Team:DTU-Denmark/Design
Design
Engineering fungal mycelium to create viable building materials for Mars is not a small challenge. This project, which was inspired by conversations with Lynn J. Rothschild from NASA, and the Stanford-Brown-RISD iGEM team, whom we owe thanks both for the inspiration and subsequent sparring on ideas, has relied on several scientific fields to try to tackle the problems we have found along the way. Being inspired by the idea of simple and cost-effective construction on Mars, our design focuses on the fungal aspect of how a cyanobacteria/fungal production system can be used to create building materials. As we explored this idea, we were presented with many problems to be solved and even more questions to be answered.
The first and most pressing one, how does one cultivate fungus on Mars? Moreover, which fungal species should we use? Can we determine and model growth characteristics of our fungi? Is the strength of fungal materials determined by any distinct genes and is it possible to regulate these in a way that will make our materials even tougher? And not least, how do we make the system cost-effective? We set out to answer these questions to guide the creation of our final design.
Growing Fungus on Mars
Growing live fungus on Mars will be challenging as the environment on Mars is extremely hostile compared to Earth. In addition to the extreme cold, the atmosphere is very thin at less than 1% of the density of Earth’s atmosphere and consists of 96% carbon dioxide and virtually no oxygen (1). Moreover, our fungus needs organic compounds as a source of carbon and energy. Bringing substrates from Earth is out of the question due to the costs of transportation, thus in situ production of materials is vital. To solve the problem of available carbon sources, one can introduce cyanobacteria to the production system. Cyanobacteria can utilize the carbon dioxide in the Martian atmosphere, albeit, like the fungus, they, too, are unable to grow directly in the extreme conditions of Mars. To solve the problem of the Martian hostility it is necessary to have a production facility. Such a production facility for cyanobacteria has been theorized by researchers at NASA (2). Using covered thermally insulated ponds with compressed air and water from the Martian ice, it would be possible to grow cyanobacteria on Mars. Supporting this idea is the fact that all of the essential elements need for cyanobacterial and even plant growth can be found on Mars (2). Based on this system theorized by NASA, we suggest appending it with the capabilities of using the cyanobacterial biomass to produce fungal materials.
However, non-destructive ways of harvesting nutrients could possibly lead to a more effective process. So instead of using lysed-dead cyanobacteria there is also the possibility of using biomass produced by them as a secreted substrate. Cyanobacteria can provide heterotrophic organisms with both nitrogen and oxygen through their photosynthetic and nitrogen-fixing pathways. Specifically, they can convert carbon dioxide, the most abundant gas in Mars’ atmosphere, into oxygen and at the same time provide NH4+ to the feeding organisms by utilizing N fixation (2). These problems are beyond the scope of our project, but provides an interesting starting point for future projects.
Choice of Organism
The choice of organism used in our project was based on an inductive strategy. We had two variables needed to be fulfilled in our attempt to achieve the goals we had set. The first was to find a fungus species that, given the right substrate, would have the ability to form firm brick structures. The second was to use a safe microorganism with a sequenced genome and established genetic methods, thus enabling us to do genetic modifications. The project started with three candidate-organisms: Pleurotus ostreatus, Schizophyllum commune and Aspergillus oryzae. These organisms have previously been grown on a variety of substrates and some have been used for creating bricks. We were also recommended to use a Fusarium by researchers at Novozymes, but opted to focus on the ones we had multiple recommendations on. For various reasons, our choice ended up being A. oryzae; A. oryzae is comparatively easy to grow on various substrates, it grows fast, gives consistently good results and is a well-established model organism. Though the other fungal species have previously shown to have desirable characteristics, when it comes to creating fungal materials, the results of our initial experiments made us decide on doing our research in A. oryzae with the possibility of transferring the discoveries to other fungal species at a later point in the project.
