Advanced Nitrogen Recovery Strategies from Urine in Bioregenerative Life Support Systems (BLSS)

Abstract

This article provides a comprehensive analysis of nitrogen recovery strategies from human urine for Bioregenerative Life Support Systems (BLSS), essential for long-duration space missions. It explores the foundational principles of closing the nitrogen loop, compares physicochemical and biological methodologies, and examines advanced biotechnological applications. The content details operational challenges, including process stability and pathogen management, and offers optimization techniques for enhanced efficiency. A comparative assessment of various technologies validates their performance in system integration and plant growth applications, providing researchers and scientists with a critical resource for developing robust, sustainable life support systems for future space exploration.

The Critical Need for Nitrogen Loop Closure in BLSS

Bioregenerative Life Support Systems (BLSS) are advanced, closed-loop ecosystems under development for long-duration space missions. These systems aim to sustainably provide crews with essential life support elements—food, water, and oxygen—through the biological recycling of waste streams [1] [2]. Effective nitrogen management is a critical cornerstone for BLSS functionality, as it is an indispensable component of crew nutrition (as a fundamental element in proteins and nucleic acids) and plant growth (as a key mineral nutrient) [2]. Without efficient nitrogen recovery and recycling, in-situ food production becomes impossible, jeopardizing mission self-sufficiency.

The current Environmental Control and Life Support System (ECLSS) on the International Space Station (ISS) relies on physicochemical processes. While it successfully recovers water and oxygen, it lacks a pathway for nitrogen recovery from waste streams, creating a major logistical burden for long-duration missions [1] [3]. It is estimated that a crew member's life support requires approximately 1.83 kg of food and 2.50 kg of water per day [1] [3]. For a 3-year mission to Mars with a crew of four, this translates to a payload of over 25,000 kg for food and water alone, with current launch costs exceeding $10,000 per kilogram [1] [3]. Furthermore, human urine is the primary source of recoverable nitrogen in a BLSS, contributing about 85% of the total nitrogen (averaging 7-16 g of nitrogen per crew member daily), mostly in the form of urea [1] [3]. Therefore, developing robust technologies to convert this urine-nitrogen into forms usable by plants is a central research focus for BLSS development, enabling the closure of the nitrogen cycle and dramatically reducing the need for resupply from Earth [1] [2].

Quantitative Analysis of Nitrogen in BLSS

Nitrogen Mass Balance and System Reliability

A fundamental understanding of nitrogen flows and storage is crucial for designing stable BLSS. Mathematical modeling reveals that even in "closed" systems, atmospheric leakage of nitrogen gas can represent the largest nitrogen loss pathway, underscoring the need for robust system sealing and pressure management [4]. Sensitivity analyses further highlight that plant nitrogen uptake is a dominant factor influencing overall system dynamics and stability [4].

Long-term system reliability is paramount. Ground-based testing, such as the 370-day closed human experiment in the Lunar Palace 1 (LP1) facility in China, provides critical data. Reliability analysis of LP1, which integrated higher plants, animals, microorganisms, and humans, estimated an average BLSS lifespan of approximately 52.4 years [5]. The failure rates of key units from this experiment are summarized in Table 1, offering insights for designing more reliable systems.

Table 1: Reliability Data from Lunar Palace 1 370-Day Experiment

System Unit	Impact on Overall Failure	Key Reliability Findings
Temperature & Humidity Control (THCU)	High	High failure probability; major impact on overall system reliability.
Water Treatment Unit (WTU)	High	High failure probability; major impact on overall system reliability.
Mineral Element Supply (MESU)	High	Significant influence on system reliability and lifetime.
LED Light Source (LLSU)	High	Significant influence on system reliability and lifetime.
Atmosphere Management (AMU)	High	Significant influence on system reliability and lifetime.
Solid Waste Treatment	Moderate	Lower failure rate compared to THCU and WTU.

Nitrogen Content in Waste Streams and Plant Requirements

The efficient refinery of waste into fertilizer requires a precise accounting of nutrient sources. Human urine is the most concentrated liquid waste stream, with nitrogen concentrations typically ranging from 4 to 9 grams per liter [1] [3]. The annual nutrient contribution per person is substantial, as detailed in Table 2. For plant production, the target is to convert this nitrogen into a form that can be assimilated. Most plants preferentially take up nitrogen as ammonium (NH₄⁺) or nitrate (NO₃⁻) [2]. The dietary protein requirement for a crew member directly influences the needed plant biomass production. An average daily protein requirement of 70-100 g per person necessitates the recovery and provision of significant amounts of bioavailable nitrogen to the plant cultivation system [2].

Table 2: Annual Nutrient Production per Capita in Human Waste

Waste Stream	Nitrogen (kg/person/year)	Phosphorus (kg/person/year)	Potassium (kg/person/year)	Reference
Urine	3.7 - 4.0	0.34 - 0.40	1.2 - 0.90	[6]
Faeces	0.50 - 0.55	0.18	0.36	[6]
Greywater	0.60	0.10	0.60	[6]

Experimental Protocols for Nitrogen Recovery and Utilization

Protocol: Nitrification of Source-Separated Urine for Fertilizer Production

This protocol outlines the procedure for converting urea and ammonium in human urine into nitrate, a preferred nitrogen source for many plants, using nitrifying bacteria [1] [2].

1. Principle: The process involves two sequential microbial reactions: first, ureolytic bacteria hydrolyze urea to ammonium and carbon dioxide; second, chemolithoautotrophic bacteria (e.g., Nitrosomonas and Nitrobacter) oxidize ammonium to nitrite and then to nitrate.

