Towards synthetic biological approaches to resource utilization on space missions

This paper demonstrates the significant utility of deploying non-traditional biological techniques to harness available volatiles and waste resources on manned missions to explore the Moon and Mars. Compared with anticipated non-biological approaches, it is determined that for 916 day Martian missions: 205 days of high-quality methane and oxygen Mars bioproduction with Methanobacterium thermoautotrophicum can reduce the mass of a Martian fuel-manufacture plant by 56%; 496 days of biomass generation with Arthrospira platensis and Arthrospira maxima on Mars can decrease the shipped wet-food mixed-menu mass for a Mars stay and a one-way voyage by 38%; 202 days of Mars polyhydroxybutyrate synthesis with Cupriavidus necator can lower the shipped mass to three-dimensional print a 120 m3 six-person habitat by 85% and a few days of acetaminophen production with engineered Synechocystis sp. PCC 6803 can completely replenish expired or irradiated stocks of the pharmaceutical, thereby providing independence from unmanned resupply spacecraft that take up to 210 days to arrive. Analogous outcomes are included for lunar missions. Because of the benign assumptions involved, the results provide a glimpse of the intriguing potential of ‘space synthetic biology’, and help focus related efforts for immediate, near-term impact.


S1 Tabular Supporting Information
summarizes the mechanisms of action and the outputs produced by organisms that take in carbon dioxide, as detailed by [1]. Table S2 provides a similar summary of organisms that utilize and produce various nitrogen compounds; these organisms also play a role in the microbial nitrogen cycle [2][3][4][5][6]. Table S3 details the methane-oxygen propellant production mechanisms and mass requirements for each of the Mars scenarios described in [7]. The associated mass costs for two different production options of a similar fuel mix for a lunar module ascent are also listed in Table S3. Table S4 summarizes the Mars ISRU mass cost of strain KN-15 of Methanobacterium and strain Marburg of Methanobacterium thermoautotrophicum when a flow rate constraint of 3,000 g of water/h is imposed by incorporating a 6 kg electrolyzer that was recently validated in [8]. Table S5 provides a similar summary of the Moon ISRU mass cost, based on complete carbon dioxide extraction from lunar regolith that has been excavated by a 108 kg excavator (scaled up from [9]). Table S6 presents the unoptimized mass cost of nutrients and growth media, when extrapolated from the literature requirements, for Mars-based methane bioproduction over the anticipated manufacture period of 205 days. 3-phosphate, which is useful for terpenoid biosynthesis [10].
Nine ATP equivalents and six NADPHs are required to synthesize one glyceraldehyde-3phosphate molecule.
Acetyl-CoA, which must be converted to other intermediates of the carbon metabolism: pyruvate, phosphoenolpyruvate, oxaloacetate, and 2-oxoglutarate. It is a useful starting point for a number of high value chemicals and biofuels [11].
Reductive Citric Acid (Arnon-Buchanan) Cycle, which reverses the reactions of the oxidative citric acid cycle (Krebs cycle) and forms acetyl-CoA from two CO 2 s.
In Chlorobium, two ATP equivalents are required to form pyruvate, and three additional ATPs are required to convert it to triose phosphates.
Acetyl-CoA, which can be used to generate acetate or methane in the process of energy conservation, and also used for the assimilation of a variety of C 1 compounds like carbon monoxide, formaldehyde, methanol, methylamine, methylmercaptane, and methyl groups of aromatic Omethyl ethers/esters.

Reductive
Acetyl-CoA (Wood-Ljungdahl) Pathway, which requires strict anoxic conditions and has high energetic efficiency.
In methanogens, no additional ATP input is required, but in bacteria, an additional ATP equivalent is required.
Acetyl-CoA, which is used to synthesize pyruvate and then other central precursor molecules.
Mostly anaerobic autotrophic representatives of Thermoproteales and Desulfurococcales, and a facultative aerobe of the Desulfurococcales, Pyrolobus fumarii at very low O 2 concentrations.
which is anaerobic.
Five ATP equivalents are required to synthesize one pyruvate, and three additional ATP equivalents are required to synthesize one triose phosphate from pyruvate. Acetyl-CoA, which is used to produce succinyl-CoA, which is then oxidatively converted to oxaloacetate, pyruvate and phosphoenolpyruvate.
Nine ATP equivalents are required to generate three molecules of pyrophosphate, and three additional ATP equivalents are required to synthesize one triose phosphate from pyruvate. Glyoxylate, which is converted to the cellular building blocks in a second cycle. The bi-cycle allows coassimilation of fermentation products such as acetate, propionate, and succinate, or 3-hydroxypropionate, an intermediate in the metabolism of the osmoprotectant dimethylsulfoniopropionate.
The green non-sulfur phototrophs of the Chloroflexaceae family, which grow preferentially under photoheterotrophic conditions. The only autotrophic representative of this family found so far is Chloroflexus aurantiacus.
Seven ATP equivalents are required for pyruvate synthesis, and an additional three ATPs are required for triose phosphate.

