Relying on Halomonas spp. in combination with acetate as substrate may allow very rapid production of the required bio-plastic, but substrate availability constraints are higher than for CH4 or CO2/H2. A terminal electron acceptor is required in all cases, which will almost certainly be O2. Supplying O2 safely without risking explosive gas mixtures, or wasting the precious resource, is again a question of reactor design and operation. Certain purple non-sulfur alphaproteobacteria and Rhodopseudomonas palustris also feature remarkable substrate flexibility and can produce PHAs . Bioplastic recovery and purification is a major challenge. To release the intracellular compound, an osmolysis process may be employed with the halophile . However, the transfer of cells into purified water and separation of the polyesters from the cell debris, potentially through several washing steps, may require substantial amounts of water. An alternative and/or complement to the common process for extraction of PHAs with halogenated organic solvents, is to use acetate or methanol as solvents . This is applicable independent of the organism and the inputs can be provided from other bio-manufactory modules. The high crystallinity of pure PHB makes it brittle and causes it to have a narrow melting range, resulting in warp during extrusion and 3D-printing. Such behavior places operational constraints on processing and hampers applications to precision manufacturing . Workarounds may be through additives, bio-composite synthesis, and copolymerization. However, this ultimately depends on what biology can provide . There is a need to advance space bio-platforms to produce more diverse PHAs through synthetic biology. ISM of bio-materials can reduce the mission cost, increase modularity,growing hydroponically and improve system recyclability compared to abiotic approaches. In an abiotic approach, plastics will be included in the payload, thereby penalizing up-mass at launch.
As with elements of FPS and ISRU, ISM increases flexibility and can create contingencies during surface operations, therefore reducing mission risk. The high modularity of independent plastic production, filament formation, and 3D-printing allows for a versatile process, at the cost of greater resources required for systems operations. Overall, this maximizes resource use and recyclability, by utilizing mission waste streams and byproducts for circular resource management.Biomanufacturing on Mars can be supported by flexible biocatalysts that extract resources from the environment and transform them into the complex products needed to sustain human life. The Martian atmosphere contains CO2 and N2 .It is very likely that the expensive and energy-intensive Sabatier plants for CH4 production will be available per Design Reference Architecture . While a HaberBosch plant could be set up for ammonia production, this is neither part of the current DRA nor exceptionally efficient. Thus, for a biomanufactory, we must have carbon fixation reactors to fix CO2 into feed stocks for nonmethanotrophs, and have nitrogen fixation reactors to fifix N2 to fulfill nitrogen requirements for non-diazotrophs. Trace elements and small-usage compounds can be transported from Earth, or in some cases extracted from the Martian regolith. In the case where power is provided from photocollection or photovoltaics, light energy will vary with location and season, and may be critical to power our bioreactors. Although photosynthetic organisms are attractive for FPS, a higher demand for carbon-rich feed stocks and other chemicals necessitates a more rapid and efficient CO2 fixation strategy. Physicochemical conversion is inefficient due to high temperature and pressure requirements. Microbial electrosynthesis , whereby reducing power is passed from abiotic electrodes to microbes to power CO2 reduction, can offer rapid and efficient CO2 fixation at ambient temperature and pressure . MES can produce a variety of chemicals including acetate , isobutanol , PHB , and sucrose , and therefore represents a flexible and highly promising ISRU platform technology . Biological N2-fifixation offers power- and resource-efficient ammonium production. Although photoautotrophic N2 fixation with, for example, purple non-sulfur bacteria, is possible, slow growth rates due to the high energetic demand of nitrogenase limit throughput .
Therefore, heterotrophic production with similar bacteria using acetate or sucrose as a feed stock sourced from electromicrobial CO2-fifixation represents the most promising production scheme, and additionally benefits from a high degree of process redundancy with heterotrophic bioplastic production. Regolith provides a significant inventory for trace elements and, when mixed with the substantial cellulosic biomass waste from FPS processes, can facilitate recycling organic matter into fertilizer to support crop growth. However, regolith use is hampered by widespread perchlorate , indicating that decontamination is necessary prior to enrichment or use. Dechlorination can be achieved via biological perchlorate reduction using one of many dissimilatory perchlorate reducing organisms . Efforts to reduce perchlorate biologically have been explored independently and in combination with a more wholistic biological platform . Such efforts to integrate synthetic biology into human exploration missions suggest that a number of approaches should be considered within a surface biomanufactory.A biomanufactory must be able to produce and utilize feed stocks along three axes as depicted in Figure 5: CO2-fifixation to supply a carbon and energy source for downstream heterotrophic organisms or to generate commodity chemicals directly, N2-fifixation to provide ammonium and nitrate for plants and non-diazotrophic microbes, and regolith decontamination and enrichment for soil-based agriculture and trace nutrient provision. ISRU inputs are sub-module and organism dependent, with all sub-modules requiring water and power. For the carbon fixation sub-module , CO2 is supplied as the carbon source, and electrons are supplied as H2 or directly via a cathode. Our proposed biocatalysts are the lithoautotrophic Cupriavidus necator for longer-chain carbon production [e.g., sucrose ] and the acetogen Sporomusa ovata for acetate production. C. necator is a promising chassis for metabolic engineering and scale-up , with S. ovata having one of the highest current consumptions for acetogens characterized to date . The fixed-carbon outputs of this sub-module are then used as inputs for the other ISRU sub-modules in addition to the ISM module . The inputs to the nitrogen fixation sub-module include fixed carbon feedstocks, N2, and light. The diazotrophic purple-non sulfur bacterium Rhodopseudomonas palustris is the proposed biocatalyst, as this bacterium is capable of anaerobic, light-driven N2 fixation utilizing acetate as the carbon source, and has a robust genetic system allowing for rapid manipulation . The output product is fixed nitrogen in the form of ammonium, which is used as a feed stock for the carbon-fixation sub-module of ISRU along with the FPS and ISM modules.