2. Reagents and Equipment:

• Source-separated human urine
• Nitrifying bioreactor (e.g., packed-bed, membrane bioreactor)
• Air pump and diffuser for aeration
• pH meter and controller
• Alkalinity source (e.g., K₂CO₃ or NaOH solution)
• Peristaltic pumps for feed and harvest

3. Procedure:

• Step 1: Urine Collection and Stabilization. Collect urine in a dedicated system to prevent faecal contamination. To prevent urea hydrolysis and ammonia volatilization during storage, acidify urine (e.g., with H₃PO₄ to pH ~4) or add a chemical inhibitor (e.g., Cr⁶⁺) [1] [3].
• Step 2: Bioreactor Inoculation and Operation.
  • Inoculate the bioreactor with a robust nitrifying culture, such as those developed for the MELiSSA project's Compartment III [1].
  • Feed the stabilized urine into the bioreactor after neutralization. Dilution may be necessary to avoid microbial inhibition due to high ammonium concentrations.
  • Maintain dissolved oxygen at >2 mg/L through continuous aeration.
  • Maintain a pH between 7.5 and 8.0 using an automated controller that adds an alkalinity source [1].
  • Operate at a controlled temperature, typically 25-30°C.
• Step 3: Process Monitoring and Harvest.
  • Monitor the conversion process by measuring the disappearance of ammonium (NH₄⁺) and the appearance and subsequent disappearance of nitrite (NO₂⁻), and the accumulation of nitrate (NO₃⁻).
  • The product is a nitrate-rich liquid fertilizer that can be directly supplied to hydroponic plant growth systems after nutrient balancing.

Protocol: Field-Scale Agronomic Trial of Urine-Based Fertilizer

This protocol describes a field-scale experiment to evaluate the fertilizer efficacy of processed human urine on a crop like barley (Hordeum vulgare), comparing it to conventional mineral fertilizer [6].

1. Principle: The experiment tests the hypothesis that the nitrogen in source-separated and treated urine is as plant-available as nitrogen in synthetic mineral fertilizers, resulting in equivalent crop yield and quality.

2. Reagents and Equipment:

• Stored or nitrified human urine (stored for >6 months at >20°C for pathogen reduction)
• Commercial mineral fertilizer (e.g., NPK blend)
• Test crop seeds (e.g., Barley varieties 'Gloria' and 'Harbinger')
• Experimental field plots
• Standard agricultural equipment for sowing, harvesting, and sample processing

3. Procedure:

• Step 1: Experimental Design.
  • Select a field and divide it into multiple plots.
  • Implement a randomized complete block design with the following treatments:
    • Urine Fertilizer: Apply urine at a target nitrogen rate (e.g., 54 or 100 kg N ha⁻¹).
    • Mineral Fertilizer: Apply an equivalent amount of N using a mineral fertilizer.
    • Control: No nitrogen fertilizer applied.
  • Replicate each treatment multiple times to ensure statistical power.
• Step 2: Application and Cultivation.
  • Apply the fertilizers evenly to the respective plots at sowing. A second (top-dressing) application can be made during the growing season (e.g., at flowering) [7].
  • Grow the crop following standard agronomic practices for irrigation, pest, and weed control.
• Step 3: Data Collection and Analysis.
  • At harvest, measure the grain yield (e.g., in Mg ha⁻¹) and straw yield for each plot.
  • Analyze quality parameters: Thousand Grain Weight (TGW), grain protein content, and germination rate.
  • Perform statistical analyses (e.g., Analysis of Variance - ANOVA) to determine if significant differences exist between the treatments.

The Scientist's Toolkit: Key Research Reagents and Materials

Table 3: Essential Reagents and Materials for BLSS Nitrogen Research

Item Name	Function/Application	Specific Example/Note
Nitrifying Bioreactor	Core unit for converting ammonium to nitrate.	MELiSSA Compartment III; requires robust bacterial consortia [1].
Nitrifying Bacteria Consortia	Biological agents for nitrification process.	Cultures of Nitrosomonas spp. (ammonium oxidizers) and Nitrobacter spp. (nitrite oxidizers) [1].
Source-Separating Sanitation Hardware	Collects human urine without faecal contamination.	Urine-diverting dry toilets or piping systems [6].
pH Controller & Dosing Pump	Maintains optimal pH (7.5-8.0) in nitrification reactor.	Critical for autotrophic bacterial activity [1].
Hydroponic Plant Growth System	Cultivates crops using recovered nutrient solutions.	System for growing higher plants (e.g., wheat, potatoes, lettuce) in BLSS [5] [2].
Analytical Instrumentation	Monitors nitrogen species in liquid and gas phases.	Flow Injection Analyzer (FIA) for NH₄⁺, NO₂⁻, NO₃⁻; CHN Analyzer for Particulate N [8].

Nitrogen is the linchpin of a successful Bioregenerative Life Support System, directly connecting waste recycling to the production of food and the support of crew life. The research protocols and data synthesized here demonstrate that nitrogen recovery from urine is not only feasible but can be highly effective, producing fertilizers that support crop yields on par with conventional methods [6]. The MELiSSA system's nitrification compartment exemplifies the advanced bioengineering required for this process [1], while long-term experiments like those in Lunar Palace 1 provide crucial data on the reliability needed for space missions [5].

Future research must continue to address the challenges of system stability and the effects of the space environment (e.g., microgravity, radiation) on biological nitrogen cycling processes [1] [2]. Closing the nitrogen loop through these sophisticated biological and physicochemical systems is a critical step toward achieving the self-sufficiency required for humanity's long-term presence in space.

Within Bioregenerative Life Support Systems (BLSS) designed for long-duration space missions, the closure of material loops is paramount. Human urine represents a critical target for resource recovery, as it is the largest contributor of nitrogen to wastewater streams [9]. With an average daily excretion of 7–16 grams of nitrogen per crew member, urine accounts for approximately 85% of the total potentially recoverable nitrogen in a BLSS [3]. Effective recovery of this nitrogen, along with water and other nutrients, is essential to reduce resupply mass and achieve a high degree of system closure [10] [3]. This application note details the composition of human urine, quantifies its resource potential, and provides detailed protocols for nitrogen recovery technologies applicable to BLSS research.

Urine Composition and Resource Potential

Physical and Chemical Characteristics

Fresh human urine is an aqueous solution, comprising approximately 95% water [11] [12]. Its pH is typically slightly acidic, ranging from 5.5 to 7.0, but can become alkaline during storage due to urea hydrolysis [9] [11]. The specific gravity ranges from 1.002 to 1.037, and osmolarity can vary widely between 50 and 1200 mOsmol/kg, influenced by diet and hydration [11].

