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 to 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 a fungus on Mars? Moreover, which fungal species should we use? Can we determine and model the 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 needed 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.
Fig 1: - Creating bricks in unconventional molds.
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
When wanting to erect livable habitats on Mars made of fungal materials, an obvious question is: What physical parameters will the fungal bricks have to withstand for them to be used as building material on Mars? Martian gravity is about 38% of Earth’s (3) makes it easier to erect self-sustainable structures on Mars than on Earth. However, the greater challenge is enabling structures to sustain the internal pressure generated by the pressure difference between the liveable habitat and that of the Martian atmosphere. The average pressure on Earth is 1.013 bar with 21% oxygen. The lowest human life-sustaining pressure is 0.121 bar, assuming a 100% oxygen concentration (4). Though, this is not desirable due to extreme fire hazards. At these oxygen concentrations, everything becomes combustible. Near the lower border of what is safe/healthy and sustainable for human life is an atmosphere containing 19.5% oxygen at approximately 0.65 bar (5). As stated earlier, the average Martian pressure is 0.006 bar. This is only 0.6% of the average barometric pressure on earth (6).
What building design/geometry is the best at containing pressures? For simulation purposes, the internal pressure is 1 atmosphere. Humans can live at lower pressures, but lab-equipment and plant growth are sensitive to lower pressures (7). This is why the International space station maintains an internal pressure of 1 ATM. Looking at a dome and a box design, the COMSOL simulations on Fig(2) and Fig(3), strongly indicate, that the dome design is the better choice. We assume that: Equal internal volume, an internal pressure of 1 bar and an external pressure of 0.006 bar. In order for the box, to be able to contain this pressure, the wall thickness would have to be 1.3 m thick, compared to the domes 0.3 m wall thickness. The pressure distribution is also clearly more uniformly distributed in the dome, compared to the box. The relations between “volume to area” of the dome and box are as follows:
\begin{equation}
Area_{circle} = r^2 \cdot \pi
\end{equation}
\begin{equation}
Area_{dome} = Area_{circle} + \frac{r^2 4 \pi}{2} = 3 r^2
pi
\end{equation}
\begin{equation}
Area_{box} = 2a^2 + 2a^2 + 2a^2 =6 a^2
\end{equation}
\begin{equation}
\frac{Area}{Volume} -relations :
\end{equation}
\begin{equation}
\frac{a^3}{Area_{box}} = a \cdot \frac{1}{6}
\end{equation}
\begin{equation}
\frac{\frac{4}{3} \pi r^ 3}{Area_{dome}} = \frac{2}{9} \cdot r
\end{equation}
What becomes apparent from this, is that the “volume to area”- relation is lower for a box. This means that you would need more building material to construct a box, compared to a dome of equal volume.
Fig 2: - The picture shows how the internal stresses accumulate in a dome, simulated in COMSOL. It also shows the minimal wall thickness (0.3 m) when using Aspergillus as building material in Mars, if the dome has to be able to contain 1 bar.
Fig 3: - The internal stress accumulation of a box is a lot higher than a dome. In order for an box, of same volume as Fig(2), the wall thickness would have to be 1.3 m in order to contain 1 bar against the Martian atmosphere.
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 would not be an advantageous evolutionary strategy. By making the fungus a little worse at surviving, we aim to make it better for a building. In our project, we ended up focusing primarily on two genes; melA and gfaA.
Protection from UV radiation and Melanin
One part of what makes the surface of Mars a hostile environment is its radiation. Unlike the terrestrial atmosphere, the Martian atmosphere does not shield well against cosmic radiation, making radiation a serious concern on the red planet (10). Part of the radiation issue is short wavelength UV radiation such as UVB and UVC, which is not just harmful to living tissue but also causes radiation damage to solid materials (11). Nature has evolved systems that give the protection against UV radiation, most well-known is the pigment melanin. Overexpressing melanin might make fungal materials more resilient against radiation damage. Studies have shown that melanotic bacteria experience increased protection from the full spectrum of UV compared to counterparts without melanin (12). To slow down the potential erosion from UV radiation, we wanted to express the gene melA from Rhizobium etli CFN42 (13) in our fungal host. A previous iGEM team from the Technical Institute of Tokyo have shown that overexpression of melanin in E. coli can been achieved using the melA gene. melA encodes this for a tyrosinase that catalyses the enzymatic steps converting tyrosine into dopaquinone. We investigated the biosynthetic pathway of melanin in A. oryzae and discovered the same metabolic step as encoded for by melA (14). Heterologous expression of MelA in A. oryzae may thus also increase the production of melanin.
