1

Final Summary:

Using microbes in bioelectric reactors to extract oxygen out of

Martian soils

Van Den Berghe1, M., West, A. J.1, Nealson, K. H.1 1. Dept. of Earth Sciences, University of Southern California, 3651 Trousdale Pkwy, Los Angeles, 90089, CA, USA.

Executive Summary

Logistical and time constraints inherent to interplanetary travel render any human visit to Mars

a deeply committing and isolating enterprise, punctuated at best by very sporadic resupply events.

The rapid development of self-sufficiency is therefore critical to any human presence, and highly

dependent on in situ resource utilization (ISRU) systems. Oxygen will be one of the most fundamental

necessities for human presence on Mars, to both sustain human life and provide a propellant for

spacecrafts. One existing oxygen-generating ISRU technology being developed (i.e. the Mars oxygen

ISRU experiment, MOXIE) makes use of atmospheric carbon dioxide to generate oxygen gas. This

process has very high energy requirements and produces large amounts of carbon monoxide as a by-

product (Meyen et al., 2016), constraints that may not be viable for supporting human crews, let alone

entire communities. Yet very large amounts of oxygen are contained in Martian soil, bound in the

crystal lattice of globally distributed, clay-sized ferric oxide minerals (Morris et al., 2004; Rubie et

al., 2004), and could be made available for human use.

In this work, we developed a bioelectrochemical system that successfully dissolved iron

oxide minerals and converted products into molecular oxygen. These results act as a proof of

concept, showing that Martian soils could be used as a substrate in an ISRU system designed to

generate oxygen. This was made possible by making use of the combined metal-reducing, and

bioelectric capabilities of bacteria in a novel bioelectrochemical reactor. By using specific gene

deletion strain of S. oneidensis in a cathode-oxidizing, iron-reducing environment, we have been able

to produce molecular oxygen. By doing so we have demonstrated for the first time that microbial

activity can catalyze the electron transfer between two solid substrates (specifically between a cathode

and an insoluble mineral oxide), and in the process generate useful molecular oxygen. This system

requires minimal energy inputs and has no toxic byproducts.

At a scientific level alone, these findings significantly expand our understanding of the limits

and metabolic capabilities of life. By using the forefront of scientific knowledge in microbiology this

project further addresses one of the most critical requirements for the human exploration and

inhabitation of Mars: to secure an accessible source of oxygen that could be used for life support and

spacecraft propellant resupply. This project received seed funding from the Dubai Future Foundation

through Guaana.com open research platform.

Main results or outputs

Introduction

Planetary geochemistry

The distinctive color of the Red planet has been shown to be in large part due to abundant and

ubiquitous ferric oxide minerals present in Mars’ topsoil. Martian coarser regolith and sand dunes are

generally composed of detrital, crystalline to amorphous basaltic minerals (i.e., olivine, pyroxene,

feldspar), formed as a result of the physical weathering of parent magmatic crustal material (Downs

and MSL Science Team, 2015; Ehlmann et al., 2017). However, topsoils also contain copious

amounts of globally distributed, poorly crystalline nanophase ferric minerals, which include hematite,

maghemite, magnetite, goethite, jarosite and schwertmannite (Bell et al., 2000; Morris et al., 2006,

2

2004). Furthermore, large, coarsely crystalline hematite deposits and spherules have been found

exposed in a number of surficial, water-altered bedrock formations across the planet, with hematite

accounting for up to 45 vol.% of some outcrops (Christensen, 2004; Christensen et al., 2001, 2000;

Fergason et al., 2014; Lanza et al., 2016). Moreover, wind-borne magnetic dust, the matrix of

infamous global year-long Martian storms, is in large part composed of magnetite, hematite and

goethite, further contributing to a widespread geographical distribution of nanocrystalline (~3µm)

ferric oxide particles (Goetz et al., 2005; Hamilton et al., 2005). These mineral phases contain an

abundance of oxygen which, if liberated and transformed, could provide an almost limitless source

of O2.

