Designing the bioproduction of Martian rocket propellant via a biotechnology-enabled in situ resource utilization strategy

Challenges in producing rocket propellant on Mars

While chemical bioproduction unit operations are similar on Mars and on Earth, special considerations must be made for implementation on Mars. First, the average Martian surface temperature is −55 °C compared to 15 °C on Earth³. While temperatures as low as −60 °C do not affect microbial viability, microbes do not grow at those temperatures. Our analysis assumes a 25 °C process temperature. Although not optimal for microbial growth (cyanobacteria: 35 °C; E. coli: 37 °C), it allows reasonable growth rates. Temperature control is a crucial assumption for our process design, though it remains an unsolved challenge. It could be achieved via reactor jacketing, use of a transparent dome-like structure to trap radiant solar heat akin to a greenhouse¹², temperature control systems, or some combination thereof¹³. Second, the thinner Martian atmosphere results in higher ionizing and gamma radiation levels reaching the surface as compared to Earth. Fortunately, many cyanobacteria, including Arthrospira platensis, and E. coli are resistant to Martian radiation levels, maintaining viability for centuries^14,15. Of more concern is the level of UV radiation that reaches the Martian surface, which poses a risk for genetic mutations. Thus, bags for cyanobacteria growth and/or the greenhouse dome should be made of a UV reflecting material while allowing transmission of photosynthetically active radiation (PAR). For example, flexible polymers, including polyvinyl alcohol or poly(dimethyl)siloxane doped with UV absorbent nanoparticles (ZrO₂, SiO₂¹⁶) or chemicals (sepia eumelanin¹⁷). Importantly, the PAR intensity that reaches the Martian surface is 57% lower than on Earth due to the increased distance from the sun³. This decrease in photon flux will result in a decreased cyanobacteria photosynthetic growth rate, thus biomass productivity is modeled using light as the growth-limiting factor. Further, frequent Martian dust storms can disrupt photon flux, and reduce photosynthetic rates. Prior to the implementation of a photosynthetically driven process, detailed analysis of the mission landing site can help predict the frequency and severity of the dust storms to inform changes to the model¹⁸. Third, biological processes require water. Although recent reports suggest that Martian water exists in sufficient amounts for the proposed bio-ISRU, harvested water may need to be pretreated to reduce salt content to enable microbial growth¹⁹. Fourth, microbial growth requires nitrogen, phosphorus, and trace metals in addition to carbon. While some trace metals (e.g. Mg, Ca, K and Na) could be obtained from Mars in a bioavailable form via electrochemistry, to mitigate uncertainty and the presence of perchlorates in Martian regolith²⁰, the majority of these nutrients will need to be shipped from Earth²¹. Of note, trace metals fed to the cyanobacteria will carry through to the hydrolysate fed to E. coli²². To complete the cycle, E. coli biomass can be fed back to the cyanobacteria after rocket propellant separation to effectively recycle the trace metals and nitrogen²³. Fifth, Mars has a nitrogen (N₂) partial pressure of 0.189 hPa³, 10-fold lower than that required for cyanobacterial N₂ fixation²⁴. In our analysis, we have not considered N₂ fixation, and use a cyanobacteria, A. platensis, that does not fix N₂. Instead, we propose shipping diammonium phosphate ((NH₄)₂PO₄) and ammonia (NH₃) from Earth. While implementation of N₂ fixing cyanobacteria, such as Anabaena cylindrica, would reduce nutrient payload mass, it would likely require atmospheric N₂ concentration or cyanobacteria engineering to enable cyanobacterial N₂ fixation³. Finally, the entire infrastructure, including nuclear power sources, must be shipped from Earth. This has direct implications for reactor design and whole process design as payload mass directly impacts mission cost. To account for these challenges, the proposed bio-ISRU analyzes water use, power requirements, and total infrastructure payload mass as key process metrics.