Table 1: Fundamental Characteristics of Fresh Human Urine

Parameter	Typical Value or Range	Reference
Water Content	91 - 96 %	[11] [12]
pH	5.5 - 7.0	[9] [11]
Specific Gravity	1.002 - 1.037	[11]
Osmolarity	50 - 1200 mOsmol/kg	[11]
Daily Volume per Crew	1.5 - 1.8 L	[10] [3]

Primary Nitrogenous Compounds and Ionic Content

The solute composition of urine is complex, but over 99% of solutes are comprised of only 68 chemicals [11]. Nitrogen is predominantly excreted as urea, which constitutes over 50% of the total organic solids and represents 75-90% of the total nitrogen content [9] [13]. Other significant components include chloride, sodium, potassium, and creatinine.

Table 2: Chemical Composition of Human Urine

Component	Average Concentration	Notes	Reference
Urea	9.3 - 23 g/L	Accounts for 80% of Total Nitrogen (TN)	[9] [11] [12]
Creatinine	0.670 g/L	Nitrogenous compound	[11] [12]
Chloride (Cl⁻)	1.87 g/L	Major anion	[11] [12]
Sodium (Na⁺)	1.17 g/L	Major cation	[11] [12]
Potassium (K⁺)	0.750 - 1.5 g/L	Essential plant nutrient	[11] [14] [12]
Ammonium (NH₄⁺)	Variable	Increases with urea hydrolysis	[9]
Phosphorus (P)	0.3 - 1.0 g/L	As phosphates	[9] [14]
Calcium (Ca²⁺)	Variable	Affected by protein & sodium intake	[11]

Global Variation and Stability

The composition of urine is not constant. It is significantly influenced by dietary intake, water consumption, age, gender, and health status [9] [11]. For instance, high-protein diets and high meat consumption lead to elevated urea and nitrogen levels [11]. A critical challenge for recovery is the instability of fresh urine. Urea is rapidly hydrolyzed by urease-producing bacteria into ammonia and bicarbonate, raising the pH to 8.9-9.45 [9]. This leads to ammonia volatilization (causing nitrogen loss and odor) and mineral precipitation (e.g., struvite), which can clog piping and deplete phosphorus [9].

Nitrogen Recovery Technologies and Protocols

Several technologies have been developed to recover nitrogen and water from urine, each with distinct mechanisms and efficiencies. The following section outlines key experimental protocols.

Reduced Pressure Distillation with Urea Hydrolysis Pretreatment

This two-step method aims to recover both water and nitrogen simultaneously [10] [15].

Principle: Urea in urine is first hydrolyzed to ammonia, which is then recovered via reduced pressure distillation under alkaline conditions [10] [15].

Detailed Protocol:

• Urine Collection and Characterization: Collect real human urine and analyze for initial urea and Total Nitrogen (TN) concentration [10].
• Urea Hydrolysis Pretreatment (Choose one method):
  • High-Temperature Acidification Method (HTAM): Acidify urine to a defined proton concentration (e.g., [H⁺] = 2 mol/L using strong acid like H₂SO₄). Heat the mixture to a high temperature (e.g., 99°C) for a set duration (e.g., 7 hours) to catalyze non-enzymatic urea hydrolysis [10].
  • Immobilized Urease Catalysis Method (IUCM): Use immobilized urease enzyme to catalyze urea hydrolysis at a lower temperature (e.g., 60°C) and neutral pH (pH=7) for a shorter duration (e.g., 40 minutes) [10].
• Reduced Pressure Distillation: Transfer the hydrolyzed urine to a reduced pressure distillation apparatus. Under alkaline conditions and reduced pressure, distill the solution. Water vapor and ammonia gas are co-distilled [10] [15].
• Condensation and Collection: The vapor mixture is cooled and condensed. The condensate contains recovered water with dissolved ammonia, forming an ammonium hydroxide solution [15].
• Analysis: Measure the volume of the condensate and analyze its nitrogen content (e.g., as ammonium) to calculate water recovery efficiency and nitrogen recycle efficiency [10].

Performance Metrics: This method can recover virtually all water from urine. Nitrogen recovery efficiency was reported at 20.5% for direct distillation, which was improved to 39.7% with HTAM pretreatment and 52.2% with IUCM pretreatment [10].

Biological Nitrification

Biological nitrification is an effective method for stabilizing nitrogen in urine by converting volatile ammonia into nitrate [9].

Principle: Ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) are employed in a bioreactor to sequentially oxidate ammonia (NH₄⁺) to nitrite (NO₂⁻) and then to stable nitrate (NO₃⁻), a preferred plant fertilizer [9].

Detailed Protocol:

• Reactor Inoculation: Inoculate a bioreactor (e.g., a sequencing batch reactor) with a enriched culture of nitrifying bacteria [9].
• Acclimatization and Operation: Feed the reactor with source-separated urine. The high salinity and ammonia concentration can inhibit nitrifying bacteria, so a progressive acclimatization strategy may be required. The reactor is operated under controlled dissolved oxygen, temperature, and pH conditions [9].
• Process Monitoring: Regularly monitor the concentrations of ammonium, nitrite, and nitrate in the effluent to assess nitrification performance and stability [9].
• Nutrient Concentration (Post-Processing): The nitrified urine, now a nitrate-rich solution, can be concentrated using membrane technologies like reverse osmosis or forward osmosis to reduce volume and produce a more practical liquid fertilizer [9].

Performance Metrics: Biological nitrification can maintain almost all nutrient components in urine. It is valued for its high nutrient recovery efficiency, low operating costs, and reduced chemical consumption [9].

Urea Electro-Forward Osmosis System (UEFOS)

This innovative system combines forward osmosis with an electric field to enhance urea recovery while inhibiting its hydrolysis [16] [13].

Principle: An electric field is applied to a forward osmosis system to directionally regulate ion migration, enhancing the osmotic driving force for water permeation and urea recovery. Simultaneously, OH⁻ generated in situ at the cathode migrates to the feed solution, inhibiting urease activity and minimizing urea hydrolysis [16] [13].