Bacterial metabolic potential and Electroactive microbiology

Historically, chemical engineering endeavors have relied on abiotic, bulk and energy-

intensive reactions to achieve a desired output. Life, particularly the microbial kind, has on the other

hand an extraordinary ability to catalyze otherwise improbable yet meaningful geochemical reactions,

which could be harnessed for geoengineering purposes with minimal energetic requirements. Yet so

far limited attention has been placed on harnessing its outstanding ability to catalyze geochemical

transformations. Mainly, efforts have been limited to biomining and bioleaching of some mineral

ores, as well as some efforts in bioremediation (Schippers et al., 2014). However, recent

developments in our scientific understanding of microbiology have informed us on some bacteria’s

extra cellular electron transport (EET) capabilities. EET-capable bacteria are indeed able to fuel their

metabolism by transferring electrons across the cell membrane to an extra-cellular substrate,

including highly insoluble mineral phases such as iron or manganese oxides. Understanding the fate

of iron has been of particular interest due to its role in modulating global biological productivity and

climactic systems (Hutchins and Boyd, 2016; Martin, 1990). However, EET-driven ferric oxide

reductive dissolution implies the congruent release of oxygen from the crystal lattice, and the fate of

oxygen after mineral dissolution still remains unknown. Yet the globally abundant presence of readily

accessible, fine-grained ferric oxides in Martian topsoils lends itself to a potentially viable and very

significant source of oxygen, a situation that warrants examining the fate of oxygen following ferric

oxide dissolution.

Furthermore, EET processes are now well known to include electro-active processes, whereby

bacteria can interact with an electrode to generate an electric current. These discoveries have

prompted some novel applications in simultaneous wastewater treatment and energy generation

(Chen, 2004; Kim et al., 2004; Nealson, 2017). Most of these bioelectric systems have however been

developed using anodes as the microbial terminal electron acceptor, and organic carbon as the

electron donor. Nevertheless, some bacteria have been shown to also be able to use cathodes as

electron donors, still broadening the potential applications in biotechnology and geoengineering

purposes (Rabaey and Rozendal, 2010; Rosenbaum et al., 2011). Thus, coupling cathode oxidation

with ferric iron oxide reduction using EET processes can offer an effective means to dissolve iron

oxide minerals present in Martian soils, liberating oxygen out of the crystal lattice. Specifically, using

a bioelectric reactor to do so offers significant advantages:

1. It can be powered by solar energy, removing the need for consumable organic electron donor

substrates, thus enhancing autonomous ISRU capabilities of the system,

2. It removes byproducts of carbon metabolism such as free protons which are otherwise likely

to interfere with molecular oxygen production,

3. A counter electrode acts as a balancing oxidant, providing the critical step in oxidizing

reduced oxygen species and ensuring molecular oxygen production.

In this context, EET-capable bacteria can act as an effective catalyst to reduce and dissolve ferric

oxide minerals, with the putative enzymatic system involving reversing electron flow through the Mtr

respiratory pathway. While it has been suggested that bacterial cathode oxidation offers scant

metabolic energy and limited growth rates (Rosenbaum et al., 2011; Ross et al., 2011; Rowe et al.,

2017), mere bacterial maintenance, survival, or even presence in a bioelectrochemical reactor may be

3

enough to catalyze the desired geochemical reactions, and certainly constitutes a branch of

geomicrobiology and geoengineering yet to be explored.

Objectives

As the demands of inter-planetary transport and exploration challenge the boundaries of our

engineering capabilities, innovative technologies that can push the limits of scientific knowledge are

required. To that end, this research project has endeavored to answer these questions:

1. Can molecular oxygen can be extracted from simulated Martian soils using bioelectric

reactors?

This question was explored by conducting experiments with cathode-oxidizing, iron-reducing

bacteria, incubated into bioelectric reactors amended with hematite as sole terminal electron

acceptor, and monitoring bioelectrochemical activity, cell density and iron reduction.

2. Can microbial activity or growth be sustained by the transfer of electrons between two solid

substrates?

The ability of bacteria to catalyze and benefit from electron transfer between two solid-phases

is as yet unknown. By conducting experiments attempting to sustain bacteria by coupling

cathode oxidation with ferric oxide reduction, we could probe into the limits of life, and the

mechanisms driving bacterially-driven electrochemical reactions.

Here, we hypothesize that selected electro-active bacteria can effectively catalyze cathode-

mineral oxide electron transfer, helping achieve iron oxide reduction and molecular oxygen

production. We further propose to quantify the growth associated with such activity, measuring

microbial growth as a function of electrical throughput and mineral substrate dissolution.