Martian rocket propellant design

Based on energy density, phase behavior, and biological reachability, we propose C₃–C₄ hydrocarbons with two oxygen atoms (diols) as potential Martian rocket propellants. The oxygen content within the propellant structure reduces the amount of LOX needed for combustion, i.e. the stoichiometric O₂/propellant mixture ratio (Fig. 2a). A lower ratio is advantageous on Mars due to its low atmospheric O₂ content, making oxygen-containing compounds (e.g. alcohols, diols) preferable for Martian application. Compared to alcohols and C₁–C₂ diols, C₃–C₄ diols have the required heating value (LHV, 20–25 MJ/kg) and specific impulse (I_sp, ~400 s) to propel a human Mars Ascent Vehicle (MAV) with lower oxidizer need (Fig. 2b). Triols require less oxidizer, but their LHV is too low to propel a human MAV. Diols require less than half the LOX mass per mass of fuel when compared to methane. Additionally, C₃–C₄ diols have appropriate boiling and melting points to remain liquid or solid over Martian temperature (−153 to 20 °C), and thus have lower volumetric and energetic storage requirements (Fig. 2c). Therefore, the energy needed to compress, or refrigerate, gaseous methane to a liquid for storage and propulsion is sidestepped. Finally, with respect to bioreachability, several natural and engineered microbes produce C₃–C₄ diols in high titers and yields²⁵, whereas C5 diols production is low²⁶, and >C5 diols production has been elusive. In particular, we consider 1,2-propanediol (1,2-PDO) (C₃)²⁷, 1,3-butanediol (1,3-BDO) (C₄)²⁸, and 2,3-BDO (C₄)²⁹ as Martian rocket propellants, all of which can be biologically synthesized today (Fig. 2d).

2,3-BDO as a Mars-specific rocket propellant

To assess the feasibility of each of the three diols, we calculate the E. coli maximum theoretical yield from glucose using the Constraint-Based Reconstruction and Analysis (COBRA) toolbox³⁰ and the iML1515 E. coli genome-scale model³¹. Theoretical yields for 1,2-PDO, 1,3-BDO, and 2,3-BDO are 0.615, 0.543, and 0.538 g diol/g glucose, respectively. Although 1,2-PDO and 1,3-BDO have the highest theoretical yields, the yields are limited by NADH requirements. While 1 mol of 2,3-BDO generates one mol of NADH, one mole of 1,2-PDO or one mol of 1,3-BDO results in the net consumption of one mol of NADH (Fig. 2d). NADH is a redox balance cofactor required by many cellular processes, and higher NADH demand often results in reduced cell growth and carbon flux through the desired pathway. Furthermore, NADH generation relies on O₂ during glycolysis and thus can be limited by poor O₂ transport at an industrial scale.

Among the three diols, 2,3-BDO is at the highest technology readiness level, produced in E. coli at high titer (73.8 g/L), yield (0.432 g/g glucose), and productivity (1.17 g/L/h)²⁹. That is, 2,3-BDO is produced at 80% of the theoretical maximum. The experimental yield of 2,3-BDO is 2.4 times higher than that of 1,2-PDO (0.178 g/g glucose)²⁷ and 3.9 times higher than that of 1,3-BDO (0.11 g/g glucose²⁸). For these reasons we focus our analysis on 2,3-BDO, which will lead to a smaller cyanobacteria farm footprint, microbial fermenter size, and fewer fermentation byproducts, streamlining 2,3-BDO purification. Additionally, separation processes for 2,3-BDO from fermentation broth are well established³² whereas separation processes for 1,2-PDO and 1,3-BDO are still being explored.

To set the 2,3-BDO mass production target for a 500-day human surface visit to Mars mission, we applied the ideal rocket equation using the theoretical I_sp value and the oxygen fuel ratios for 2,3-BDO (Eq. (4)). As there are no experimental I_sp values for diols, we calculated the theoretical I_sp of 2,3-BDO to be 420 s. A total of 8.4 tons of 2,3-BDO and 16.5 tons of LOX is required to power a MAV. The total required propellant plus LOX is 18% lower when using 2,3-BDO instead of methane (Table 1). Of note, the theoretical I_sp values for methane and 2,3-BDO are in the same range (~400 s). Importantly, as the literature value for CH₄ I_sp is ~369 s², it is possible that 2,3-BDO has a lower experimental value. Therefore, we have increased the 2,3-BDO mass production target estimate to 10 tons, which assumes a very conservative 2,3-BDO I_sp of 383 s. The presented process metrics are based on producing 10 tons of 2,3-BDO, requiring 19.6 tons of LOX.

