Lichen-Mediated Self-Growing Construc8on Materials for Habitat Ou<i=ng on Mars

Nisha Rokaya a, Erin C. Carr b, Richard A. Wilson a, Congrui Jin c*

a
Department of Plant Pathology, University of Nebraska–Lincoln, Lincoln, NE 68583
b
School of Biological Sciences, University of Nebraska–Lincoln, Lincoln, NE 68588
c
Department of Engineering Technology and Industrial DistribuMon, Texas A&M University,
College StaMon, TX 77843
*
Corresponding author. E-mail addresses: jincongrui@tamu.edu

Abstract

As its next step in space exploration, the National Aeronautics and Space Administration (NASA)
revealed plans to establish a permanent human presence on Mars. To build the centrally located,
monolithic habitat, NASA has a history of experimenting with lightweight inflatable habitats to
reduce mass and volume. However, the physical structures used to outfit the inflatable must
generally be launched by a second spacecraft. This study proposes that, rather than shipping
prefabricated outfitting elements to Mars, habitat outfitting can be realized by in-situ
construction using cyanobacteria and fungi as building agents. A synthetic lichen system,
composed of diazotrophic cyanobacteria and filamentous fungi, can be created to produce
abundant biominerals (CaCO3) and biopolymers, which will glue Martian regolith into
consolidated building blocks. These self-growing building blocks can be assembled into various
structures, such as floors, walls, partitions, and furniture. Diazotrophic cyanobacteria are mainly
responsible for 1) fixing CO2 and N2 from the atmosphere and converting them into O2 and
organic carbon and nitrogen sources to support filamentous fungi; and 2) giving rise to high
concentrations of CO32- as a result of photosynthetic activities. Filamentous fungi are mainly
responsible for 1) binding Ca2+ onto fungal cell walls and serving as nucleation sites to promote
CaCO3 precipitates; and 2) assisting the survival and growth of cyanobacteria by providing them
water, minerals, additional CO2, and protection. We have tested and confirmed that such co-
culture systems can be created, and they grow very well solely on Martian regolith simulants, air,
light, and an inorganic liquid medium without any additional carbon or nitrogen sources. The
cyanobacterial and fungal growth in such co-culture systems were significantly better than their
axenic growth, demonstrating the importance of mutual interactions. This is the first study that
unlocks the potential of creating a stable phototrophic-heterotrophic system to build habitat
outfitting on Mars.

Keywords: Self-Growing Materials; Construction Materials; Martian Construction; Space

Exploration; Synthetic Biology; Mutualistic Co-Culture

1. Introduction

As its next step in space exploration, the National Aeronautics and Space Administration (NASA)
revealed plans to establish a permanent human presence on Mars. To build the centrally located,

1
monolithic habitat, NASA has a long history of experimenting with lightweight inflatable habitats
to reduce mass and volume. Compared with habitat construction, habitat outfitting, i.e., the
technology to provide the crew with the necessary equipment to perform mission tasks as well
as a comfortable, safe, and livable habitable volume, is much less explored. Inflatable habitats in
particular require significant outfitting, since many elements will not launch pre-integrated as
they would in a metallic habitat. Currently, the outfitting elements must generally be launched
by a second spacecraft. This research proposes that, rather than shipping prefabricated outfitting
elements to Mars, habitat outfitting can be realized by in-situ construction using cyanobacteria
and fungi as building agents. A synthetic lichen system, composed of diazotrophic cyanobacteria
and filamentous fungi, can be created to produce abundant biominerals (CaCO3) and biopolymers,
which will glue Martian regolith into consolidated building blocks. These building blocks can later
be assembled into various structures to outfit the inflatable, such as floors, walls, partitions, and
furniture, as shown in Fig. 1.

Figure 1. Utilizing only in-situ resources on Mars, such as sunlight, water, CO2, N2, and trace
minerals, cyanobacteria and fungi can work together to form a synthetic community to produce
biominerals and biopolymers which will glue Martian regolith particles into a consolidated
building block.

The synthetic community must include three components: 1) photosynthetic microorganisms

that synthesize carbohydrates from CO2, satisfying the community’s energy and carbon
requirements; 2) nitrogen-fixing microorganisms that convert atmospheric N2 into organic
nitrogen, satisfying the community’s nitrogen requirements for essential cellular processes; and
3) Ca2+ attractors that deposit CaCO3 to bind regolith particles into building blocks. In this study,
the goal is to construct the simplest synthetic community that satisfies all the three requirements,
so only two species are included in the system, i.e., diazotrophic cyanobacteria and filamentous
fungi. In this self-sustaining system, each participant supports different functionalities, as shown

2
in Fig. 2. Diazotrophic cyanobacteria are mainly responsible for 1) fixing CO2 and N2 from the
atmosphere and converting them into O2 and organic carbon and nitrogen sources to support
filamentous fungi; and 2) giving rise to high concentrations of CO32- as a result of photosynthetic
activities, which is an essential process for CaCO3 deposition. Filamentous fungi are mainly
responsible for 1) binding Ca2+ onto fungal cell walls and serving as nucleation sites to promote
CaCO3 precipitates; and 2) assisting the survival and growth of cyanobacteria by providing them
water, minerals, additional CO2, and protection. In addition, both participants secrete
extracellular polymeric substances that enhance the adhesion between regolith particles and
calcium carbonate precipitates and the cohesion among precipitated particles.