Methods

1. Apparatus

We have developed a custom-built 2-chambered, three-electrode bioelectrochemical reactor

in which a biotic chamber coupled microbially-mediated cathode oxidation with iron reduction, and

an abiotic chamber oxidized reduced oxygen species and generated molecular oxygen. The chambers

were separated by a Nafion XL proton-selective membrane (PSM) (Fuel Cell Store, College station,

TX). The biotic chamber contained a working electrode made of conductive indium tin oxide (ITO)

coated glass, and a custom-built reference electrode of Ag-Ag/Cl in saturated KCl solution, both

connected to a potentiostat (eDAQ Inc., Colorado Springs, CO), with a working potential set at -

351mV vs. standard hydrogen electrode (SHE). The biotic chamber further contained 0.1g of crushed

(<63µm), UV-sterilized red ochre hematite (Ward’s Science, Rochester, NY), acting as a simulated

Martian soil substrate and indirect source of oxygen. During biotic experiments, this chamber also

contained a Cyo-A gene-deletion mutant strain of Shewanella oneidensis MR-1, chosen for its EET

capabilities and reduced oxygen metabolism (Bretschger et al., 2007). Thus the biotic chamber carried

reaction 1, electrons being introduced into the reactor by the ITO cathode:

Fe2O3 + 2e + 2H2O +2H+ = 2Fe2+ + H2O + 2OH- (1)

The abiotic chamber contained autoclaved 18.2MΩ-cm water, a dissolved oxygen (DO)

microsensor, and a custom-built titanium and platinum counter electrode (also connected to the

potentiostat). Thus the abiotic chamber was designed generated molecular oxygen through reaction

2, electrons being taken up from the reactor by the balancing counter electrode:

H2O = 1

2O2 + 2e + 2H+ (2)

4

This system therefore promoted molecular oxygen production through a stepped reaction,

whereby microbially-mediated iron reduction through cathode oxidation was balanced out by the

oxidation of water at the counter electrode. The PSM allowed the balancing diffusion of free protons

while blocking the flow reactants and microbes between chambers (Figure 1).

Figure 1: Conceptual drawing of the bioelectric system. Cathodic oxidation and iron oxide reduction occur in the biotic chamber while

water oxidation and molecular oxygen generation occurs in the abiotic chamber. The two are separated out by a proton-selective

membrane allowing to maintain charge balance in the bioreactor.

The 2-chambered reactor was built of borosilicate glass, and moieties were held together by

a clamped flange, sealed by a pair of custom-built Viton® fluoroelastometer chemical-resistant

rubber sheet O-rings (McMaster-Carr, Santa Fe Springs, CA). The PSM was cleaned and prepared

as previously described (Chae et al., 2008) prior to use. To further ensure airtight connections, the

working and reference electrodes were emplaced in a rubber stopper and sealed with airtight silicone

gasket as well as high vacuum grease. The apparatus was confirmed airtight with DO readings in

static tests. Sampling ports and the counter electrode were placed through crimped butyl rubber septa.

All reactor chamber components were acid-washed, sonicated in 10% liquinox, and autoclaved prior

to use. Electrodes were washed in acetone and 70% ethanol prior to autoclaving and use. DO

microsensors were acid washed and sterilized with 70% ethanol prior to use.

2. Culturing conditions

Preparing for biotic experiments, a single colony of Shewanella oneidensis MR-1 Cyo-A

deletion bacteria was grown in an oxygenic, 18mM lactate minimal medium (Bretschger et al., 2010,

2007) to an optical density (OD) at 600nm of ~1.0 at 30ºC. To induce a preliminary cell attachment

to the working ITO electrode and promote electro-active pathways, 1mL of this culture was triple-

washed and injected into a 3-electrode chamber containing the same 18mM lactate minimal medium,

kept anoxic through constant N2 purging and a set potential of 699mV vs. SHE.

After about 36 hours of anode-reducing growth, the working and reference electrodes were

removed from this chamber, rinsed with experimental minimal medium, and introduced into the biotic

chamber of the 2-chamber reactor, thus transferring only biofilm-attached cells into the experimental

reactor. The minimal medium in the biotic chamber lacked a carbon source, buffer, and had a starting

pH of 8 in order to promote cross-PSM proton transfer. Hematite was later introduced in the biotic

chamber, settling in part directly on the working electrode. Both chambers of the reactor were then

purged with filtered N2 gas for up to two hours, with DO readings ensuring anoxic conditions. The

experiments began when the potential was set (-351mV vs. SHE) on the working electrode. Abiotic

5

experiments were performed by simply introducing autoclaved electrodes into a sterile experimental

reactor, bypassing all preparatory growth steps, and both chambers containing 0.4% formaldehyde.