How to Build Structures on Mars
In determining material properties of our fungal materials, experiments are critical. Depending on how many parameters you want to examine, the number of experiments tend to exponentially increase. Using the Fedorov exchange algorithms, we were able to reduce the number of experiments in the first tests from 216 to 64 and in the second run from 648 to 68, which was a huge time saver. It became clear to us that we needed to produce a lot of fungal sample cubes. Initially, we fabricated some acrylic boxes. These were fast and easy to make. It turns out that acryl does not withstand the pressures and temperature when the form gets autoclaved. We then turned to metal. These metal boxes took a long time to make, and after having fabricated 11, it was decided that the effort would not be worth it. After having done some research, silicone ice cube trays turned out to be the perfect alternative to our metal boxes. Silicone can easily withstand autoclaving and allowed for very uniformly shaped samples. They can also be purchased prefabricated cheaply. The sample cubes were all prepared in slightly different ways according to our experiment optimization such as baking times, compound composition, substrates etc. All the fungal samples were subjected to a destructive compressional test, where the stress/strain-curve was determined for compressional strength. In order to obtain the Young’s modulus of the fungus, the tensile strength would also have to be measured. If Young’s modulus is known, the entire materials’ properties and characteristics can be inferred.
Improving the fungal materials through synthetic biology
When using fungal mycelium and organic materials as the basis for building materials, a new venue of possibilities suddenly appear. Fungi are genetically modifiable. This allows for tuning of different external parameters or even adding properties such as biosensors etc. Thus, the interesting question to answer in this aspect is; what genetic modifications are beneficial when it comes to construction on Mars? Like any other organism, any fungus is optimized for the survival of its species in its niche. No fungal strain is solely optimized for creating strong materials, as that wouldn’t be a advantageous evolutionary strategy. By making the fungus a little worse at surviving, we aim to make it better for building. In our project, we ended up focusing primarily on two genes; melA and gfaA.
Protection from UV radiation and Melanin
When using fungal mycelium and organic materials as the basis for your building materials, a new venue of possibilities suddenly appear. The fungi is genetically modifiable, which allows for tuning of different external parameters or even adding properties such as biosensors etc.
The interesting question to answer is this respect is: What genetic modifications are beneficial when it comes to construction on Mars?
Like any other organism, any fungi is optimized for the survival of its species in its niche. No fungal strain is solely optimized for creating strong materials, as that wouldn’t be a advantageous evolutionary strategy. By making the fungi a little worse at surviving we aim to make it better at building
Understanding morphology and the environment under which it lives is pivotal for identifying potential gene candidates
(Briefly on fungal morphology, The strength of fungal cell walls is provided by its a)
In our project we ended up focussing especially two genes, melA and gfaA.
Protection from UV radiation and Melanin
One part of what makes Mars as harsh environment is radiation. Unlike the terrestrial atmosphere, the martian atmosphere does not shield well against cosmic radiation. Among such kind of radiation in UV light (higher amount of damaging UVC and UVB radiation on surface. (https://mars.jpl.nasa.gov/mgs/sci/fifthconf99/6128.pdf))
Melanotic bacteria increased protection for the full spectrum of UV (We want to transfer this. (https://www.sciencedirect.com/science/article/pii/S0165022X0700190X?via%3Dihub)
“melA encodes for the first step in the melanin biosynthetic pathway. it encodes a tyrosinase that catalyzes the reaction dopaquinine”.
Using the tokyo tech melA gene (https://2009.igem.org/Team:Tokyo_Tech/BlackenedEcoli#Protection_from_UV_ray) (refitted to fungi).
Melanin is a pigment and a well-known UV-protectant. It is a senseful choice both in the sense it fits our theme and furthermore it gives us a selectable genotype. By UV-irradiating the transformants vs. non-transformants it will be possible to show the effect of melanin on the growth rate/or ability to grow.