Figure 2. SchemaMc illustraMon of the self-sustaining system, in which each participant supports
different functionalities.
2. Background and Mo8va8on
Self-growing technology engineers microorganisms to produce bonding materials, via
biologically or biotechnologically mediated routes, which will autonomously glue fine granular
parMcles into a cohesive structure. Its applicaMon in self-strengthening soil [1], self-stabilizing sand

3
[2], and self-healing concrete [3-5] has been extensively studied in the past decades. Dosier et al.
took one step further by invesMgaMng the uMlizaMon of bacterial biomineralizaMon to consolidate
sand parMcles into masonry units [6], as shown in Fig. 3(a). She founded a company called
bioMASON based on her patented process [7], i.e., certain species of ureolyMc bacteria are first
blended into a scaffold made from gelaMn and sand parMcles; the bacteria promote the
producMon of CaCO3 minerals via the ureolysis pathway that bond loose sand parMcles into a
cohesive structure at room temperatures; and the mixture of bacteria and scaffold is molded into
various shapes to form masonry units.
Besides biominerals, fungal mycelium has also been uMlized as bonding materials. Bayer and
McIntyre founded a company called EcovaMve Design based on their patented process [8]. First,
agricultural waste is cleaned and inoculated to create the growth of fungal mycelium. Mycelium
acts as a self-replicaMng glue that bonds together the waste parMcles, forming a solid structure
and filling any void space. During growth, the material can be molded into various shapes to
create packaging and insulaMon foams, as shown in Fig. 3(b). Another company, MycoWorks,
employs a similar process using fungal mycelium to create building blocks and leather-like fabrics,
as shown in Fig. 3(c) and Fig. 3(d), respecMvely.

Figure 3. (a) Cylinder samples produced by bioMASON. Image provided by bioMASON. (b)
Mycelium-based packaging materials produced by EcovaMve Design. Image provided by
EcovaMve Design. (c) Mycelium-based building blocks produced by MycoWorks. Image
provided by MycoWorks. (d) Mycelium-based leather-like fabrics produced by MycoWorks.
Image provided by MycoWorks. (e) MarMan regolith simulants consolidated by microbially
induced CaCO3 precipitaMon aier 5 days of incubaMon [9]. From “Microbial induced calcite
precipitaMon can consolidate marMan and lunar regolith simulants” by Dikshit, R., Gupta, N.,
Dey, A., Viswanathan, K., Kumar, A. PLoS ONE 2022; 17: e0266415. CC BY.
The potenMal applicaMons of self-growing technology in enabling long-term human space
exploraMon and colonizaMon are stunning. Whereas on Earth it will have to prove that it is more
economical than tradiMonal approaches, on Mars it may be the only opMon, due to its
autonomous nature which requires minimal human intervenMon and material replenishment.