Detailed Protocol:

• System Setup: Construct a three-chamber cell, with the central chamber containing the source-separated urine (Feed Solution, FS), flanked by two draw solution (DS) chambers. Anion and cation exchange membranes separate the chambers. Electrodes (anode and cathode) are placed in the DS chambers [16].
• Solution Preparation: The FS is source-separated urine. The DS is a high-concentration solution (e.g., 2 mol/L NaCl) providing the osmotic driving force. Electrode chambers are filled with an electrolyte like Na₂SO₄ [16].
• System Operation: Apply a constant current (e.g., 10 mA) across the electrodes. The electric field enhances ion migration, increasing the effective osmotic pressure difference. Water spontaneously moves from the FS to the DS, concentrating the urine and recovering urea in the FS [16] [13].
• Hydrolysis Inhibition: The alkaline environment (OH⁻) generated at the cathode and migrated to the FS suppresses urease activity, preserving urea [16] [13].
• Sampling and Analysis: Periodically sample the FS to measure urea concentration and calculate urea recovery efficiency and kinetics [16].

Performance Metrics: The UEFOS achieved a urea recovery efficiency of 55.15% in 180 minutes, a 90.36% improvement over an open-circuit (no electric field) system. It also successfully slowed the kinetic rate of urea hydrolysis [13].

Logical Workflow for Technology Selection

The following diagram illustrates a decision-making workflow for selecting an appropriate nitrogen recovery technology based on mission priorities and constraints.

Technology Selection Workflow

The Scientist's Toolkit: Key Research Reagents and Materials

Table 3: Essential Research Materials for Urine Nitrogen Recovery Experiments

Research Reagent / Material	Function / Application	Example Use Case
Immobilized Urease	Enzyme catalyst for controlled urea hydrolysis.	Urease Catalysis Pretreatment Method [10].
Strong Acids (H₂SO₄, H₃PO₄)	Acidification to inhibit urea hydrolysis, chemical catalyst.	High-Temperature Acidification Method; urine stabilization [10] [3].
Nitrifying Bacteria Consortium	Mixed culture of AOB and NOB for biological nitrogen stabilization.	Biological Nitrification Reactors [9].
Anion & Cation Exchange Membranes	Selective ion transport in electrochemical systems.	Urea Electro-Forward Osmosis System (UEFOS) [16] [13].
Forward Osmosis Membranes	Selective water transport driven by osmotic pressure.	UEFOS and other osmotically-driven concentration processes [16].
Draw Solutes (e.g., NaCl)	Create high osmotic pressure draw solution.	Used as Draw Solution in UEFOS and FO systems [16].
Chemical Stabilizers (e.g., Cr⁶⁺)	Oxidizing agent to prevent urea hydrolysis during storage.	Urine stabilization in ECLSS (e.g., ISS UPA) [3].

The current Environmental Control and Life Support System (ECLSS) aboard the International Space Station (ISS) represents the state-of-the-art in physicochemical (PC) life support technology for near-Earth missions [3] [1]. While this system successfully recovers water and oxygen, it operates as a partially open-loop system for nitrogen, failing to recover this crucial nutrient from liquid waste streams for food production [1]. This limitation presents a significant obstacle for long-duration space exploration missions beyond Earth's orbit, where resupply from Earth becomes impractical and cost-prohibitive [3]. Within the context of Bioregenerative Life Support System (BLSS) research, the ISS ECLSS serves as a critical case study in the limitations of purely physicochemical approaches to nitrogen management, particularly highlighting the challenge of nitrogen recovery from urine [3]. This document details these limitations and provides associated experimental protocols for investigating alternative nitrogen recovery strategies.

The ISS ECLSS is comprised of two primary subsystems: the Water Recovery System (WRS) and the Oxygen Generation System (OGS) [3] [1]. The system is designed to address the crew's metabolic needs for water and oxygen, achieving up to a 96.5% reduction in water payload mass through recycling [3]. However, its handling of nitrogenous waste, primarily from urine, reveals its fundamental constraints as a closed-loop system.

Table 1: Primary Components of the ISS ECLSS Relevant to Waste Processing

System Component	Primary Function	Role in Nitrogen Management
Water Recovery System (WRS)	Recovers potable water from various waste streams, including urine and condensate [3].	Manages but does not recover nitrogen; stabilizes urea to prevent its breakdown.
Urine Processor Assembly (UPA)	Processes urine via distillation to recover water [3].	Concentrates nitrogenous waste (urea, ammonium) for disposal, not recycling [3].
Oxygen Generation System (OGS)	Produces breathable O₂ via electrolysis of water [3].	Indirectly connected; uses water from WRS but does not process nitrogen.
Carbon Dioxide Reduction System (CRS)	Converts metabolic CO₂ and H₂ into water and methane via the Sabatier reaction [3].	No nitrogen processing function; methane (CH₄) is vented overboard [3].

The Fate of Nitrogen in the ECLSS

Urine is the most significant source of recoverable nitrogen in a life support system, with a crew member excreting 7–16 grams of nitrogen per day, accounting for approximately 85% of the total potentially recoverable nitrogen [3]. In the current ISS system, urine is first collected and chemically stabilized in the Wastewater Storage Tank Assembly (WSTA). The stabilization process involves adding a solution of phosphoric acid (H₃PO₄) and hexavalent chromium (Cr⁶⁺) [3] [1]. The acid serves to convert volatile ammonia into non-volatile ammonium ions, while the chromium acts as an oxidizing agent to inhibit microbial ureolysis (the hydrolysis of urea) [3]. This stabilized urine is then fed to the Distillation Assembly (DA), where water is evaporated, separated from the waste brine, and further purified. The resulting nitrogen-rich brine is currently considered a waste product and is stored for later disposal, rather than being processed for nutrient recovery [3]. This represents a fundamental linear throughput of nutrients in an otherwise partially closed-loop system.

Figure 1: Nitrogen Pathway in the ISS ECLSS. The diagram illustrates the linear flow of nitrogen from crew waste to disposal, highlighting the lack of recovery and recycling within the system.

Quantitative Limitations and Research Implications

The operational parameters of the ISS ECLSS reveal specific quantitative limitations that directly impact mission design and resource logistics for long-duration exploration.