Melanin thermally stable (https://pdfs.semanticscholar.org/42b5/d1c95260addbfdf54a8632a771f16f43c40f.pdf)
Melanin (Codon optimized for A. oryzae)
(Codons is a key concept when it comes to working with fungi, and a wisely codon optimized gene for a specific species of fungi and see yield increases on heterologous genes by factors - e.g: http://www.pnas.org/content/113/41/E6117 ). Expressing the codon optimized gene alongside the original gene will show the relative expression level and thus the effect of codon optimization.
Cell wall structure and GfaA
GfaA was chosen having structural strength of the materials in mind. The structural strength fungal cells walls come from β-Glycans and Chitin, (Chitin being the most important of the two). Gfa encodes for an enzyme ??? that catalyzes the rate limiting step in the production of chitin, and overexpression. (mutants overexpressing Gfa in yeast have been shown to have I higher wall chitin content) and we theorized that overexpression of this enzyme in aspergillus oryzae might lead to a higher-than-normal Chitin content of the fungal cell walls and henceforth hopefully stronger materials
rate limiting - transferase , etc...
(http://mic.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.27249-0#tab2).
Using Hph or NAT1 as a selection marker.
Colors and selectable markers - AmilCP
AmilCP is a blue Chromoprotein. We wanted to use the expression of a color protein as a “Hello World” gene. AmilCP is a blue color protein? (pigment?), and by such can be used as an easily distinguishable marker.
Mission Architecture
So how do we propose going about this entire endeavor? When a finalized version of our GMO fungi has been produced, the idea for astronauts to bring a container with our fungi spores and a container with cyanobacterium. These are brought to Mars. On their way, the astronauts will cultivate the cyanobacteria in a growing vessel, which is shaped like a long triangle. As you can see on the cross-section drawing, the vessel is a lightweight, collapsible container that can fitted with a gas pump. All the sides are zippable and on the inside, there are sown in spacers that makes the inside volume of the vessel, carve out our brick. The cyanobacteria will fill out the volume of the growing vessel while being fed CO2 through a pump, into the vessel. When the growing vessel is full with the bacterium, the astronauts will plant the GMO fungi spores onto the bacteria. After a short while the fungi will have “eaten” the cyanobacteria and taken its place, filling out the entire volume of the vessel. The astronauts can then open and start building their habitat from fully formed fungi bricks.
(1) Williams, M. What is the Weather like on Mars? (accessed Oct 15, 2018).
(2) Verseux, C.; Baqué, M.; Lehto, K.; Vera, J.-P. P. D.; Rothschild, L. J.; Billi, D. Sustainable life support on Mars – the potential roles of cyanobacteria. International Journal of Astrobiology 2015, 15 (01), 65–92. (Accessed 16/10-18)
(3)Michael E., John K. Expediting Factors in Developing a Successful Space Colony. [online] 2011, Worcester Polytechnic Institute. p. 22.
(4) Oxygen and Human Requirements.
http://www.geography.hunter.cuny.edu/tbw/wc.notes/1.atmosphere/oxygen_and_human_requirements.htm. (Accessed Oct 12, 2018).
(5) Mars Fact Sheet. https://nssdc.gsfc.nasa.gov/planetary/factsheet/marsfact.html. (Accessed Oct 14, 2018).
(6) What Are the Limits of Human Survival? https://www.livescience.com/34128-limits-human-survival.html. (Accessed Oct 12, 2018).
(6.5) proven here: http://academic.uprm.edu/pcaceres/Courses/MMII/IMoM-6A.pdf
(7) Zip Tie Domes. Geodesic Dome Kits that are Easy to Build!
https://www.ziptiedomes.com/faq/What-Is-Geodesic-Dome-Frequency-Explained.htm. (Accessed Oct 9, 2018).
(8) Andre, M.; Massimino, D. Advances in Space Research 1992, 12 (5), 97–106.
https://ac.els-cdn.com/027311779290015P/1-s2.0-027311779290015P-main.pdf?_tid=368dbac9-af6e-4343-abcf-9d97c861638c&acdnat=1539794447_304c6b3ffbe763c679c91c5960db27ee