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Without recovery, capture of N must come directly and entirely from the atmosphere. On Earth, this process is split between microbial production and Haber-Bosch industrial production. The energy and mass requirements to fix N using the Haber-Bosch process are likely prohibitively expensive. A more efficient approach, especially on a small scale, is to use bacterial fixation through the enzyme nitrogenase. This can be accomplished by fixing N from the atmosphere of an enclosed life support system or from the Martian atmosphere, although the lower partial pressure of N₂ must be considered (Klingler et al., 1989; Mahaffy et al., 2013).

The N from protein degradation is converted into urea, which is the dominant N form in the waste stream. Urea comprises over one half of the total dissolved solids and 75–90% of the discarded N in human urine (Rose et al., 2015; Simha and Ganesapillai, 2017). The remaining N is bound in other N-containing compounds such as creatinine and ammonium salts. Urea excretion from the human body varies based on diet, with the average adult human producing about 500 L of urine per year, equating to about 20 g urea CM⁻¹ d⁻¹ (Lind et al., 2001; Amtul et al., 2002). Urea can be fed directly to plants in the root zone, but it is taken up very slowly. Unaided urea hydrolysis is slow, but will eventually proceed due to bacterial contamination (Elliot, 1986). Supplementing the growing media with small amounts of urease has the potential to significantly increase the rate of hydrolysis (Figure 3).

Urine exits the body as a sterile liquid (Simha, 2013). Pathogens from unhealthy humans and fecal contamination from incomplete source separation compromise sterility (Höglund et al., 2002; Santos et al., 2004). Sterilization involves processes to eliminate pathogens from the waste stream to reduce contamination of downstream products. Urine may be sterilized by heat, pressure, or ultraviolet light, but these methods require energy. Alternatively, storage at low pH for weeks to months can inactivate viruses, inhibit pathogens, combat the rise in pH due to urea hydrolysis, and reduce ammonia volatilization (Hellström et al., 1999; Patel et al., 2020). If acidic conditions are maintained, no N is lost during the sterilization process.

The volume of urine must be condensed following sterilization to both recycle water and collect the valuable metabolites. Evaporation energy requirements to remove water can be minimized by vapor-compression distillation to generate additional heat for evaporation and to recover 85% of the energy used compared to standard evaporation (Wood, 1982). Urea can also be separated from the liquid phase through freeze/thaw cycles. When urine begins to freeze, the ice that forms has minimal solutes. These solutes, such as urea, remain dissolved in the unfrozen portion. Removal of the distilled frozen water effectively concentrates the dissolved solids while concurrently purifying water for other uses. Lind et al. (2001) found that freezing and melting urine samples could concentrate more than 80% of the original N into a much lower volume. Reverse osmosis may also be used to remove water from urine and concentrate solutes. Ammonia recovery during reverse osmosis has been found to be about 70%, but higher recoveries may be possible if acidification is used to prevent volatilization (Thörneby et al., 1999). An 80% recovery rate means 16 g urea CM⁻¹ d⁻¹ can be recovered as ammonium following volume reduction.

Stabilization of condensed urine is essential to minimize volatilization of ammonium to ammonia gas. Released ammonia causes unpleasant odors and becomes difficult to capture in the gas phase. When urea is hydrolyzed in water, ammonium, CO₂, and hydroxide are released, causing the pH of the solution to increase. Alkaline pH causes ammonium to de-protonate and volatilize to ammonia gas. Acidification is therefore necessary to both maintain neutral pH for optimal microbial activity in an aqueous system and prevent the loss of ammonia gas as shown in Figure 4 (Hellström et al., 1999; Maurer et al., 2006). CO₂ must be vented or additionally recycled during this step to prevent build-up to toxic levels.

Although energy consumption is high for recovery, volume reduction of urine can reduce the energy requirement by 58% (Maurer et al., 2003). Tun et al. (2016) studied filters for reducing water volume and concentrating ammonium from urine using membrane distillation following acidification. They found a combination PTFE/PP filter reduced the amount of ammonium transferred in relation to water to as low as 6.91 × 10^–5 g-N/g-H₂O. Some of these processes have been implemented in long-term studies to quantify N recovery from urine for simulated space missions. Beler-Baykal et al. (2011) utilized the natural conversion of urea to ammonium over six weeks to transform the N in urine into a bioavailable form before passing it through a column of clinoptilolite for separation to recover about 86% of the original N. An additional benefit of the use of clinoptilolite was the elimination of salinity from the waste stream, a valuable attribute to eliminate salt stress in plants. Simha et al. (2018) used a regenerative activated carbon column to achieve 90% recovery of urea from urine concentrate after multiple passes through their system. Fu et al. (2016) studied a 105 days crewed simulated closed ecosystem and were able to recover 20.5% of N from urine using simple distillation. However, this recovered N was stored instead of fed to plants, which were fertilized with pre-formed plant minerals stored from the beginning of the study.