4
During the self-growing process, every microbial cell funcMons as a nanomachine that can
replicate and repair itself and perform a wide variety of tasks, i.e., it constantly senses its
environment, uMlizes available sources of energy and maqer, and directs precise assembly of
complex structures across mulMple length scales that ulMmately enable the adopMon of
macroscale morphologies and funcMons to opMmally respond to the changing environment. The
Indian InsMtute of Science demonstrated the feasibility of using ureolyMc bacteria to induce CaCO3
precipitaMon to consolidate MarMan regolith simulants into brick-like structures with promising
structural strength [9], as shown in Fig. 3(e). NASA’s Ames Research Center has an ongoing project
“Myco-Architecture off planet”, which explores the feasibility of using fungal mycelium to bond
MarMan regolith and grow them into a fully funcMonal human habitat [10].
As a very young but promising technology, microbe-mediated self-growing technology has
achieved a significant level of success in various applicaMons. However, none of the current self-
growing pracMces is fully autonomous, since the building agents used in the exisMng studies, very
oien restricted to a single species or strain, are all heterotrophs and thus their survivability
depends on a constant external supply of organic carbon and nitrogen sources.
In natural environments, microorganisms typically do not live or funcMon opMmally in isolaMon,
and most of them live in interacMng heterogeneous communiMes. Self-sustained large-scale
CaCO3 producMon mediated by microbial acMviMes is well illustrated in natural environments by
stromatolites, thrombolites, and whiMng deposits. Field-based characterizaMon of these
calcareous deposits revealed that the complex interacMons among different microbial species
have important ramificaMons for self-sustained CaCO3 precipitaMon. For example, it has been
found that the largest known microbialite on Earth in the highly alkaline Lake Van, Turkey [11],
and the marine stromatolites at Shark Bay, Australia, and the Bahamian islands [12] are formed
as a consequence of the complex interacMons between photoautotrophic cyanobacteria and
heterotrophic bacteria. The process can be briefly described as follows.
First, cyanobacterial photosynthesis captures CO2 and converts it to organic compounds, and
such acMviMes give rise to a high concentraMon of CO32- [13]. However, CaCO3 precipitaMon is
actually inhibited by cyanobacterial growth, because large amounts of Ca2+ are trapped by the
negaMvely charged extracellular polymeric substances of cyanobacterial mucilage and sheaths
[14]. In the oligotrophic environments where most microbialites are found, acMvely growing
heterotrophic bacteria uMlize cyanobacterial exudates, including mucilage and sheaths, as carbon
and energy sources [11]. As the exudates are metabolized by the heterotrophs, the large amounts
of Ca2+ accumulated in cyanobacterial mucilage and sheaths are released into the soluMon. The
heterotrophic bacteria then bind the Ca2+ onto their cell walls and serve as nucleaMon sites for
the CaCO3 deposiMon [12]. In addiMon, the extracellular polymeric substances secreted by both
of them help enhance the cohesive property of the precipitated parMcles [14].
Inspired by nature, the innovaMon of this study is to explore a fully autonomous self-growing
technology by creaMng a syntheMc lichen system and making use of the mutualisMc interacMons
between diazotrophic cyanobacteria and filamentous fungi. Filamentous fungi, instead of
bacteria, are used as CaCO3 producers, due to their remarkable survivability against harsh
condiMons and extraordinary capability to promote large amounts of CaCO3 within short periods
of Mme [15, 16]. Filamentous fungi are heterotrophs whose survivability depends on a constant

5
external supply of organic carbon and nitrogen sources, and thus a photosyntheMc organism is
needed to funcMon as the interface between the natural resources available on Mars and the
nutrient sources for the biomaterial-producing fungi. Cyanobacteria are aerobic autotrophs
whose life processes require only light, water, CO2, nutrients, and trace minerals, many of which
are diazotrophic cyanobacteria that can assimilate atmospheric N2 as nutrients. In this study, a
symbiosis between diazotrophic cyanobacteria and filamentous fungi will be created, just like
natural lichens. These co-culture systems will uMlize the capabiliMes of two species
simultaneously, leading to a fully autonomous self-growing process.
The structure of this study is shown in Fig. 4. Since the selected fungal strains need to be mixed
with MarMan regolith in a photobioreactor, the pH of MarMan regolith is a key factor influencing
fungal growth and metabolic acMviMes. Because the pH of all the available MarMan regolith
simulants is moderately or highly alkaline, alkaliphilic fungal strains are collected for the first
tesMng, i.e., the survivability tesMng. The fungal strains that demonstrate opMmal survivability in
the growth medium containing MarMan regolith simulants and potato dextrose agar (PDA) are
selected for the second tesMng, i.e., the mutualism tesMng. A series of co-culture systems are
created, and their growth solely based on MarMan regolith simulants, air, light, and an inorganic
liquid medium, i.e., a modified Bold’s Basal Medium (BBM) amended with MOPS, with or without
addiMonal carbon or nitrogen sources is observed. For each monoculture or co-culture, four
different cases are tested, including C+N+, C+N-, C-N+, and C-N-. To assess microbial growth in
each well, three quanMtaMve characterizaMon methods are applied, i.e., resazurin assay, fungal
plaMng on selecMve medium, and measurement of phycocyanin autofluorescence. The co-culture
systems with the opMmal survivability and mutualism without any addiMonal carbon or nitrogen
sources are selected as candidates for habitat outiung on Mars.

Figure 4. Alkaliphilic fungal strains are collected for the first testing, i.e., the survivability testing.
The fungal strains that demonstrate optimal survivability in the growth medium containing
Martian regolith simulants and PDA are selected for the second testing, i.e., the mutualism
testing. Co-culture systems are created, and their growth solely based on Martian regolith

6
simulants, air, light, and an inorganic liquid medium with or without additional carbon or nitrogen
sources is observed. For each co-culture well, the number of live fungal cells, live cyanobacterial
cells, and live cells are quantitatively characterized.