S2 Alternative Propellant and Generation Cost
There exists another rocket fuel candidate with specific impulse and density-specific impulse properties that are comparable to a methane-oxygen combination: the currently uncommon blended monopropellant, nitrous oxide-hydrocarbon, where the hydrocarbon is propane, ethane, ethylene, acetylene, etc. Nitrous oxide (N 2 O) [19] has been analyzed for spacecraft propulsion as a monopropellant [20,21] and as a blended monopropellant, for instance with propane (abbreviated as NOP) [22,23], ammonia [24], and paraffin when mixed with oxygen (known as Nytrox) [25]. Nitrous oxide's appeal lies in its specific impulse properties (which are particularly high as a blended monopropellant), its lack of toxicity, and its relative ease of storage and handling. NASA has also studied nitrous oxide monopropellant rockets and their potential applications [26,27], and is interested in proprietary blends of nitrous oxide and hydrocarbons for potential use as a Mars ascent vehicle propellant. These blends are collectively known as Nitrous Oxide Fuel Blends X (NOFBX) [28][29][30][31], where each fuel blend has X replaced by a two-digit number that designates the hydrocarbon used and its mixture ratio. These hydrocarbons include ethane, ethylene and acetylene [32].
The mass of a nitrous oxide-hydrocarbon blended monopropellant that is required to lift a Mars ascent vehicle to orbit can be determined as follows. Since the amount of thrust that is required for ascent is ∆V = 5.625 km/s [7], and the specific impulse of the methane-oxygen propellant is I sp = 371 s (at a nozzle expansion ratio of 200 and a chamber pressure of 68 atm) [33] with a fuel mass m f = 31, 458 kg (Table S3), the ideal rocket equation, where g = 9.80665 m/s 2 suggests that the mass of the Mars ascent vehicle, m 1 , is about 8,518 kg. Using the ideal rocket equation again, along with a specific impulse of I sp = 325 s taken from that of NOFBX [31] (which has a theoretical maximum I sp of 345 s; NOP to vacuum has a comparable I sp of 312 s [23]), the mass of a nitrous oxide-hydrocarbon blended monopropellant that is required for ascent computes to about 41,237 kg. It is anticipated that this entire required mass will be launched from Earth if this blended monopropellant is selected for Mars ascent, although the "potential for in situ top-off" has also been identified [30]. It is possible to convert carbon dioxide to ethylene with the electrochemical approach in [34].

S3 Biological Nitrous Oxide-Hydrocarbon Generation Methods and Costs
There is a significant potential fuel benefit to producing nitrous oxide biologically on Mars, perhaps in combination with a hydrocarbon to achieve a mixture that has a high specific impulse. This benefit results from reducing the launch mass cost of complete nitrous oxide-hydrocarbon propellant delivery. As stated in Section 2.4, we seek a manufacture process that utilizes a feedstock of biologically-produced acetate, which can be made with hydrogen electrolyzed from water that is extracted from the Martian soil. Such a process exists: the biological manufacture of ethane and propane from acetate can proceed with the organisms and conditions described in [35]; alternatively, ethane can be electrolyzed from acetate; and lastly, the so-called Glycogen Accumulating Organisms (GAOs) [36] take up acetate anaerobically to produce nitrous oxide via denitrification at an efficiency of either 90% with a nitrate nitrogen (NO 3 -N) load, or 95% with a nitrite nitrogen (NO 2 -N) load. The latter efficiency is the highest efficiency known [37]. Other biological nitrous oxide production mechanisms are also available [38], and the conditions for producing nitrous oxide are well-studied [39][40][41][42][43][44][45][46][47]. There is evidence that the idea of combining biologically-produced nitrous oxide with a rocket engine is actively being researched, for instance to power a wastewater treatment plant [48]. At an 8.0:10.0 mixture ratio of nitrous oxide to ethane [32], about 18,328 kg of the blend is nitrous oxide, and the remainder 22,909 kg is ethane. This ratio is selected from among the possibilities in [32] because it maximizes the percentage content of nitrous oxide, which has a greater acetate conversion efficiency than ethane. That is, the Kolbe electrolysis stoichiometry of 2CH 3 COO − → CH 3 CH 3 + 2CO 2 + 2e − requires 2 mol of acetate for every 1 mol of ethane produced, while GAOs require 17.20 mol of acetate for every 14.78 mol of N 2 O produced with an NO 2 -N load [36], and are therefore more efficient at acetate conversion. A total of 100,589 kg of acetate is required to produce the necessary ethane and nitrous oxide for the blended monopropellant. However, even with two 2,000 L bioreactors (that, together with a soil processing plant, weigh about as much as the shipped mass for the "Bring Hydrogen" case in Table S3), only 29.6 kg of acetate can be produced per day at the experimental autotrophic production rate of 7.4 g/L/day; hence, 3,398 days are required to biomanufacture the requisite acetate. Because additional reactor(s) are also needed to accomplish nitrification upstream of GAO nitrous oxide production, the overall mass and time costs render nitrous oxide-ethane biomanufacture impractical for now.