Recent work has used synthetic microbial communities to complete nitrification directly in fresh diluted urine. This method bypasses the separate aerobic digestion step where nitrification may normally be present. Though previous stabilization steps still apply, Christiaens et al. (2019) were able to achieve N production rates of 29 mg nitrate L⁻¹ d⁻¹ in 10% diluted urine even under high sodium pressures. Udert and Wächter (2012) developed a system combining distillation with nitrification to completely recover nutrients from urine. Controlling pH for optimal nitrification led to 97% N recovery in the form of ammonium nitrate. Feng et al. (2008) showed up to 95% of the original ammonium could be converted to nitrate under high dissolved oxygen levels with rigorous solution pH control. These processes present alternative methods that may save space and resources in comparison to separate dedicated bioreactors.

Significant nitrification occurs in all agricultural soils and there is great potential to enhance nitrification in the root zone in a regenerative system. Controlled watering would optimize aeration, and the process could be supplemented by adding nitrifying organisms.

We are currently optimizing small scale bioreactors to convert the urea in urine into ammonium. A peristaltic pump is used to move solution from a bottom reservoir to the top of the column where it flows through perlite under continuous recirculation (Figure 5). Initial results show that ammonia volatilization is minimized when the pH is controlled near neutral (Figure 6). Our diagram assumes that we can achieve 100% recovery of ammonium from urine (Figure 2).

Aerobic digestion utilizes aerobic microorganisms to break down proteins and oxidizes N to nitrate while releasing CO₂ and water. The digestion process requires constant agitation and aeration to maintain aerobic conditions and promote microbial degradation of waste (Layden et al., 2007). Aerobic digestion is commonly used in waste water treatment facilities on Earth to stabilize fecal matter, food wastes, and microbial bio-solids (Khalili et al., 2000). Additional volatile losses of nitrous oxide present a considerable recovery challenge and can add an additional 20% daily loss of N (Figure 2). The product is a stable material (no objectionable odor, acceptable and slow decay rate, disinfected) that can facilitate conversion of Martian regolith into soil. During NASA’s Breadboard project, potato yield of plants grown with liquid effluent from aerobic digestion of inedible plant biomass were within 10% of control yields (Mackowiak et al., 1997). Recalcitrant nutrients accumulated over time, but final potato composition was comparable to control tubers. While N was not specifically studied, this project illustrates the compatibility of aerobic digestion effluent with crops in regenerative life support systems.

Anaerobic digestion also uses anaerobic microorganisms to break down wastes into ammonium while releasing methane as a byproduct. The process can be dry or wet depending on the level of moisture. Dry fermentation anaerobic digestion has higher methane yields, lower water inputs, and reduced energy requirements compared to wet fermentation (Luning et al., 2003). The generated methane can be used as a clean energy source and the ammonium can be fed to plants or into additional nitrification digesters to be converted to nitrate. Microbes, much like plants, often show toxicity symptoms and reproductive challenges at high levels of ammonium. This feedback loop poses challenges for anaerobic digestion if it is to sustain a continuous ammonium output without negatively impacting microbial populations (Shapovalov et al., 2020). The remaining bio-solids from digesters in advanced life support systems can be added to plant substrates for slow, extended release of nutrients or tilled into the Martian regolith to begin developing a soil base for future in situ agriculture. This recalcitrant N can be 10% from anaerobic digestion on a daily basis that must be replaced through bacterial fixation.

A visual comparison of aerobic and anaerobic digestion is shown in Table 2. Aerobic digestion is much faster than anaerobic digestion, but it requires higher energy inputs to maintain adequate oxygen levels. A combination of these two systems allows production of both nitrate and ammonium for plant fertilizers. Their input quantities can be manipulated to produce the desired ratios of both N species. An important consideration in both anaerobic and aerobic digestion is the use of batch or continuous throughput methods. Batch digestion involves adding wastes, processing through digestion over a set time period, and removing the desired byproducts before restarting the process. Continuous throughput methods would have an initial waste supply and continue adding additional waste while simultaneously removing byproducts for fuel or fertilizers. Most research has focused on the efficiency of methane produced from both systems. Batch systems with dual solid and liquid phases have shown increased methane production compared to continuous systems when unwanted contaminants were separated from bulk waste prior to digestion (Chowdhury and Fulford, 1992; Zhang et al., 2013). Ammonium is likely to follow this trend as another valuable microbial byproduct.