3. Results and Discussion

3.1. Results of Survivability Testing of Fungal Strains

The tested fungal strains were originally purchased from the American Type Culture CollecMon
(ATCC), the Centraalbureau voor Schimmelcultures (CBS), and the Fungal GeneMcs Stock Center.
They include Trichoderma reesei (ATCC13631), Aspergillus crystallinus (ATCC16833), Aspergillus
restrictus (ATCC16912), Penicillium atramentosum (ATCC10104), Hemicarpenteles paradoxus
(ATCC16918), Penicillium s9pitatus (ATCC10500), Penicillium inflatum (ATCC48994), Penicillium
hirayamae (ATCC18312), Aspergillus malodoratus (ATCC16834), Aspergillus niger (ATCC16888),
Aspergillus flavus (ATCC9643), Penicillium brefeldianum (CBS235.81), Trichoderma viride
(HF3MP), and Trichoderma viride (HF3TV).
Their growth at the end of the 14-day incubaMon period in PDA with the presence of sterilized
Enhanced Mojave Mars Simulant MMS-2 (MarMan Garden, AusMn, TX, USA) is shown in Fig. 5. The
growth rates and pH measurements at the end of the 14-day incubaMon period in PDA with the
presence of MMS-2 are shown in Table 1.

It can be seen that ten fungal strains grew very well with the presence of MMS-2, and they
were selected for the mutualism tesMng. Under opMcal microscopy, abundant conidia and
conidiophores were observed from the plates with the presence of MMS-2, and they had similar
morphology compared to those produced on the plates without MMS-2. The highest growth rate
reached 6.03 mm/day for T. viride (HF3MP). The other four strains had limited growth with the
presence of MMS-2, although they grew very well without the presence of MMS-2. Agar plug
controls without any inoculum showed no fungal growth.

The pH of the base medium was measured to be 8.8. During the incubaMon, all the plates had
a decreased pH value. The highest pH value aier the 14-day incubaMon was 8.7 in the case of P.
inflatum due to no fungal growth and the lowest value was 5.9 in the case of A. niger which
demonstrated excellent fungal growth.

7
 Figure 5. Fungal growth at the end of the 14-day incubaMon period in PDA with the presence of
MMS-2.

8
 Table 1. The growth rates and pH measurements at the end of the 14-day incubaMon period.
Tested Fungal Strain Growth Rate (mm/day) pH Selected?
Aspergillus crystallinus (ATCC16833) 3.09 7.8 No
Aspergillus restrictus (ATCC16912) 5.32 8.1 Yes
Penicillium atramentosum (ATCC10104) 4.07 8.4 Yes
Hemicarpenteles paradoxus (ATCC16918) 2.38 7.8 No
Penicillium stipitatus (ATCC10500) 3.67 6.8 Yes
Penicillium inflatum (ATCC48994) 0.00 8.7 No
Penicillium hirayamae (ATCC18312) 1.97 6.1 No
Aspergillus malodoratus (ATCC16834) 3.17 7.5 Yes
Penicillium brefeldianum (CBS235.81) 4.33 7.8 Yes
Aspergillus niger (ATCC16888) 6.00 5.9 Yes
Aspergillus flavus (ATCC9643) 5.03 7.7 Yes
Trichoderma reesei (ATCC13631) 6.01 8.1 Yes
Trichoderma viride (HF3MP) 6.03 8.2 Yes
Trichoderma viride (HF3TV) 6.02 8.5 Yes
Agar Plug Control 0.00 8.8 N/A

3.2. Results of Mutualism Testing of Co-Culture Systems

Each of the selected fungal strains was paired with the diazotrophic cyanobacterium Anabaena
sp. (UTEX2576), which was purchased from the Culture Collection of Algae at the University of
Texas at Austin (UTEX). Their growth solely based on Martian regolith simulants, air, light, and an
inorganic liquid medium, i.e., a modified BBM amended with MOPS, with or without additional
carbon or nitrogen sources was observed. Note that cellulose and (NH4)2SO4 were used as carbon
and nitrogen sources, respectively, but the protocol can be used to assess the effect of any
carbon or nitrogen sources. NH4+ is a preferred nitrogen source in cyanobacteria [17], and it has
been found that cyanobacteria benefit from low concentration of sulphates [18], for which
gypsum on Mars could be used as a supplement. Cellulose can be rapidly utilized as carbon
sources by many of the tested fungal strains [19].

The color of the base medium was red due to the presence of MMS-2, and the dense growth
of the cyanobacterium often turned the growth medium into green or blackish green, and thus
the presence of cyanobacterial growth could be directly observed, as shown in Fig. 6. Abundant
bubbles were observed from all the cyanobacterial and co-culture wells due to photosynthetic

9
activities. White mycelial clusters were observed from some of the fungal wells, such as in the P.
brefeldianum well.

Figure 6. The picture of each well at the end of the 28-day incubation period.
The pH of the control well, the modified BBM with MMS-2 and MOPS but without any
cyanobacterial or fungal strains, was measured to be 8.37. During the incubation, some wells had
a decreased pH value, and the others had an increased pH value, but none of them changed
significantly, as shown in Table 2. The highest pH value after the 28-day incubation period was
8.50 from two co-culture wells whose fungal component is P. s9pitatus and A. niger, respectively.
The lowest value was 8.29 from a co-culture well whose fungal component is T. viride (HF3TV).
Table 2. The pH of each well at the end of the 28-day incubaMon period.