In order to test different electrochemical conditions and promote activity, a filter-sterilized

and anaerobic anthraquinone-2,6-disulfonate (AQDS) solution was injected into 100µM in the biotic

chamber during experiments. Additionally, purging the biotic chamber with 0.22µm-filtered N2 gas

(~40cc/min) was also performed in order to test the added effect of increased medium flow across the

working electrode.

3. Fluorescence microscopy and activity

Samples were extracted from the biotic chamber of experiments every ~22h, with aliquots

separated out for total cell count using acridine orange (1mg/mL stock) to quantify total planktonic

cell. Redox Sensor Green (RSG, prepared as specified by the manufacturer, Molecular Probes, Life

Technologies Inc.) was also used to quantify cell count based on activity in the electron transport

chain, both in the plankton and on the working electrodes. Images and counts were performed under

a Zeiss Axio optical microscope. Iron(II) concentrations were measured from duplicate sample

aliquots (n=2) of the biotic chamber of experiments following an established ferrozine protocol

(Myers and Nealson, 1988) using a FLUOstar Optima (BMG Labtech, Cary, NC) micro plate reader.

Bioelectrochemical activity in the form of electrical current was monitored and recorded at 1

minute intervals with the eDAQ potentiostat. DO concentrations were measured every 1 minute using

a microsensor connected to a multichannel amplifier/multimeter (Unisense, Aarhus, Denmark).

4. Imaging

Scanning electron microscope (SEM) secondary electron imaging was performed on electrode

materials following experiments with a Nova NanoSEM 450 (FEI, Hilsboro, OR). Elemental

mapping was performed on the same instrument with energy dispersive X-ray analysis using a

TEAM™ software system (EDAX, Draper, UT). Operating conditions involve a working distance of

5-10mm and an accelerating voltage of 10kV All samples were prepared using a 7-step ethanol

dehydration process, then fixed with a critical point dryer (Tousimis, Rockville, MD), and coated

with a Pt/Pd Cressington 108 sputter-coater.

Results and discussion

Three biotic experiments were performed, with the cathodic working ITO electrode supported

an electro-active Shewanella biofilm coupling hematite reduction in the biotic chamber of the

bioreactor. These conditions alone promoted limited current production (~0.1-0.5µA), yielding no

detectable oxygen production in the abiotic chamber (Figure 2). However, these conditions did

support an increase in planktonic cell density in the biotic chamber, a surprising finding since the

minimal medium contained no carbon source for respiration. This could be explained by latent cell

growth due to stored lactate from the preceding anode-reducing growth conditions, or by biofilm-

attached cells reverting to a planktonic phase.

6

Figure 2: Dissolved oxygen and current measurements (A), along with planktonic cell counts and dissolved Fe(II) measurements (B)

in the biotic experiment (shown here are the representative results of 1 of 3 experiments).

In order to help increase bioelectric activity, AQDS was injected into the biotic chamber to a

concentration of 100µM (below known toxic concentrations as per Hong et al., 2007; Shyu et al.,

2002) to act as an electron shuttle and catalyst. This increased current by an order of magnitude, and

initiated rising and detectable oxygen concentrations in the abiotic chamber. This addition also led to

the detection of dissolved Fe(II) in the biotic chamber. All these effects were further accentuated

when the biotic chamber was purged with nitrogen, which served to increase medium flow over the

working electrode, increasing electrochemical activity. Current increased by another order of

magnitude, and corresponding oxygen concentrations up to 40µM. Planktonic cell density increased

still in that time, consistent with increased biofilm detachment. Planktonic cells were furthermore

shown to support by the end of the experiments an active electron transport chain (RSG stain) in

densities comparable to those of total cell count (acridine orange), signifying that planktonic cells

were actively catalyzing redox reactions. RSG staining of the working electrodes shows pervasive

colonization of electro-active cells on hematite particles (Error! Reference source not found.), with

an estimated density of ~105cells/mm2 by the end of experiments. Corresponding Fe(II)

concentrations increased to ~5µM (Figure 2). SEM imaging showed ITO working electrodes

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Acridine orange cell count

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Fe(II) concentration

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populated by Shewanella and nanowire-like structures connecting iron oxide minerals and the

electrode. These results are consistent with a bioelectrochemical system which supports the coupling

of cathode oxidation and iron

reduction facilitated by microbial

electron transport.