Bio-ISRU for 2,3-BDO process design overview

The bio-ISRU for 2,3-BDO is composed of four modules: (1) cyanobacteria cultivation using Martian CO₂ and sunlight, (2) cyanobacteria biomass preprocessing, consisting of biomass concentration and enzymatic digestion to release sugars and trace nutrients for use by the heterotrophic microbe, (3) microbial fermentation to upgrade the sugars into 2,3-BDO, and (4) 2,3-BDO extraction and separation to ~95% purity from the microbial broth (Fig. 3).

Cyanobacteria growth

To fix Martian CO₂, we will use A. platensis (i.e. spirulina), which under nitrogen limitation maximizes glycogen production, up to 60% of dry cell weight (DCW)⁹. A. platensis grows well at 0.38 hPa of CO₂, thus the 6.67 hPa CO₂ present on Mars will enable robust A. platensis growth. Indeed, higher CO₂ levels improve cyanobacteria growth³³. Beyond CO₂ concentration, a process concern is the negative effect of the <10 hPa total atmospheric pressure on Mars versus 1013 hPa on Earth. Elevated CO₂ levels should help to mitigate this effect, with 50 hPa of CO₂ in a 100% CO₂ atmosphere showing uninhibited growth³⁴. However, the Martian atmosphere still has a 5-fold lower pressure than previously studied. Thus, CO₂ pressurization may be required to achieve optimal growth, and further study at these low pressures is needed to determine the full effects and validate our assumption that growth is indeed light limited and not CO₂ limited.

Cyanobacteria culturing requires the addition of nitrogen and phosphorus, as well as trace elements (Ca, Cu, Fe, K, Mg, Mn, Na, and Zn)³⁵. To reduce mission risk, all the required nitrogen and phosphorus to produce enough cyanobacterial biomass to reach the 15 tons of 2,3-BDO mass target will be shipped from Earth. Required nitrogen and phosphorus mass were calculated based on the elemental composition of cyanobacteria (C_4.5H_8.2N_0.129O_2.219S_0.006P_0.007 ref. ³⁶). Nutrients will be provided in 20% excess to enable adequate uptake concentrations in the growth media³⁷. Under these requirements 0.75 tons of (NH₄)₂PO₄, and 1.20 tons of anhydrous NH₃ are included in payload mass calculations. Alternative sources of nitrogen and phosphorus include fixing N₂ in situ via sunlight-driven chemical catalysis³⁸ or harvesting phosphorus from Martian regolith³⁹. However, regolith harvesting requires additional time and infrastructure. Further, the harvested regolith contains harmful perchlorates²⁰, and the nutrients may not be readily dissolvable for microorganism consumption. Trace elements are provided based on their previously reported minimum requirements to support unhindered cyanobacterial growth and are recycled through the process³⁵.

Cyanobacteria cultivation

While Martian water is sufficient to sustain the bio-ISRU, harvesting and keeping it in liquid form will require 1–2 kW per ton of water². With this in mind, we explored two cyanobacteria cultivation methods: water-intensive suspended growth and less water-intensive biofilm growth. We did not explore an open pond strategy due to contamination of the Martian environment and water evaporation concerns. (Fig. 4a–c).