Melanin (Codon optimized for A. oryzae)
As a part of the project we set out to codon optimize, the Tokyo Tech melA gene from Rhizobium etli CFN42 to increase the expression in A. oryzae. Several studies have shown that expression of heterologous genes in some cases can be largely increased (15). The effect of the codon optimization could potentially be measured by comparing expression levels of the optimized and the original gene.
Fig 4: - Working hard on our transformants.
Cell wall structure and GfaA
The choice of building materials is always based on a set of parameters like strength, weight, thermal conductivity, durability, and price. Fungal materials are typically lightweight, good thermal insulators, have a good compressive strength (16), and in the context of Martian settlement, they could likely be made comparatively cheaper than building materials brought from Earth. Long-term durability is still somewhat an open question - especially in the context of Martian use. Biodegradability, which is a potential issue on Earth, is not a significant problem on Mars due to its extreme environment, thus suggesting that long-term stability might be achievable.
Fungal materials have shown to be sufficiently strong to make structures and various fibreboards. Given the lower gravity on Mars, fungal structures should be able to sustain themselves. We have models which suggest that fungal materials might also be able to sustain the internal pressure of habitats, yet it seems that making the materials stronger would be a desirable phenotypic characteristic to attain. Research suggested that the gene gfaA might be of interest having materials in mind. The structural strength of fungal cells walls comes from $\beta$-Glycans and chitin. gfaA encodes for a fructose-6-phosphate transferase that catalyzes the first and rate-limiting step in the UDP-N-acetylglucosamine biosynthetic pathway, the immediate precursor of chitin (17).
Studies have shown that after induced cell wall stress in A. niger, there are higher levels of gfaA mRNA present and that this is accompanied with higher levels of chitin deposition in the cell wall (17). We theorized that over-expression of this enzyme in Aspergillus oryzae might lead to a higher-than-normal chitin content of the fungal cell walls and hence, hopefully, stronger materials.
Colors and selectable markers - AmilCP
Finally, we have also been working on with the amilCP gene. amilCP encodes a blue chromoprotein and exhibits strong blue color when expressed in E. coli, and we wanted to use amilCP constructs as a reporter system to test transformation success of different fungi.
Mission Architecture
So how do we propose going about this entire endeavor? When a finalized version of our GMO fungus has been produced, the idea for astronauts is to bring a container with our fungus spores and a container with cyanobacterium to Mars. On their way, the astronauts will cultivate cyanobacteria in a growing vessel that is shaped like a long triangle. As you can see on the cross-section drawing, the vessel is a lightweight, collapsible container that can be fitted with a gas pump (pump not included in Fig(5)). The sides are zippable and on the inside, they are sown in spacers that make 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 filled with the bacterium, the astronauts will put the GMO fungal spores on top of the bacteria. After a short while, the fungi will have consumed the cyanobacteria and filled out the entire volume of the vessel. The astronauts can then open and start building their habitat from fully formed fungal bricks.
Fig 5: The figure illustrates the growth vessels that the astronauts could utilise to grow shaped building material from fungus. They start by cultivating Cyanobacteria,. Hereafter, they plant fungal spores into the bacterium, letting it grow into fully formed fungi bricks.
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(13)Tokyo Tech iGEM Team. 2009. Protection from UV ray.
(14)Kanehisa Laboratories. 2018. Tyrosine Metabolism - Aspergillus oryzae.
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