An abiotic control was

performed, whereby all components,

particularly the working electrode,

were sterile. Results show a similar

pattern, with starting conditions

producing limited current (~1µA)

and undetectable oxygen or Fe(II).

The addition of AQDS in the “biotic”

chamber (here sterile) increased the

current by almost an order of

magnitude, and coincided with the

onset of detectable oxygen signal.

Purging the biotic chamber with

nitrogen further increased the current

4-fold, and an increase in Fe(II)

concentrations, though oxygen

concentrations failed to increase and

remained steady at ~5µM. The

system remained sterile throughout

the experiment with no cell count

(Figure 4). This abiotic control shows

that while abiotic electrochemical

activity did occur in the system,

distinctly lower oxygen

concentrations were produced in the

absence of electro-active bacteria.

This likely reflects the reality of a

more complex electrochemical

process than presumed in Figure 1.

Explanations might involve

limitations in proton diffusion rates across the PSM affecting the rates of equation 2 potentially due

to bacterially-mediated pH control in the biotic chamber, or AQDS oxidation/reduction rates (likely

relating to bacterial respiration) and potential reaction with protons crossing the PSM.

A

B

Figure 3: SEM image of the working electrode in the biotic experiment, showing

Shewanella cells connecting iron oxides and the ITO electrode with nanowire-like

structures (A), and optical microscope image of Shewanella cells attached to the

working electrode and stained with RSG having colonized hematite particles

(blurred areas point to unfocused cell fluorescence along the z axis due to relief

along hematite grains) (B). Scale bars are 5µm.

8

Figure 4: Dissolved oxygen and current measurements (A), along with planktonic cell counts and dissolved Fe(II) measurements (B)

in the abiotic control.

At this stage, the bioelectrochemical mechanisms involved in this reactor, coupling cathode

oxidation with iron reduction and oxygen production, remain imprecisely understood. Similarly, the

exact biochemical pathways used by Shewanella to catalyze the coupling of cathode oxidation and

iron reduction (two solid substrates) remains putative. However these results clearly show that this

bioelectrochemical reactor did succeed at pairing electrode oxidation with iron reduction, and in the

process generating and isolating molecular oxygen. This system also showed that electro-active

bacteria can thrive in a system where the electron donor and acceptor are solid extra-cellular

substrates, and in the process accentuate oxygen production.

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Cu

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(µ

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Time (h)

A

Dissolved oxygen

Current

AQDS addedPurging with N2

0

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/mL

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Redox Sensor Green cell count

Fe(II) concentration

B

9

Obstacles or changes of direction during the project

The original design for the bioreactor did not involve two chambers separated by a PSM.

Instead it was a single chamber in which all the stepped reactions as described in Figure 1 would

occur. Such a design had the disadvantage of mixing all the oxidized (i.e. molecular oxygen) and

reduced (i.e. dissolved, reduced iron)

products in the same chamber—thus

likely causing the two to react with

each other. However it was hoped

that these products would still have a

residence time in the chamber high

enough to be detected by sensors.

Additionally, a single-chamber

system was significantly easier to

build and operate.

However, assumptions about

the residence time of products were

proven to be wrong, something five

successive experiments showed quite

clearly (data not shown here).

Nonetheless, repeated evidence of

bioelectric activity at the working

electrode (Figure 6), coupled with

metal oxide precipitation at the

counter electrode (Figure 7) suggested an active, functional system, though not optimized for

molecular oxygen production and isolation. Thence, a 2-chambered design was developed inspired

by previous microbial fuel cell designs (Wang and Coates, 2017). This new design was meant to

separate reduction at the working electrode and oxidation counter electrode reactions, avoiding metal

oxide precipitation and allowing to isolate molecular oxygen.

Figure 6: SEM image and EDAX elemental maps of the counter electrode in single-chamber

biotic experiments, showing the ubiquitous precipitation of Mn and Fe oxide minerals over the

Pt electrode, suggesting in-situ oxidation activity.

Figure 5: SEM image of the working electrode in a single-chamber biotic

experiment, showing Shewanella cells colonizing iron oxide particles with

nanowire-like structures. Scale bar is 5µm.

10

Potential impact and opportunities for implementation of the results

These findings act as a proof of concept, supporting the viability of a Mars-based ISRU system

that could use this novel bioelectrochemical system in order to derive molecular oxygen from ferric

oxide minerals. This has the potential to enable the human exploration and colonization of Mars by

securing access to one of the most important resources needed, whether as a component of life support

systems, or as a vital propellant for spacecrafts allowing people to explore other worlds or return to

Earth. Using extremely abundant and ubiquitous metal oxides as substrate could provide an almost

limitless source of oxygen. This system also offers the added benefit, compared to an existing oxygen-

generating system like MOXIE, of requiring minimal amounts of energy and producing no hazardous

byproducts.