Table 2: Key Limitations of the ISS ECLSS for Long-Duration Missions

Parameter	ISS ECLSS Performance	Implication for Long-Duration Missions
Nitrogen Recovery	None (nitrogen is stored as waste brine) [3].	Precludes in-situ food production; requires full food payload or alternative system.
Food Production	Not implemented [3].	Total reliance on terrestrial resupply or pre-positioned food.
Resupply Dependency	High (for food and some consumables) [3].	Logistically challenging and cost-prohibitive for missions to Mars or beyond [3].
Estimated Mission Payload	~25,287 kg for food/water for a 4-person, 3-year mission [3].	Mass that must be launched from Earth, representing a significant cost.
Water Recovery Efficiency	Up to 85% from urine [3].	Effective for water, but does not address the nutrient loop.
System Input Consumables	Requires steady supply of chemicals (e.g., H₃PO₄) [3].	Adds to the mass of required resupply cargo.

Experimental Protocol: Assessing Microbial Nitrogen Recovery as a BLSS Alternative

The limitations of the ISS ECLSS have accelerated research into Bioregenerative Life Support Systems (BLSS), which utilize biological and physicochemical processes to create a closed-loop system. A promising approach involves using microbial communities to convert urine-derived nitrogen into forms usable by plants. The following protocol outlines a methodology for assessing the viability of nitrogen cycle microorganisms after exposure to simulated space conditions, a critical step for BLSS feasibility [17].

Aim

To evaluate the post-preservation activity of key nitrogen cycle microorganisms (ureolysis, nitritation, nitratation, denitrification, anammox) following exposure to simulated space conditions (microgravity and radiation).

Materials

• Test Organisms: Axenic cultures and defined communities of ureolytic bacteria (e.g., Cupriavidus pinatubonensis), ammonia-oxidizing bacteria (AOB, e.g., Nitrosomonas europaea), nitrite-oxidizing bacteria (NOB, e.g., Nitrobacter winogradskyi), denitrifiers, and anammox bacteria [17].
• Growth Media: Specific liquid media for each functional group (e.g., mineral media with ammonium for AOB, nitrite for NOB).
• Preservation Conditions: Facilities for simulated microgravity, radiation exposure, and controlled temperature (e.g., 4°C, ~20°C).
• Analytical Equipment: Spectrophotometer, ion chromatography system (or equivalent) for measuring NH₄⁺, NO₂⁻, NO₃⁻ concentrations.

Methodology

Step 1: Culture Preparation and Preservation

• Grow each microbial culture to its late exponential phase.
• Divide each culture into aliquots for three preservation conditions:
  • Experimental (F): Exposure to defined space conditions (e.g., low gravity ~10⁻⁴ g, radiation ~700 µGy d⁻¹, ~20°C) for a set duration (e.g., 44 days) [17].
  • Ground Control (G23): Storage on Earth at ambient temperature (~23°C) for the same duration [17].
  • Refrigerated Control (G4): Storage on Earth at 4°C for the same duration [17].

Step 2: Reactivation and Activity Measurement

• After the preservation period, inoculate a small volume of each preserved sample into fresh, specific growth medium.
• Incubate under optimal conditions (temperature, aeration as required) to allow for reactivation.
• Monitor the conversion of key nitrogen species over time:
  • Ureolysis: Measure the decrease in urea and increase in NH₄⁺.
  • Nitritation (AOB activity): Measure the decrease in NH₄⁺ and increase in NO₂⁻.
  • Nitratation (NOB activity): Measure the decrease in NO₂⁻ and increase in NO₃⁻.
  • Denitrification: Measure the decrease in NO₃⁻ and/or NO₂⁻ under anoxic conditions.
  • Anammox: Measure the simultaneous decrease in NH₄⁺ and NO₂⁻.

Step 3: Data Analysis

• Calculate the volumetric conversion rates (mg N L⁻¹ d⁻¹) for each process.
• Compare the rates between the three preservation conditions (F, G23, G4) using statistical analysis (e.g., t-test) to determine the effect of space conditions on microbial resilience.

Expected Outcomes

Previous research has shown that all mentioned nitrogen cycle functionalities can be reactivated after actual space exposure, with rates often similar to or even higher than ground controls stored at a similar temperature. Refrigerated storage (4°C) typically yields the highest reactivation rates [17].

Figure 2: Experimental workflow for testing microbial nitrogen recovery under space conditions.

The Scientist's Toolkit: Key Research Reagents for Nitrogen Cycle Studies

Table 3: Essential Research Reagents for Microbial Nitrogen Recovery Experiments

Reagent / Material	Function / Role in Experimentation
Ureolytic Bacteria (e.g., Cupriavidus pinatubonensis)	Converts urea in urine into ammonium (NH₄⁺), making nitrogen available for other processes [17].
Ammonia-Oxidizing Bacteria (AOB) (e.g., Nitrosomonas europaea)	Performs nitritation: oxidizes ammonium (NH₄⁺) to nitrite (NO₂⁻) as part of nitrification [17].
Nitrite-Oxidizing Bacteria (NOB) (e.g., Nitrobacter winogradskyi)	Performs nitratation: oxidizes nitrite (NO₂⁻) to nitrate (NO₃⁻), a preferred plant nutrient [17].
Anammox Bacteria (e.g., Candidatus Kuenenia stuttgartiensis)	Converts ammonium (NH₄⁺) and nitrite (NO₂⁻) directly into dinitrogen gas (N₂) under anoxic conditions, potentially for gas balance [17].
Denitrifying Bacteria	Reduces nitrate (NO₃⁻) or nitrite (NO₂⁻) to nitrogen gases (N₂, N₂O), closing the nitrogen loop [17].
Defined & Reactor Communities	Used to study microbial interactions and community resilience, which is often higher than in axenic cultures [17].
Ion Chromatography System	Essential analytical equipment for precise measurement of nitrogen species (NH₄⁺, NO₂⁻, NO₃⁻) in solution [17].
Synthetic Urine Formulation	Provides a standardized, reproducible substrate for nitrogen recovery experiments, mimicking astronaut urine [3].