The inputs for the regolith enrichment sub-module include regolith, fixed carbon feedstocks, and N2. Azospira suillum is a possible biocatalyst of choice due to its dual use in perchlorate reduction and nitrogen fixation . Regolith enrichment outputs include soil for the FPS module , H2 that can be fed back into the carbon fixation sub-module and the ISM module, chlorine gas from perchlorate reduction, and waste products. Replicate ISRU bioreactors operating continuously in parallel with back-up operations lines can ensure a constant supply of the chemical feed stocks, commodity chemicals, and biomass for downstream processing in ISM and FPS operations. Integration of ISRU technologies with other biomanufactory elements, especially anaerobic digestion reactors,grow strawberries hydroponically may enable complete recyclability of raw materials, minimizing resource consumption and impact on the Martian environment .Waste stream processing to recycle essential elements will reduce material requirements in the biomanufactory. Typical feed stocks include inedible crop mass, human excreta, and other mission wastes. Space mission waste management traditionally focuses on water recovery and efficient waste storage through warm air drying and lyophilization . Mission trash can be incinerated to produce CO2, CO, and H2O . Pyrolysis, another abiotic technique, yields CO and H2 alongside CH4 . The Sabatier process converts CO2 and CO to CH4 by reacting with H2. An alternate thermal degradation reactor , operating under varying conditions that promote pyrolysis, gasification, or incineration, yields various liquid and gaseous products. The fact remains however, that abiotic carbon recycling is inefficient with respect to desired product CH4, and is highly energy-intensive. Microbes that recover resources from mission wastes are a viable option to facilitate loop closure. Aerobic composting produces CO2 and a nutrient-rich extract for plant and microbial growth . However, this process requires O2, which will likely be a limited resource. Hence, anaerobic digestion, a multi-step microbial process that can produce a suite of end products at lower temperature than abiotic techniques , is the most promising approach for a Mars biomanufactory to recycle streams for the ISM and FPS processes. Digestion products CH4 and volatile fatty acids can be substrates for polymer-producing microbes . Digestate, with nutrients of N, P, and K, can be ideal for plant and microbial growth , as shown in Figure 6. Additionally, a CH4 and CO2 mixture serves as a biogas energy source, and byproduct H2 is also an energy source . Because additional infrastructure and utilities are necessary for waste processing, the extent of loop closure that is obtainable from a treatment route must be analyzed to balance yield with its infrastructure and logistic costs. Anaerobic digestion performance is a function of the composition and pretreatment of input waste streams , as well as reaction strategies like batch or continuous, number of stages, and operation conditions such as organic loading rate, solids retention time, operating temperature, pH, toxic levels of inhibitors and trace metal requirements . Many of these process parameters exhibit trade-offs between product yield and necessary resources. For example, a higher waste loading reduces water demand, albeit at the cost of process efficiency. There is also a potential for multiple co-benefits of anaerobic digestion within the biomanufactory. Anaerobic biodegradation of nitrogen-rich protein feed stocks, for example, releases free NH3 by ammonification. While NH3 is toxic to anaerobic digestion and must thus be managed , it reacts with carbonic acid to produce bicarbonate buffer and ammonium, decreasing CO2 levels in the biogas and buffering against low pH.
The resulting digestate ammonium can serve as a fertilizer for crops and nutrient for microbial cultures.FPS and ISM waste as well as human waste are inputs for an anaerobic digester, with output recycled products supplementing the ISRU unit. Depending on the configuration of the waste streams from the biomanufactory and other mission elements, the operating conditions of the process can be varied to alter the efficiency and output profile. Open problems include the design and optimization of waste processing configurations and operations, and the identification of optimal end-product distributions based on a loop closure metric against mission production profiles, mission horizon, biomanufacturing feed stock needs, and the possible use of leftover products by other mission elements beyond the biomanufactory. A comparison with abiotic waste treatment strategies is also needed, checking power demand, risk, autonomy, and modularity benefits.Biomanufactory development must be done in concert with planned NASA missions that can provide critical opportunities to test subsystems and models necessary to evaluate efficacy and technology readiness levels . Figure 7 is our attempt to place critical elements of a biomanufactory road map into this context. We label critical mission stages using Reference Mission Architecture -S and RMA-L, which refer to Mars surface missions with short and long durations, respectively. Reliance on biotechnology can increase the risk of forward biological contamination . Beyond contamination, there are ethical issues that concern both the act of colonizing a new land and justifying the cost and benefits of a mission given needs of the many here on earth. Our road map begins with the call for an extensive and ongoing discussion of ethics . Planetary protection policies can provide answers or frameworks to address extant ethical questions surrounding deep-space exploration, especially on Mars . Critically, scientists and engineers developing these technologies cannot be separate or immune to such policy development.The upcoming lunar exploration missions, Artemis and Gateway , provide additional opportunities for integration with Earth-based biomanufactory development. Early support missions will provide valuable experience in cargo predeployment for crewed operations, and is likely to help shape logistics development for short-term as well as long-term Mars exploration missions when a biomanufactory can be deployed. Here we present a subset of Artemis efforts as they relate to mission elements with opportunities for testing and maturing biomanufacturing technology. Although ISRU technologies for the Moon and Mars will be sufficiently distinct due to different resource availabilities, crewed Artemis missions provide a testing ground for crewed Mars bio-process infrastructure. Later Artemis missions also provide a suitable environment to test modular, interlocked, scalable reactor design, as well as the design of compact molecular biology labs for DNA synthesis and transformation.