Looking forward, one important consideration of this system for implementation in the field

will need to be the effects of known high perchlorate concentrations in Martian soils. While

perchlorate can be highly toxic to most living organisms, some bacteria have evolved to learn to live

by reducing it, a process also know to produce oxygen gas (Coates and Achenbach, 2004). Perchlorate

concentrations in Martian soils are typically much lower than those of ferric oxides (~1wt.% for the

former, and up to 45wt.% for the latter; Christensen, 2004; Christensen et al., 2001, 2000; Hecht et

al., 2009; Morris et al., 2004; Sutter et al., 2017), and therefore contain less total oxygen than ferric

oxides. They still however create both a risk and an opportunity for the purposes of

bioelectrochemical ISRU systems. Therefore, a similar bioelectrochemical reactor will need to be

developed that can use perchlorate-reducing bacteria in order to simultaneously produce some usable

oxygen from perchlorate reduction, and remove its toxic concentrations from soils (Wang and Coates,

2017). To that effect further developments can be performed with more realistic Martian soil

simulants (Zeng et al., 2015) whereby a multi-step process can separate out perchlorate reduction and

metal oxide reduction, generating oxygen all along the way.

In the context of long-term colonization, human efforts will likely include terraforming, which

will require incubating microbial life in particularly harsh environments. Ideally suited for such

environments are bacteria like Shewanella, used in these experiments, which are capable of

performing EET, using solid substrates such as metal oxides and electrodes to respire. Incubating

such bacteria in Martian regolith using bioelectric reactors analogous to the one described here may

help in promoting an active microbial ecosystem on Mars. Establishing such metal oxide and

perchlorate-respiring microbiome would likely be the critical first step in an ecological transgression,

establishing a fertile organic substrate in an otherwise barren soil, and encouraging the further

establishment of plant forms. Furthermore, promoting an electro-active, metal-reducing bacterial

presence could help in accelerating oxygen production at a regional or global scale, hastening

terraforming efforts.

To conclude, the experimental results from this study illuminate some of the ways that

establishing advanced microbiological laboratories and bioreactor systems on Mars could allow a

human settlement to catalyze geochemical transformations and secure a source of oxygen, as well as

provide the necessary basis to experiment with planetary bioengineering. Few systems could be more

important and critical to the success of any human settlement on Mars.

Conclusion and next steps

There is an enormous amount of oxygen on Mars: contained not in the atmosphere but in soils.

In order to support human efforts of all kinds on the red planet, we will need to access resources in

situ, and it would make little sense to look anywhere else for oxygen than in its soils. To that effect,

results presented here have successfully shown that a bioelectrochemical system such as that designed

in this study can leverage cutting-edge developments in microbiological sciences to efficiently extract

oxygen out of common, easily accessible, Martian base materials.

11

With a proof of concept completed, the reactor design should be improved in order to

maximize experimental efficiency, purity and repeatability. From there further testing will need to be

conducted in order to improve our mechanistic understanding of the bioelectrochemical system. Chief

among this is the need to understand and characterize the precise biochemical pathways bacteria use

to catalyze oxygen production. This could be done through a wide range of methods, from various

incubation experiments to detailed geochemical analyses, the use of genetic tools (such as testing a

range of deletion mutants, transcriptomics, etc.), and digital holographic microscopy. With this

mechanistic knowledge, the system could be then be optimized to maximize bacterial growth, activity

and reaction rates in order to enhance oxygen production. Further funding would need to support

glass and machine shop costs, all laboratory analyses, microscopy time, genetic tool development and

analyses, research assistantships, laboratory supplies, travels to conferences and institutional

overhead. Such a funding cycle would have a period of about 18 to 24 months and be in the range of

USD 150,000 to 200,000.

In the longer term, this system is intended to be used effectively on Mars and therefore

requires further development and optimization on Martian soil simulants. The ultimate goal of course

would be to use a proofed system on the red planet itself and test it in situ, as part of a scientific

mission. Such an experiment would constitute the first use of biotechnology on another world, will

safeguard a source of oxygen for life support as well as a propellant for spacecrafts, and will pave the

way for long-term human missions and settlements.

12

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