmed.ncbi.nlm.nih.gov/39747236/

[16] R. Volger, G. M. Pettersson, S. Brouns, L. J. Rothschild, A. Cowley, and B. Lehner, "Mining moon & mars with microbes: Biological approaches to extract iron from Lunar and Martian regolith," Planetary and Space Science, vol. 184, p. 104850, 2020, doi: 10.1016/j.pss.2020.104850.