The ISS ECLSS successfully demonstrates the feasibility of recovering water and oxygen in a microgravity environment. However, its inability to close the nitrogen loop by recovering nutrients from urine for food production constitutes a major limitation for long-duration space exploration [3]. This case study underscores the necessity of integrating biological processes, such as microbial nitrogen cycling, into future life support systems. Research, as outlined in the provided protocol, confirms the resilience of key nitrogen-converting microorganisms to space conditions, providing a promising pathway toward developing the fully closed-loop, bioregenerative systems required for human presence beyond Earth orbit [17].

Bioregenerative Life Support Systems (BLSS) represent a fundamental shift in how life support is managed for long-duration space missions, moving from resource consumption to resource recycling. Unlike the current Environmental Control and Life Support System (ECLSS) on the International Space Station (ISS), which relies primarily on physicochemical processes and requires regular resupply from Earth, a BLSS aims to create a closed-loop, self-sustaining ecosystem [1]. This paradigm transition is critical for missions to the Moon and Mars, where resupply is impractical. A core challenge in this transition is the efficient recovery and recycling of essential elements, particularly nitrogen, from waste streams. This document details application notes and protocols, framed within a broader thesis on nitrogen recovery from urine, to guide researchers in developing robust BLSS technologies.

Core Concepts: Open-Loop vs. Closed-Loop Systems

Definitions and Key Characteristics

• Open-Loop Control System: An open-loop system operates without feedback. Its control actions are independent of the system's output. It is simpler, more cost-effective, and often faster but lacks accuracy and adaptability because it cannot self-correct [18] [19]. In a life support context, this corresponds to a system that consumes resources without recycling them.
• Closed-Loop Control System: A closed-loop system incorporates feedback to adjust its control actions based on the output. It continuously monitors performance and makes necessary adjustments to maintain a desired state. This leads to greater accuracy, adaptability, stability, and efficiency, albeit with increased design complexity [18] [19]. In life support, this is the principle of a BLSS, where waste products are processed and reused.

Table 1: Comparison of Open-Loop and Closed-Loop Control Systems

Feature	Open-Loop System	Closed-Loop System
Feedback	No feedback mechanism [18].	Continuous feedback loop is essential [18].
Accuracy	Lower; cannot correct for disturbances [19].	Higher; self-correcting based on output [18].
Adaptability	Poor; cannot adapt to changing conditions [18].	High; can adapt to environmental changes [18].
Complexity & Cost	Simple design and cost-effective [18].	More complex and expensive to implement [18].
Example	A washing machine with a timer [18].	A thermostat-controlled heating system [18] [19].
LSS Analogy	ECLSS (stores/ventes waste, requires resupply) [1].	BLSS (recycles waste in a closed loop) [1].

The BLSS as a Closed-Loop System

A BLSS is a prime example of a complex closed-loop system. It combines biological components (plants, bacteria, algae) and physicochemical processes to regenerate air, water, and food from crew waste [1]. The system's stability relies on multiple interconnected feedback loops. For instance, human respiration produces CO₂, which is consumed by plants for photosynthesis, producing O₂ for the crew. Similarly, organic waste and urine are broken down by microorganisms into minerals that fertilize plant growth, which in turn provides food. The MELiSSA (Micro-Ecological Life Support System Alternative) project by the European Space Agency is a leading example of this engineering approach, modeling a lake ecosystem in a closed loop [1].

Nitrogen Recovery: The Linchpin of the BLSS Paradigm

The Critical Role of Nitrogen

Nitrogen is an essential element for amino acids and proteins. In a BLSS, the primary source of recyclable nitrogen is human urine, which accounts for approximately 85% of the total recoverable nitrogen, mostly in the form of urea [1]. An average crew member excretes 7–16g of nitrogen per day via urine. Efficient recovery of this nitrogen is non-negotiable for the in-situ production of food crops and edible microbial biomass, making it a critical research focus for BLSS development.

Table 2: Key Quantitative Data on Nitrogen in BLSS Context

Parameter	Value / Range	Context / Significance
Daily N excretion per crew	7 - 16 g [1]	Highlights the volume of nitrogen available for recovery from urine.
Urine volume per crew/day	~1.80 L [1]	Indicates the physical volume of the primary waste stream.
Nitrogen in urine	4 - 9 g/L·day⁻¹ [1]	Concentration of nitrogen in the collected urine stream.
Contribution of urine to recoverable N	~85% [1]	Establishes urine as the most significant nitrogen source.
Target N form for plants/algae	Ammonium (NH₄⁺), Nitrate (NO₃⁻)	Urea must be converted to these forms for assimilation by producers.

Experimental Protocols: Nitrogen Recovery from Urine

Protocol: Nitrification Performance in a Bioreactor (Compartment III of MELiSSA)

This protocol outlines the methodology for establishing and monitoring a nitrifying bioreactor, a key component for converting ammonia into nitrate.

• 1. Objective: To evaluate the viability, metabolic activity, and ammonia detoxification capacity of nitrifying bacteria within a simulated BLSS bioreactor.
• 2. Materials:
  • Bioreactor: Configured based on design specifications (e.g., cylindrical vs. flat-bottom, see [20]).
  • Biological Component: Nitrifying bacterial culture (e.g., Nitrosomonas, Nitrobacter).
  • Synthetic Urine Feed: A solution containing urea and ammonium salts to simulate pretreated urine.
  • Peristaltic Pumps: For controlled flow of liquids.
  • Dissolved Oxygen Probe and Meter.
  • pH and Temperature Sensors.
  • Sampling Ports: For aseptic collection of liquid samples.
  • Analytical Equipment: Spectrophotometer, HPLC, or equivalent for metabolite analysis.
• 3. Procedure:
  • Bioreactor Setup and Sterilization: Assemble the bioreactor and associated tubing. Sterilize in-place or autoclave removable parts.
  • Inoculation: Aseptically introduce the nitrifying bacterial culture into the bioreactor's biological compartment.
  • System Perfusion: Initiate flow of the synthetic urine feed medium through the system using peristaltic pumps. Maintain a defined hydraulic retention time.
  • Environmental Control: Continuously monitor and control dissolved oxygen (>2 mg/L), pH (7.5-8.0), and temperature (25-30°C).
  • Sampling: At predetermined intervals (e.g., T = 0, 60, 120 minutes), aseptically collect samples from the inflow and outflow streams.
  • Analysis:
    • Viability: Measure Lactate Dehydrogenase (LDH) release as a marker of cell integrity [20].
    • Functionality:
      • Oxygen Consumption: Calculate from inflow and outflow dissolved oxygen values [20].
      • Ammonia Detoxification: Quantify ammonia (NH₄⁺) and nitrate (NO₃⁻) concentrations in inflow vs. outflow samples via colorimetric assays (e.g., spectrophotometry).
  • Data Collection: Record all parameters for the duration of the experiment (e.g., 120 minutes).