A combination of both methods will likely be the best approach due to the inevitable buildup of recalcitrant bio-solids that must eventually be eliminated from the digesters. This method is termed semi-continuous and involves coordinated input of waste and removal of byproducts over a set time frame before cleaning and restarting the digester. The process is similar to industrial aquaponics systems on Earth that use continuous throughput mineralization tanks to convert organic fish wastes into inorganic N forms useable by plants.

The N fixation rates of most PNSB are high. Rhodopseudomonas palustris is a diazotroph that possesses three different nitrogenase enzymes and photoheterotrophic metabolic capability. It also has high metabolic versatility and is capable of photoheterotrophic, diazotrophic, photoautotrophic, and non-diazotrophic metabolism (Soundararajan et al., 2019). Since CO₂ fixation and N fixation compete for ATP, carbon sources can be provided to maximize N fixation (Larimer et al., 2004). Rhodopseudomonas palustris NifA* has been genetically engineered to have reduced inhibition of nitrogenase at high ammonium concentrations (Adessi et al., 2012). This strain is typically cultured under photoheterotrophic conditions to maximize biomass N content. Although photons provided from sunlight are an option to drive bacterial photosynthesis, this would require a system that utilizes solar fiber optics and concentrating mirrors, which has a higher equivalent system mass than a system that utilizes light-emitting diodes (LEDs) and photovoltaics (Hardy et al., 2020). LED systems can be designed to output specific wavelengths of photons, and R. palustris appears to be able to use photons with wavelengths between 400 and 900 nm for photosynthesis with relatively similar efficiencies (Soundararajan et al., 2019). Acetate or wastewater organics are provided as a readily available food source along with ample N₂ gas. Maximizing N biomass output will raise demand for available organic compounds, but these compounds can be obtained from waste, heterotrophic metabolism of R. palustris may stand to be an overall benefit.

Rhodopseudomonas palustris can obtain N through urease-facilitated urea hydrolysis or N fixation via nitrogenase (Malofeeva, 1979). This suggests that N remediation and fixation may occur simultaneously. In non-engineered strains, nitrogenase activity would decrease during urease catabolism as ammonium levels rise and prevent nitrogenase expression (Adessi et al., 2012). It is unlikely that urease or its activity inhibits R. palustris NifA* given its nitrogenase desensitization to elevated ammonium concentrations. Since urease activity is not limited by ATP, whereas nitrogenase activity is, it is likely that nitrogenase would be deprioritized under energy limiting conditions. Urea degradation can occur if urea is readily available. Once urease catalysis ceases, N fixation will become the primary means of N acquisition, though both processes can occur simultaneously if urea and N₂ are present. Remediation of N via urea hydrolysis can directly serve to treat wastewater, and N losses can be compensated by nitrogenase activity. This forms a valuable part of the Martian N cycle in which N fixation and remediation can occur in tandem. The major barrier to this process and the implementation of such a system is the removal of inhibitory components, such as those present in sludge or wastewater soap content. Preparing wastewater for integration with N fixation and recovery systems will therefore likely demand considerable upstream processing.

In regenerative life support systems, N-fixing bacterial biomass would serve as a principal N source for crops. Within cells, most of the N is contained in amino acids. Bacterial cells must be broken down by other microbes which mineralize the organic N to ammonium. The pH of this process must be controlled to both allow for an optimal bacterial environment and prevent the volatilization of ammonia gas. Culturing R. palustris is optimized when it is provided with an input of N₂ (80%) and CO₂ (20%) to facilitate N fixation and pH control. The system must also account for lighting conditions, stirring mechanisms, heating, and the energy required to extract and replenish media during and after harvest. Our current culture system (Figure 7.) has a total power demand of approximately 100 WL⁻¹ of media to provide these conditions, from which approximately 0.1 and 0.05 g of N per liter day can be produced from acetate and wastewater media, respectively. We have demonstrated the N recovery process from bacteria can take about 6 weeks without the intentional inoculation of additional microbes and recover 80% of the N. Eventual recovery systems will use both anaerobic and aerobic digesters to achieve desired products and implement specific bacterial strains with high digestion capabilities to increase both the speed and recovery efficiency of biological N fixation.