Team:DTU-Denmark/Results-choosing-organism
Choice of organism
Growing conditions between fungal species can vary depending on how they grow in nature. Factors could be temperature, amount of light and the substrate, among other things. With the goal of growing fungal bricks in mind, choosing a suitable organism for our project was one of our very first objectives.
Choice of organism
We initially focused on the research of the fungal species Pleurotus ostreatus, Aspergillus oryzae and Schizophyllum commune. S. commune has been investigated and used for the generation of bricks by the company Ecovative Design (1), whom we collaborated with. Furthermore, S. commune also allowed us to study genes affecting the structure of mycelium, by using mutants of SC3. This strain is characterized by a deletion of the SC3 gene, which is responsible for the production of hydrophobins. Hydrophobins are small proteins present in filamentous fungi that have the ability to repel water and create a coating on hydrophobic surfaces (2).
P. ostreatus is a common edible mushroom also known as the oyster mushroom that has been widely cultivated. Except from being characterized by strong mycelia, it is also used in industrial processes and mycoremediation (3).
A. oryzae has been used for fermentation processes for at least 2000 years (4). Moreover, its genome has been sequenced (5), thus making it an optimal choice for the generation of a genetic toolbox.
As our project requires the eventual growth of fungi on Mars, the species used should ideally be able to grow under the conditions there. Besides temperature, radiation is a key element to consider. $\alpha$- and $\beta$-radiation are fairly easily blocked, so even though there is an increased background radiation on Mars (6), the primary concern will be the increased levels of $\gamma$-radiation, since this type of radiation can penetrate very thick walls and inhibit growth of fungi or bacteria (7). To simulate the background gamma-radiation dose that fungi would receive on Mars, which is in the range of 12 rads/year (8), A. oryzae together with another fungal species, Ganoderma resinaceum was exposed to this level of radiation during the first three days of growth. The results suggested that the radiation did not inhibit the growth of any of the species.
Results
In the first experiment, the species were seeded at 3 different concentrations of MEA (47,5 g, 50 g and 52,5), PDA (19,5 g, 39 g and 58,5 g) and YPD. Thereafter, growth pictures were taken every 24 hours and the size of the fungal colonies was compared. Furthermore, different growth conditions (temperature and light) were tested for each of the species. S. commune was incubated 30ºC at light and 27ºC at light, P. ostreatus at 28ºC at dark and 25ºC at light, and A. oryzae at 28ºC at dark and 30ºC at dark.
In addition, we also investigated the growth of a mutated S. commune strain, SC3.
Fig. 1: Growth of A. oryzae on PDA 58.5g (A), S. commune on PDA 39g (B) and P. ostreatus on PDA 19.5g (C) plates after 24, 48 and 72 hours respectively after inoculation.
S. commune, P. ostreatus and A. oryzae grew at a similar rate on all media. However, the mutant strain of S. commune, ΔSC3, was able to grow only at PDA 39 g (fig. 2).
Fig. 2: Growth of S. commune Δsc3 on PDA 39g plates after 24, 48 and 72 hours respectively after inoculation.
Fig. 3: Growth of A. oryzae on rice to create bricks after 7 days of incubation at 28ºC.
This preliminary experiment indicated several species were interesting for our research, as they were able to grow in common media, producing mycelium that could form fungal bricks possibly of use for making fungal materials. After incubation at the following conditions: S. commune at 30ºC at light, P. ostreatus at 28ºC at dark and A. oryzae at 28 ºC at dark, only A. oryzae showed sporulation despite a similar growing speed. As the protoplastation and transformation protocol available to us relied on spores to produce the protoplasts A. oryzae seemed like the best choice of species to continue work on for the genetic engineering part. Furthermore, comparing the bricks it quickly became clear that A. oryzae produced the most coherent bricks (fig. 3), with the other species producing extremely brittle materials. This might be a feature of the substrates chosen for growth, as they were chosen based on species’ preference in terms of growth rate, so for A. oryzae it was rice and for P. ostreatus and S. commune it was sawdust. Addition of coffee grounds did nothing for the texture of the bricks but a mixture of rice and sawdust made the A. oryzae bricks harder. As shown in Table 1, once again it seemed that A. oryzae was the best choice for further work.
Table 1: Summary of the properties investigated according to every species. 
In addition to being the only one to sporulate and to form coherent bricks (fig. 4), A. oryzae is a well-studied species that are used in fermentation processes and has been engineered previously. Therefore, we decided to focus the project on forming bricks and doing genetic engineering of genes of interest on A. oryzae.
Fig. 4: Incubator containing the boxes used to grow the bricks. 
Eventually, radiation tests were performed on A. oryzae and Ganoderma resinaceum (fig. 5). The irradiated samples were kept at a temperature of 20° Celsius, controls were prepared and set to grow at the same temperature. The irradiated samples got a combined dose of 63.85 mSv from 28.09.2018, 13.10 to 01.10.2018, 10.45. The irradiated samples had growth radii which were on average twice as large as the controls (fig. 6, 7). These results are thought to be spurious, and the most likely candidate is that the sample room was kept at a higher temperature than 20° Celsius. The primary result is thus that the fungi were able to grow, but it is unknown if the increased amount of radiation caused an increase in mutations.
Fig. 5: Agar plates inoculated with species of interest, placed at 4 m from the gamma radiation source at DTU Risø. 
Fig. 6: A. oryzae growth after 3 days incubated under a gamma radiation dose of 63.85 mSv 
Fig. 7: G. resinaceum growth after 3 days incubated under a gamma radiation dose of 63.85 mSv
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