Protocol: Evaluation of Bioreactor Design on LMO Performance

This protocol, adapted from [20], tests how bioreactor architecture impacts the function of a more complex biological component: Liver Microorgans (LMOs).

• 1. Objective: To determine the influence of bioreactor configuration (cylindrical vs. flat-bottom) on the ammonia detoxification capacity of LMOs.
• 2. Materials:
  • LMOs: Thin slices (approx. 338 µm) of liver tissue preserving native cellular organization [20].
  • Bioreactor Designs: Cylindrical BAL and "flat bottom" BAL models.
  • Perfusion System: With integrated peristaltic pump.
  • Ammonia-Overloaded Blood Analog: A solution containing ~1 mM NH₄⁺.
  • KH-Base (KHB) Solution: Standard incubation buffer.
• 3. Procedure:
  • LMO Preparation: Obtain LMOs from model organisms (e.g., Wistar rats) and pre-incubate in KHB solution for 15 minutes [20].
  • Control Assay (NRS): Incubate LMOs in the Normothermic Reoxygenation System (a shaken suspension) with ammonia-overloaded medium to establish baseline functionality [20].
  • Experimental Assay (BAL): Load LMOs into the two different BAL prototypes.
  • Perfusion: Perfuse both BAL systems with the ammonia-overloaded blood analog for 120 minutes.
  • Sampling: Collect fluid from the biological compartment at T=0, 60, and 120 minutes.
  • Analysis:
    • Measure ammonia concentration in all samples.
    • Calculate the percentage of initial ammonia detoxified.
    • Monitor additional parameters: LDH release, oxygen consumption, and gene expression (e.g., CPSI, OTC) [20].
• 4. Expected Results: As demonstrated in [20], LMOs may show significant ammonia detoxification in a flat-bottom bioreactor (e.g., 49.3%) but fail to do so in a cylindrical design, highlighting the critical impact of design on biological performance.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials and Reagents for BLSS Nitrogen Recovery Research

Item	Function / Application
Liver Microorgans (LMOs)	Complex biological component retaining liver structure and multiple cell types; used to test advanced ammonia detoxification pathways [20].
Nitrifying Bacterial Cultures	The workhorse microorganisms for the biological conversion of toxic ammonia to nitrate in dedicated bioreactor compartments (e.g., MELiSSA III) [1].
Synthetic Urine Formulation	A standardized, chemically defined solution used to simulate human urine for reproducible experimentation, avoiding variability and ethical constraints of real urine.
KH-Base (KHB) / KHA Solutions	Buffered salt solutions for the obtainment, pre-incubation, and maintenance of biological components like LMOs, ensuring physiological pH and ion balance [20].
Lactate Dehydrogenase (LDH) Assay Kit	A standard tool for quantifying LDH enzyme released from damaged cells, serving as a key metric for assessing the viability of the biological component [20].
Colorimetric Assay Kits (Ammonia, Nitrate)	Enable precise and relatively simple quantification of key nitrogen species in liquid samples to monitor process efficiency.

Workflow and System Diagrams

The following diagrams, generated using Graphviz DOT language, illustrate the core concepts and experimental workflows described in these application notes.

BLSS Nitrogen Cycle

Nitrification Bioreactor Workflow

ECLSS vs BLSS Paradigm

In the context of Bioregenerative Life Support Systems (BLSS) for long-duration space missions, the recovery of nitrogen from crew urine is paramount for achieving closed-loop resource regeneration. Nitrogen recovery is essential for in-situ fertilizer production to support plant growth, which in turn provides food, oxygen, and water recycling [1] [3]. Among the key challenges identified for the robust operation of these systems are the management of high salinity derived from urine electrolytes, the control of urea hydrolysis and its subsequent effects, and maintaining overall process stability against inhibitory conditions [21] [10]. This document outlines the core challenges and provides detailed protocols for researching and mitigating these issues, framed within the European Space Agency's MELiSSA (Micro-Ecological Life Support System Alternative) initiative, one of the most advanced BLSS concepts [1].

Core Challenges in Nitrogen Recovery

High Salinity Stress

The high salt content of human urine, primarily sodium chloride, poses a significant threat to biological treatment processes and subsequent plant cultivation [10].

• Impact on Microbial Activity: Elevated salinity can destabilize microorganisms, reduce metabolic activity, and impair the kinetics of nitrification—the biological conversion of ammonia to nitrate.
• Differential Inhibition: Nitrite-oxidizing bacteria (NOB) are more sensitive to salinity than ammonia-oxidizing bacteria (AOB). Studies show full nitrification can be maintained up to a conductivity of 9.5 mS/cm, while partial nitrification persists up to 63.3 mS/cm. Beyond this threshold, severe inhibition of NOB occurs [21].
• System Implications: High salinity in recycled water streams can lead to soil salinization in plant growth modules, negatively impacting crop yields [10].

Urea Hydrolysis and Free Ammonia Inhibition

Urine is rich in urea, which rapidly hydrolyzes to ammonia (NH₃) and carbonate ions. This process can lead to the accumulation of free ammonia (FA), a potent inhibitor of nitrifying bacteria [21] [3].

• Inhibition Thresholds: FA concentrations as low as 0.7-6 g N/m³ can inhibit NOB, while Ammonia-Oxidizing Bacteria (AOB) are inhibited at higher concentrations of 10-150 g N/m³ [21].
• pH and Temperature Dependence: The FA concentration is highly dependent on pH and temperature, with higher values shifting the ammonium/ammonia equilibrium toward the inhibitory FA form [21].
• Process Instability: Uncontrolled urea hydrolysis and FA buildup can lead to complete nitrification failure. Incidents with extreme FA concentrations (e.g., 280 g N/m³ for 19 hours) have been shown to cause severe and prolonged inhibition, requiring significant recovery time [21].