For the suspended cyanobacteria growth model, we use low density polyethylene (LDPE) photobioreactors (PBRs)⁴⁰, which lower contamination threats to the Martian environment, have low evaporation rates, allow highly controllable growth rates through mixing, enable CO₂ delivery via gas bubbling, and can be shipped in rolls to be inflated upon arrival. On Earth, suspended growth is used for the pilot- and demonstration-scale production of cyanobacterial biomass for food and biodiesel³⁶. Suspended growth biomass productivity was modeled using Monod growth kinetics based on lab- and pilot-scale growth of A. platensis on Earth⁴¹ with key variations to take into account Martian conditions. The model assumes that on Mars, as it is the case on Earth⁴¹, cyanobacteria growth is limited by light penetration rather than CO₂, nitrogen, or phosphorus sources. Key model variations to account for Martian conditions include: (1) Photon flux at the reactor surface (E₀), which takes into account the 57% lower photon flux on Mars than on Earth³, (2) Reactor diameter (D), which was optimized to achieve maximal light penetration on Mars, by spacing the 4.5 cm wide PBR units 1 m apart³⁶, and (3) photon flux (E_K) and cellular respiration rate (R_o) at 25 °C. Hanging bag PBRs rather than a horizontal system was modeled to increase the ratio of light absorption area, i.e. PBR surface area to land area (F ratio: 1.84; horizontal system F ratio: 1). The F ratio could be increased to 15 by reducing the PBR spacing or increasing the PBR height, at the expense of reduced light intensity at the PBR surface due to shading from surrounding bags⁴². As on Earth, biomass concentration is modeled at 1 g/L to allow for maximum light penetration into the culture. All other parameters were taken from literature⁴¹, resulting in a modeled biomass productivity of 6.54 g/m²/day. This biomass productivity is given in terms of land area footprint of the cyanobacteria farm to enable the use of published power and mass correlations³⁶. The cyanobacterial cultivation will be run as a continuous reactor where the calculated biomass productivity was used to determine the rate of constant biomass harvest to maintain the culture at the desired concentration of 1 g/L. The calculated productivity requires processing of 0.105 tons (105 kg) of biomass per day to reach our production goal and timeline. On Earth, cyanobacteria have growth productivity between 20 and 30 g/m²/day at temperatures from 20 to 30 °C for both suspended and biofilm growth^36,43.

Cyanobacteria biofilm growth holds promise in improved gas delivery, light penetration, reduced water usage, and reduced energy demand due to the elimination of culture mixing. Biofilm growth has been performed at pilot but not industrial scale⁴³. Due to the density of the biofilm and reduced water flow⁴², it has a lower contamination risk than suspended growth. Indeed, large-scale, outdoor, biofilm cultivation experiments have avoided contamination without implementation of special measures^43,44,45. If this scenario were not extended to our system, contamination risk of the open biofilm system could be mitigated by using a cyanobacteria extremophile, such as the salt-tolerant Tolypothrix sp.⁴⁶. Cyanobacteria biofilm growth was modeled using the same growth model used for suspended growth. Biofilm will be grown on a hydrophilic, porous, growth substrate⁴⁷, resembling a cotton towel⁴³, 0.3 mm thick to match the thickness of the LDPE PBR bags⁴⁰. Only light penetration (k), reactor diameter (D), i.e. film thickness, and areal chlorophyll concentration (C_o), which depend on the increased biomass concentration in biofilm growth (7.5 g/m² versus 1 g/L) were changed. Other growth parameters, including photosynthetic efficiency, respiration rate, and optimal photon flux, were assumed to be species-dependent, and thus independent of growth method and were used unchanged. For direct comparison, the 1 m spacing and F ratio of 1.84 were also applied to biofilm growth, resulting in a modeled productivity of 6.64 g/m²/day, similar to the value modeled for suspended growth.

Suspended and biofilm growth result in similar biomass productivities (~6.6 g/m²/day); however, biofilm growth uses 90% less water (6.33 × 10⁴ L) when compared to suspended growth (6.64 × 10⁵ L)⁴⁵ to produce 52.4 tons of cyanobacteria biomass over 500 sols (Fig. 4d). Based on water harvesting via soil evaporation², biofilm growth results in energy savings of up to 1200 kW. Further, the biofilm system payload mass is 14% less than that of the suspended system. The reduction is due to the mass of the suspended growth frame needed to hold the PBR bags, even considering the reduced system weight under Martian gravity (Fig. 4a, b). Of note, the cotton material used as the biofilm growth substrate accounts for 82% of the biofilm growth method mass (Fig. 4a).

Cyanobacteria biomass concentration

Cross-flow filtration was modeled to concentrate cyanobacteria due to its lower payload mass when compared to centrifugation equipment, and more rapid and efficient separation than settling tanks⁴⁸. A hydrophilic and neutrally charged membrane concentrates the biomass from 1 g/L in the PBR to 20 g/L for input into the enzymatic digester. The membrane is modeled to have a 40 kDa cut-off^49,50 with long-term membrane flux approaching 40 L/m²/h and stability of 6 weeks⁵¹. Biomass collection from biofilm growth was set at a continuous 20 g/L, eliminating the need for a concentration unit operation for biofilm cultivation mode⁹. Biofilm biomass will be harvested using low-energy mechanical scraping⁵². While continuous harvesting of biofilm biomass has not been implemented at pilot or industrial scale, approaches for scale-up include rotation of planar⁵³ or circular⁵⁴ growth substrate, or use of a high-velocity water flow. A further experimental investigation is required to fully model power and mass requirements for biofilm biomass harvesting, which will be crucial for Martian implementation.