10
 Monoculture Co-Culture
Label Fungal Strain
C+N+ C+N- C-N+ C-N- C+N+ C+N- C-N+ C-N-
F1 A. restrictus 8.45 8.40 8.42 8.42 8.42 8.34 8.33 8.46
F2 P. atramentosum 8.41 8.36 8.34 8.38 8.41 8.35 8.33 8.43
F3 P. stipitatus 8.41 8.40 8.34 8.40 8.40 8.34 8.50 8.47
F4 A. malodoratus 8.41 8.42 8.41 8.40 8.48 8.37 8.32 8.42
F5 P. brefeldianum 8.37 8.34 8.31 8.38 8.44 8.36 8.32 8.43
F6 A. niger 8.36 8.33 8.31 8.36 8.39 8.44 8.47 8.50
F7 T. reesei 8.49 8.41 8.40 8.41 8.39 8.33 8.33 8.39
F8 T. viride (HF3TV) 8.37 8.36 8.36 8.35 8.39 8.32 8.29 8.36
F9 T. viride (HF3MP) 8.35 8.34 8.31 8.33 8.36 8.39 8.41 8.43
F10 A. flavus 8.36 8.36 8.36 8.37 8.40 8.31 8.31 8.39

During the 28-day incubation, microscopic fluorescent images were periodically taken from
each well to monitor cell growth. Some representative images are shown in Fig. 7. It was found
that, at the end of the 28-day incubation period, there were only a few live cyanobacterial cells
in the C-N- co-culture wells whose fungal component is A. restrictus (F1), P. atramentosum (F2),
A. malodoratus (F4), and P. brefeldianum (F5), respectively, whereas in the other C-N- co-culture
wells, cyanobacteria exhibited robust growth, interweaving with the fungal filaments, which
indicates that they constitute a promising synthetic lichen system.

11
 Figure 7. Representative microscopic images taken at the end of the 28-day incubation period
of the C-N- co-culture wells whose fungal component is (a) F5: P. brefeldianum, (b) F9: T. viride
(HF3MP), (c) F8: T. viride (HF3TV), and (d) F7: T. reesei. Red-colored fluorescing cells are live
cyanobacterial cells, yellow arrows point to dead cyanobacterial cells, purple arrows point to
fungal filaments, green arrows point to heterocysts, i.e., the N2-fixing cells formed under
nitrogen-starvation conditions, and blue arrows point to akinetes, i.e., the enveloped, thick-
walled, non-motile, dormant cell formed under stressed conditions. There were almost no live
cyanobacterial cells in (a), whereas cyanobacteria exhibited robust growth in (b), (c), and (d). The
scale bar is 100 μm in (a) and 10 μm in (b), (c), and (d).

3.3. Quantitative Characterization of Cyanobacterial and Fungal Growth

The autofluorescence of phycocyanin was used to characterize the number of live

cyanobacterial cells, and the results after the 28-day incubation period are shown in Fig. 8. It can
be seen that the cyanobacterial growth is optimal in the C+N+ case. Note that the co-cultures
were made by mixing the two components in equal proportion, and thus initially they had only
one half of the live cyanobacterial cells in comparison with the monoculture of cyanobacteria.
Therefore, in the C+N+ co-culture wells, the cyanobacteria generally displayed comparable or
faster growth rate than those in the cyanobacterial monoculture.

12
 The C-N- case is the focus of this study, and it can be seen that the autofluorescence
measurements are very low in the C-N- co-culture wells whose fungal component is A. restrictus
(F1), P. atramentosum (F2), A. malodoratus (F4), and P. brefeldianum (F5), respectively, which is
consistent with the microscopic observation, whereas in the other C-N- co-culture wells, the
cyanobacterial growth was comparable to or better than their axenic growth, demonstrating the
benefit of mutual interactions. The results indicate that under the same nutrient-poor condition
not all fungal strains effectively support their cyanobacterial partner in the co-culture system.

Figure 8. Results of phycocyanin autofluorescence measurement after 28-day incubation.

The resazurin assay was used to assess the number of live cells, both fungal and cyanobacterial,
after the wells were incubated for 28 days, and the results are shown in Fig. 9. In general, the
fungal growth in the co-culture wells, especially in the C-N- case, was significantly better than
their axenic growth, demonstrating the advantage of nutrient-poor condition for creating
mutualistic partnership. In the C-N- co-culture wells whose fungal component is F1, F2, and F4,
in which cases the phycocyanin autofluorescence measurements are very low, the resazurin
assay generates very high values, indicating excellent fungal growth in these co-culture wells,
whereas in the other C-N- co-culture wells such as F3, F6, F7, F8, F9, and F10, both fungal and
cyanobacterial growth was better than their axenic growth.