Cyanobacteria biomass enzymatic digestion

Lysozyme, α-amylase, and glucoamylase digestion releases between 0.3 and 0.45 g of glucose/g of cyanobacterial biomass over 24 and 48 h, respectively⁹. A shorter enzymatic digester residence time requires a smaller digester volume and reduced water usage; however, it also requires more cyanobacteria biomass to achieve the desired sugar output, thus a larger cyanobacteria farm. The goal is to convert the 52.4 tons of cyanobacteria biomass to the 23.2 tons of glucose required to produce 10 tons of 2,3-BDO. Increasing biomass digestion from 24 to 48 h reduces cyanobacteria growth system biomass requirement by ~34%, resulting in a ~34% reduction in algae water requirement, and a 33% reduction in payload mass of the cyanobacterial growth system for both biofilm and suspended growth systems (Fig. 5). Taken together, decreasing the size of the cyanobacterial farm is beneficial, even at the expense of increased size of the biomass digester.

Microbial production of rocket propellant on Mars

E. coli was modeled to convert cyanobacterial glucose to 2,3-BDO, as hydrolyzed cyanobacteria have been used as the sole feedstock to drive E. coli growth⁵⁵ and chemical production¹¹. As Mars is devoid of life, using a non-pathogenic microbe reduces environmental contamination risks, while process implementation could take advantage of the high levels of germicidal UV radiation that reach the surface of Mars to sterilize process components prior to inoculation. E. coli has been engineered to produce 2,3-BDO, and for process design we used steady-state productivity of 1.17 g 2,3-BDO/L/h and a yield of 0.432 g 2,3-BDO/g glucose, matching the E. coli state-of-the-art²⁹. Of note, O₂ is important for NADH regeneration during 2,3-BDO production²⁹. Cyanobacterial cultivation produces excess O₂ to meet the ~12 tons requirement of the fermentation unit over the course of 500 sols (500 Mars days) in addition to the O₂ required for rocket propellant (19.6 tons, Table 1).

Rocket propellant extraction and separation

We modeled liquid–liquid extraction (LLE) followed by membrane-based separation to purify 2,3-BDO from the fermentation broth. In the LLE unit, hydrophilic 2,3-BDO is extracted from fermentation broth using butanol, a previously validated solvent⁵⁶, which does not theoretically reduce fuel performance if not fully removed from 2,3-BDO judging by its similar heating value (36 MJ/kg) and I_sp (439 s). LLE was modeled using the Phasepy python package⁵⁷ with binary interaction parameters regressed in Aspen Plus (AspenTech). To concentrate and dewater 2,3-BDO, a polydimethylsiloxane/polyvinyledenefluoride membrane was modeled using flux differential equations⁵⁶. Our model indicated that 0.5 m² of membrane area is required to reach 95% 2,3-BDO purity. Thus, the membrane separations unit accounts for <1% of total payload mass. As butanol must be shipped from Earth, the process includes a solvent recycle stream to minimize the overall solvent input into the system. To reduce water usage, the water-rich stream coming out of the LLE unit will be purified in the water recycling unit operation and fed back to the E. coli fermenter (Fig. 3). An important consideration will be the amount of butanol lost in this stream, as lost butanol will increase payload mass and introduce toxicity risks to E. coli. Our analysis shows that the butanol concentration in water outlet stream will be ~0.01%. Nevertheless, careful consideration must be taken to prevent butanol accumulation to not run into E. coli toxicity. To mitigate this risk, E. coli could be engineered for improved butanol tolerance⁵⁸.

Water recycling

Martian water will be sufficient to support a Martian colony as well as the bio-ISRU of 2,3-BDO¹⁹. However, maximization of process efficiency and integration requires wastewater management and recycling. Implementation of reverse osmosis for wastewater purification will prevent the buildup of salts and proteins over the course of the process. The concentrated waste stream can be reintroduced to the process in order to recycle trace nutrients, while the remainder of the rejected waste can be utilized in other ISRU applications, such as for fertilizer⁵⁹ or for astronaut nutrition⁶⁰, as the bio-ISRU does not produce toxic compounds. For this application, a reverse osmosis membrane with long-term stability and a lightweight material was modeled. Specifically, a thin-film composite membrane, 200 µM thick with water flux of 25 L/m²/h⁶¹. Based on the water-rich stream flow rate of 3.64 L/min coming out of the LLE unit, a membrane area of 8.73 m² is required, bringing the mass of the required water treatment unit to 0.008 tons, 0.03% of total payload mass. Importantly, this takes into account the mass of the membrane and housing, but not any required pumps and control systems.

Bio-ISRU full process analysis

Holistic analysis of the process was performed to determine water requirement, power use, and total payload mass for the bio-ISRU for 2,3-BDO production (Fig. 6). Cyanobacteria biofilm growth requires 89% less water, 65% less power, and 15% less payload mass than suspended growth. The stark difference in power use (17.64 vs. 50.62 kW) comes from the elimination of mixing during biofilm growth (26% of the power usage) and the removal of the biomass concentration unit operation (41% of the power usage) even with reduced power demand due to reduced Martian gravity. If the energy for water harvesting is included in the calculations, biofilm growth requires 8.5-fold less power than suspended growth.

When compared to the DRA 5.0 O₂ only strategy, the bio-ISRU for 2,3-BDO production using cyanobacteria biofilm growth requires 2.8-fold greater payload mass from Earth, yet has a 32% lower power requirement (Fig. 6, Table 1). Almost 80% of the biofilm bio-ISRU payload mass comes from cyanobacteria cultivation, due to the large mass of glucose required to feed E. coli to produce the 10 tons of 2,3-BDO. Most of the biofilm growth payload mass comes from the cotton material (82%) (Fig. 4b). Of note, the bio-ISRU evolves O₂, which is sufficient for microbial 2,3-BDO production (~12 tons) and rocket launch (19.6 tons), leaving 43.81 tons of excess O₂ over 500 sols_. The excess O₂ can be used for subsequent aircraft launches or crew life support systems. For context, a crew of 6 consumes 2 tons of O₂ for life support (e.g. breathing) over the course of 500 sols².

Bio-ISRU optimization overview

Although the bio-ISRU produces sufficient oxygen for rocket launch and even excess oxygen for use in other applications, it is not competitive to the proposed DRA 5.0 O₂ only strategy in terms of payload mass (13.42 tons heavier). We put forth a series of model-guided biological and materials improvements that should ultimately render the bio-ISRU competitive to the DRA 5.0 O₂ only strategy in terms of payload mass with a significantly lower power requirement, while still emitting more than 20 tons of excess oxygen.

Bio-ISRU biological optimization

To determine engineering targets for improving the bio-ISRU, we focused on three biological strategies: (1) increasing cyanobacterial biomass productivity, (2) increasing glucose yield in the enzyme digester, and (3) increasing 2,3-BDO yield in the fermenter.

First, we focused on improving cyanobacterial biomass productivity (6.64 g/m²/day), as 80% of the payload mass comes from the cyanobacterial growth system. Using process modeling, we analyzed water, power, and payload mass requirements for cyanobacterial growth productivities between 5 and 15 g/m²/day (Fig. 7a). Improving productivity to 13.28 g/m²/day reduces water usage by 43% and payload mass by 34%. As expected, it only reduced the power consumption by 3%, as the enzyme digester dominates power consumption, and is relatively unchanged by cyanobacterial productivity. Increasing cyanobacterial biomass productivity could be achieved by (a) improving light input by supplementing sunlight with artificial lighting, (b) improving cyanobacteria photosynthetic efficiency, e.g. by utilizing the wider range of the light spectrum that reaches Mars, or (c) increasing the growth rate of cyanobacteria by adapting A. platensis to grow faster at 25 °C (35 °C optimal) or using a cyanobacterial species with faster growth rate at low temperatures. If artificial lighting is used to double the photon flux, productivity is increased by 55% to 10.27 g/m²/day. However, this adds 190 tons of payload mass and 1150 kW of power due to the large land footprint of the cyanobacterial growth operation based on calculations for solar-powered artificial lighting⁶². Integration of organic light emitting diodes (LED)⁶³ powered by photovoltaics⁶⁴ into the dome surrounding the process could provide a lightweight, future strategy for increasing light flux to the cyanobacteria, though the technology requires further developments. Improving cyanobacteria photosynthetic efficiency (α, mol of carbon fixed/mol of photons) may be a more viable strategy. The theoretical maximum for α is 0.125, i.e. 1 mol of carbon fixed/8 mol of photons. In the model, α is set to 0.061, 49% of the theoretical maximum. To double biomass productivity, α would need to reach 0.117, or 93% of theoretical maximum. Although improving the photosynthetic efficiency of autotrophic organisms has been difficult⁶⁵, promising strategies, such as RuBisCo overexpression and reducing the size of the light harvesting antennae, have led to improvements in cyanobacterial photosynthesis rate and biomass productivity, respectively⁶⁶.