It can be concluded that in the C-N- co-culture wells, extremely fast fungal growth often
suffocates cyanobacterial growth as shown by the cases of F1, F2, and F4, although this is not
always true as shown by the cases of F3 and F6; whereas the fungal strains that demonstrate
moderate growth often create mutualistic partnerships with photoautotrophic cyanobacteria as
shown by the cases of F7, F8, F9, and F10, although this is not always true as shown by the case
of F5.

13
 Figure 9. Results of resazurin assay after 28-day incubation.

The method of fungal plating on selective medium was used to assess the number of live fungal
cells in each well after the wells were incubated for 28 days, as shown in Fig. 10(a). 100 µL of
homogenous sample in each well was withdrawn and spread evenly on a selective medium in a
Petri dish. The colony surface area was estimated after 3 days of incubation. Each plate was
photographed and then all pixels in the picture were converted to black or white. The ratio of
white pixels to total pixels was recorded. We found that this method can only provide a rough
estimation of the live fungal cells, and a more accurate method will be explored as future work.
The colony surface area coverage was all above 25% from the C-N- co-culture wells and all above
15% from the C+N+ co-culture wells, indicating that there were significant amounts of live fungal
cells after 28 days of incubation, even when no supplemented carbon or nitrogen source was
present.

14
 Figure 10. (a) The method of fungal plating on selective medium was used to assess the number
of live fungal cells at the end of the 28-day incubation period. (b) The fine granular particles of
Martian regolith simulants were bonded together by the microbial filaments and the biomaterials
they produced.

This proof-of-concept study demonstrated the feasibility of creating mutualistic partnerships

between photoautotrophic cyanobacteria and heterotrophic fungi in the alkaline environment of
Martian regolith simulants. We observed that the fine granular particles of Martian regolith
simulants were bonded together by the microbial filaments and the biomaterials they produced,
as shown in Fig. 10(b), which was taken from the C-N- co-culture well whose fungal component
is T. viride (HF3MP). As our future work, the self-sustained production of CaCO3 precipitates by
the synthetic lichen system will be promoted by adjusting various influencing factors, such as
pressure, temperature, atmosphere, and growth medium composition. The mixture of microbes
and regolith simulants will be periodically collected and analyzed using scanning electron
microscopy (SEM) and atomic force microscopy (AFM). A series of mechanical tests will be
performed on the consolidated blocks.

4. Concluding Remarks

In this study, synthetic symbioses consisting of a diazotrophic cyanobacterium Anabaena sp.

and a filamentous fungus P. stipitatus, A. niger, T. reesei, T. viride (HF3TV), T. viride (HF3MP), or
A. flavus were constructed and their successful growth on Martian regolith simulants, air, light,
and an inorganic liquid medium without any additional carbon or nitrogen sources have been
tested and confirmed. The cyanobacterial and fungal growth in the co-culture systems were

15
better than their axenic growth, demonstrating the importance of mutual interactions. These
synthetic lichen systems can be utilized to enable self-growing materials for habitat outfitting on
Mars.

To realize self-growing habitat outfitting, the spores of the selected cyanobacterial and fungal
strains will be placed inside a photobioreactor, which will be shipped from Earth to Mars. The
outside of the photobioreactor will be completely cleaned and sterilized. Neither cyanobacteria
nor fungi could directly thrive under the Martian atmosphere, since it has a pressure of 7.5 mbar,
which is only 0.7% of that of Earth, incompatible with the metabolism of most microbes. In
addition, there exists only 2.8% N2 in the Martian atmosphere, and thus cyanobacteria cannot
extract enough N2 to support their diazotrophic growth [20]. Hence, to realize the proposed
technology, the photobioreactor should provide the microbes with a tightly regulated
atmospheric condition as well as other conditions, such as illumination, heating to appropriate
temperatures, and protection against damaging radiation. To prevent contamination, the
microorganisms will never be released out of the photobioreactor. The self-growing process will
cease when illumination is withdrawn or when the microbes are killed by high temperatures or
radiation.

Besides habitat outfitting, this technology can also be used for damage repair on Mars, i.e., for
load-bearing structures with cracks, the microbial spores that are injected or sprayed into the
cracks can germinate when conditions are favorable, grow, and precipitate CaCO3 to heal the
cracks in situ. This technology is not only of significant importance to the future colonizers of new
planets but also plays a critical role in tackling the challenges on our own planet by revolutionizing
military logistics and construction in remote, austere, high-risk, and post-disaster environments.
For example, in the rubble after natural or man-made disasters, this self-growing technology can
be used to bond local waste materials to build shelters. In addition, the development of
construction materials that capture atmospheric CO2 in the production process is highly aligned
with our nation’s commitment to decarbonization.

5. Materials and Methods

5.1. Survivability Tes8ng of Fungal Strains

The growth medium was prepared using sterilized MMS-2 (The Martian Garden, Austin, TX,
USA) and PDA (Difco, BD Diagnostic Systems, Sparks, MD, USA) at a concentration of 0.1 g MMS-
2 per mL of PDA. A 6 mm diameter mycelial disc from the margin of a 7-day-old culture of each
fungus was inoculated using a cork borer at the center of 30 mm Petri dish containing 15 mL
growth medium. After inoculation, the Petri dishes were incubated in natural daylight conditions
at 25 °C for two weeks. The series without MMS-2 was tested for comparison purposes. Sterile
PDA plugs were used as control. Each case was tested in triplicates.