Second, we looked at increasing the enzyme loading or implementation of undigested biomass recycle stream to increase the glucose yield in the enzyme digester. As modeled, the enzymatic digestion has a 45% yield by weight of total biomass. If the enzymatic digester reached 60 wt% of total digested biomass (Fig. 7a), the payload mass, water requirement, and power use of the entire process would be reduced by ~25%. Although we focus on sugar release, cyanobacteria can accumulate fatty acids to 40–45% of DCW⁶⁷ and in the future, those fatty acids could be extracted and fed to E. coli.

Third, the E. coli yield of 2,3-BDO from glucose has more limited room for improvement as it is already at 80% of the theoretical maximum (0.432 g/g). Even so, reaching 95% of theoretical yield (0.51 g/g) would reduce water, power and mass requirements by ~16% for all metrics (Fig. 7a). Collectively, increasing cyanobacterial biomass productivity to 13.28 g/m²/day, the enzymatic digester yield to 60 wt%, and fermentation yield to 0.51 g of 2,3-BDO/g of glucose, reduces the payload mass of the bio-ISRU to 9.17 tons, just 22% higher than the DRA 5.0 O₂ only strategy. The 56% payload mass reduction comes from a 69% reduction in cyanobacterial farm size.

Bio-ISRU materials optimization

To further reduce the bio-ISRU payload mass, we evaluated alternative materials for the cyanobacterial biofilm growth substrate, the microbial fermenter, and other units of operation. The biofilm growth substrate accounts for 64% of the total payload mass of the original bio-ISRU model. Replacing the cotton-like material with a similar density to the LDPE used for the PBR bags reduces the original bio-ISRU payload mass by 23% to 16.15 tons. In the original bio-ISRU model, steel was chosen as the material for the microbial fermenter and other unit operations based on Earth requirements, including ease of sterilization and structural stability. On Mars, the lower gravity should allow for better performance of lighter weight materials. For example, using fermenters made of aluminum or high-density polyethylene (HDPE) would further reduce process mass to 14.98 and 14.60 tons, respectively, if implemented along with the lighter biofilm growth substrate (Fig. 7b).

Fully optimized Bio-ISRU process analysis

By incorporating the proposed biological and materials improvements, the optimized bio-ISRU will produce 10 tons of 2,3-BDO over 500 Sols, evolving sufficient oxygen for fermentation and rocket launch, and leaving 20.35 tons to support other aspects of Martian exploration. The optimized bio-ISRU requires 38% less power and has a 13% lower payload mass than the DRA 5.0 O₂ only strategy (Fig. 7c). Further, subsequent bio-ISRU missions will require only 3.73 tons of resupply payload mass, which is less than the needed methane resupply for the DRA 5.0 O₂ only strategy (6.5 tons). Under Earth conditions, the lifetime of most process components is 10–20 years⁶⁸, and while Martian conditions will likely shorten this lifetime, most process components can be reused over multiple missions. If nutrients and butanol solvent can be successfully recovered, the only resupply needed will be the biofilm growth substrate, which has a lifetime of 1–2 years⁴⁰, and the digestion enzymes, which, as modeled, are only shipped for a single 500 Sol mission.