Radial growth measurements were recorded along two perpendicular diameters. The fungal
growth was also evaluated via optical microscopy (Carl Zeiss model III, Zeiss, Jena, Germany). The

16
pH of each plate was recorded by melting the PDA media and took three independent
measurements using an Orion double junction pH electrode.

5.2. Microbial Strains and Growth Medium for Mutualism Testing

The fungal strains that demonstrated optimal growth in the survivability testing were selected
for mutualism testing. Fungal spores were extracted in 20% glycerol and stored in -80 °C. Frozen
spores were thawed at 25 ± 2 °C for 1 hour prior to inoculation. The strains of the diazotrophic
cyanobacterium were propagated in a BG11 growth medium for two weeks prior to inoculation.

BBM is often used as an axenic-culture maintenance medium due to its predominantly

inorganic nature. In this study, a modified BBM was used as the base medium for both
monocultures and co-cultures, as shown in Table 3. Two modifications were made on the basis
of the standard BBM, i.e., NaNO3 was removed to eliminate the presence of additional nitrogen
source, and Bold’s Trace Stock Solution was replaced by Hutner's Trace Stock Solution to enhance
cell growth, as shown in Table 4.

Table 3. The base medium, i.e., a modified Bold’s Basal Medium, used in this study.
Component Amount Stock Solution Concentration Final Concentration
CaCl2•2H2O 10 mL/L 1 g/400mL dH2O 0.17 mM
MgSO4•7H2O 10 mL/L 3 g/400mL dH2O 0.3 mM
K2HPO4 10 mL/L 3 g/400mL dH2O 0.43 mM
KH2PO4 10 mL/L 7 g/400mL dH2O 1.29 mM
NaCl 10 mL/L 1 g/400mL dH2O 0.43 mM
EDTA Stock 0.05 mL
Iron Stock 0.05 mL
Boron Stock 0.05 mL
Hutner’s Trace Stock 0.05 mL

Table 4. Hutner’s Trace Stock Solution used in this study.

Component Amount Final Concentration
Na2EDTA•2H2O 50 g/L 0.134 mM
ZnSO4•7H2O 22 g/L 0.077 mM
H3BO3 11.4 g/L 0.184 mM

17
 MnCl2•4H2O 5.1 g/L 0.026 MM
FeSO4•7H2O 5 g/L 0.018 mM
CoCl2•6H2O 1.6 g/L 0.007 mM
CuSO4•5H2O 1.16 g/L 0.005 mM
(NH4)6Mo7O24•4H2O 1.1 g/L 0.0008 mM

Cyanobacterial monoculture was inoculated with an optical density of 0.25 at 720 nm. Fungal
monoculture was inoculated with a concentration of 5 × 105 spores/mL. A total of 150 µL was
inoculated for each monoculture and added to 24-well plates. For the co-culture cases, a total of
150 µL was inoculated by mixing the two components in equal proportion, i.e., 75 µL of
cyanobacterial monoculture with an optical density of 0.25 at 720 nm and 75 µL of fungal
monoculture with a concentration of 5 × 105 spores/mL were mixed together and added to 24-
well plates.

All the inoculums and cultures in this study were grown under the same condition, i.e., standing
cultivation, illumination of 100 μmol/m2/s with 14:10-hour light:dark cycle, temperature of 25 ±
1 °C, 60% relative humidity, and filter-sterilized air.

To study the effect of additional carbon and nitrogen sources, the base medium, i.e., the
modified BBM, was supplemented by cellulose and (NH4)2SO4, respectively. For each
monoculture or co-culture, four different cases were tested, including C+N+, C+N-, C-N+, and C-
N-. The C-N- growth medium was the base medium. The C+N- growth medium indicated that the
base medium was supplemented by cellulose with a concentration of 0.12 g per 100 mL. The C-
N+ growth medium was the base medium supplemented by (NH4)2SO4 with a concentration of
2.94 mM. The C+N+ growth medium indicated that the base medium was supplemented by
cellulose with a concentration of 0.12 g per 100 mL and (NH4)2SO4 with a concentration of 2.94
mM. Each well has 2 mL of growth medium. Sterilized MMS-2 was added to each well at a
concentration of 0.1 g/mL. The inert pH buffer MOPS (Fisher Scientific, Pittsburgh, PA, USA) was
added to each well at a concentration of 20 mM. The series with MMS-2 and MOPS but without
cyanobacterial or fungal strains was used as control. For each case, three biological replicates
were produced.

5.3. Quantitative Characterization of Fungal and Cyanobacterial Growth

The cell growth was periodically monitored by microscopic examination, which was performed
using an Axiovert 200 fluorescence microscope. To take the fluorescent images, the excitation
wavelength of fluorescent light ranged from 460 nm to 550 nm, which led to chlorophyll in cells
emitting red light. The pH of each well was periodically monitored by taking three independent
measurements using an Orion double junction pH electrode.

18
 The resazurin assay was used to assess the number of live cells in each well after the wells were
incubated for 28 days. This assay is based on the irreversible reduction of oxidized blue non-
fluorescent resazurin to a red highly fluorescent dye, i.e., resorufin, by the mitochondrial
respiratory chain in live cells. The amount of resorufin can be monitored by measuring
fluorescence, which is directly proportional to the number of live cells in the sample. This assay
was widely used to characterize the number of live cells of filamentous fungi [21]. Although not
widely documented in the exiting literature, it was also used to characterize cyanobacteria [22].
At the end of the 28-day incubation period, 100 µL of homogenous sample in each well was
withdrawn in duplicate and transferred to 48 wells on a 96-well plate. The resazurin solution was
heated to 37 oC to ensure all components were completely in solution and then 20 µL of resazurin
solution was added to each well. The plates were incubated for 4 hours at 25 ± 2 °C and then
read through a BioTek Synergy H1 hybrid spectrophotometer (Agilent Technologies, Santa Clara,
CA, USA). The relative fluorescent units (RFUs) were measured using a 560 nm excitation and 590
nm emission filter set. Prior to testing, a normalization test is performed to confirm the feasibility
and accuracy of this assay by measuring the number of live cells of the fungal and cyanobacterial
strains to be used in this study, respectively, when the number of live cells in a well is known.

The autofluorescence of phycocyanin was used to differentiate cyanobacterial cells from

fungal cells, since it is unique to cyanobacteria and does not exist in fungi. It only measures live
cyanobacterial cells since dead cells lose pigments very quickly either through decomposition or
photodegradation. After the wells were incubated for 28 days, a fluorescent area scan with 620
nm excitation and 647 nm emission of each well on the 24-well plates was performed using the
BioTek Synergy H1 hybrid spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) and
the average of the individual wells’ RFUs was calculated. Prior to testing, a normalization test is
performed to confirm the feasibility and accuracy of this assay by measuring the number of live
cells of the cyanobacterial strains to be used in this study when the number of live cells in a well
is known.

The method of fungal plating on selective medium was used to assess the number of live fungal
cells in each well after the wells were incubated for 28 days. 100 µL of homogenous samples in
each well was withdrawn and spread evenly on a selective medium in a Petri dish at 25 ± 2 °C.
The selective medium, originally developed for isolation of Trichoderma spp. from soil, was a
modified PDA through the addition of chloramphenicol at a concentration of 0.25 g/L [23]. The
colony surface area was estimated after 3 days of incubation. Each plate was photographed and
then all pixels in the picture were converted to black or white. The ratio of white pixels to total
pixels was recorded [24].
Acknowledgments
This study was funded by the NASA InnovaMve Advanced Concepts (NIAC) program of the
NaMonal AeronauMcs and Space AdministraMon (NASA) under the grant number of
80NSSC23K0584. This study was also parMally funded by the Young Faculty Award (YFA) program
of the Defense Advanced Research Projects Agency (DARPA) under the grant number of
D22AP00154. The views, opinions, and/or findings expressed are those of the authors and should
not be interpreted as represenMng the official views or policies of the Department of Defense or

19
the U.S. Government. Erin C. Carr was supported by Postdoctoral Research Fellowships in Biology
(PRFB) program of NaMonal Science FoundaMon (NSF) under the award number of 2209217.
Dr. Steven Harris at the Department of Plant Pathology, Entomology and Microbiology at Iowa
State University is thanked for useful discussion and providing some of the alkaliphilic fungal
strains. Dr. Lynn Rothschild at NASA’s Ames Research Center is thanked for useful discussion.
CRediT Authorship Contribu8on Statement
Nisha Rokaya: WriMng - original drai, WriMng - review & ediMng, Soiware, Methodology,
InvesMgaMon. Erin C. Carr: WriMng - review & ediMng, Soiware, Methodology, InvesMgaMon,
Resources. Richard A. Wilson: WriMng - review & ediMng, Resources, InvesMgaMon, Supervision.
Congrui Jin: WriMng - original drai, WriMng - review & ediMng, Methodology, Soiware,
InvesMgaMon, ConceptualizaMon, Supervision, Funding acquisiMon.
ORCID
Erin C. Carr hqps://orcid.org/0000-0001-6551-9207
Congrui Jin hqps://orcid.org/0000-0003-0606-5318
Declara8on of Compe8ng Interest
The authors declare that they have no known compeMng financial interests or personal
relaMonships that could have appeared to influence the work reported in this paper.
Data Availability
Data will be made available upon request.
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