Process Stability and Robustness

For space missions, systems must be highly reliable and able to recover from unforeseen upsets. Biological systems for nitrogen recovery are susceptible to failures from substrate overloading, pH shifts, and the combined stress of salinity and FA [21].

• Robustness Testing: System designs must be tested under extreme conditions, including high FA concentrations and salinity, to evaluate recovery potential and ensure operational continuity [21].
• Technology Selection: The choice between suspended growth systems (e.g., activated sludge) and attached growth systems involves trade-offs. Suspended growth systems often achieve higher nitrification rates (>1000 mg N/L·d) but require effective biomass separation [21].

Table 1: Key Challenges and Inhibitory Parameters in Urine Nitrification

Challenge	Key Parameter	Inhibition Threshold	Primary Effect
High Salinity	Conductivity	>9.5 mS/cm (Full Nitrification) >63.3 mS/cm (NOB Inhibition)	Reduced microbial activity; selective inhibition of nitrite-oxidizing bacteria (NOB).
Free Ammonia (FA) Inhibition	FA Concentration (g N-NH₃/m³)	0.7-6 (NOB) 10-150 (AOB)	Disruption of bacterial enzymes; complete nitrification failure at high concentrations.
Process Stability	Combined Stressors	N/A	Prolonged system recovery time after operational failures.

Experimental Protocols

Protocol: Assessing Nitrification Robustness to Salinity and FA

This protocol is designed to evaluate the resilience of a nitrification process under the combined stress of high salinity and free ammonia, simulating potential failure scenarios in a BLSS [21].

Research Reagent Solutions

Table 2: Key Research Reagents and Materials

Reagent/Material	Function/Explanation
Sequencing Batch Reactor (SBR)	A suspended growth bioreactor for activated sludge processes; allows for controlled feeding, reaction, and settling cycles.
Activated Sludge Inoculum	A diverse microbial community containing ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB).
Source-Separated Human Urine	The primary waste stream and substrate for the experiment, providing ammonium from hydrolyzed urea.
Sodium Chloride (NaCl)	Used to artificially increase the salinity of the synthetic or real urine feed.
pH probes and controllers	Essential for monitoring and controlling pH, a critical parameter that governs the equilibrium between ammonium (NH₄⁺) and inhibitory free ammonia (NH₃).
Dissolved Oxygen probes	For monitoring and maintaining aerobic conditions necessary for nitrification.

Methodology

• Reactor Setup: Operate a pilot-scale SBR (e.g., 150 L) with automated control for pH, dissolved oxygen, temperature, and mixing [21].
• Inoculation and Acclimatization: Inoculate the reactor with activated sludge. Gradually acclimate the biomass to the target salinity level by adding NaCl to the urine feed over several days.
• Inducing FA Stress: To simulate a failure, allow the pH to rise (e.g., through a controller malfunction), which will shift the ammonium equilibrium toward FA. Alternatively, pulse with a high concentration of urea/ammonium.
• Monitoring: Track key parameters throughout the experiment:
  • Ammonium (NH₄⁺-N), Nitrite (NO₂⁻-N), Nitrate (NO₃⁻-N): To monitor nitrification performance.
  • pH and Temperature: To calculate FA concentrations.
  • Mixed Liquor Suspended Solids (MLSS): To monitor biomass concentration.
• Recovery Phase: Once severe inhibition is observed, re-establish optimal pH and operational conditions. Monitor the time required for the system to recover full nitrification activity.

The logical workflow and parameter relationships for this protocol are summarized in the diagram below.

Protocol: Evaluating Urea as a Nitrogen Fertilizer in Hydroponics

This protocol assesses the feasibility of using urea, derived from processed urine, as a nitrogen source for crop production in BLSS hydroponic systems [22].

Research Reagent Solutions

• Plant Material: Soybean (Glycine max) or other candidate crops (e.g., tomato, wheat).
• Growth System: Closed-loop Nutrient Film Technique (NFT) or similar hydroponic system.
• Nutrient Solutions:
  • Control: Nitrate-based complete nutrient solution.
  • Treatment: Nutrient solution where urea is the sole nitrogen source.
• Inoculant: Bradyrhizobium japonicum suspension (for legume tests).

Methodology

• System Preparation: Set up hydroponic gullies in a controlled environment chamber. Maintain strict control over PAR, temperature, humidity, and photoperiod.
• Experimental Design: Employ a factorial design with two factors: N source (Nitrate vs. Urea) and Inoculation (With vs. Without B. japonicum).
• Cultivation: Grow plants with recirculating nutrient solutions. Monitor and adjust the pH of all solutions daily.
• Data Collection:
  • Plant Growth: Biomass, root length, plant height.
  • Physiology: Leaf chlorophyll content, photosynthetic rate.
  • Yield and Quality: Seed yield, protein content.
  • Nutrient Uptake: Analysis of N, K, Ca, Mg in plant tissue.
  • Nodulation: Nodule count and mass (for legumes).
• Data Analysis: Compare plant performance and nutrient use efficiency between the nitrate and urea treatments.

The Scientist's Toolkit

This section details essential reagents, systems, and analytical methods for research into nitrogen recovery for BLSS.

Table 3: Essential Research Tools for BLSS Nitrogen Recovery Studies

Tool Category	Specific Tool/Reagent	Function & Relevance
Bioreactor Systems	Sequencing Batch Reactor (SBR)	Allows for flexible operation and testing of suspended growth nitrification under various stress conditions [21].
Bioreactor Systems	Membrane Bioreactor (MBR)	Couples biological treatment with membrane filtration, effective for biomass retention in microgravity [21].
Hydroponic Systems	Nutrient Film Technique (NFT)	A closed-loop soilless cultivation system for evaluating plant nutrient uptake from recycled streams like nitrified urine [22].
Microbial Inocula	Adapted Nitrifying Activated Sludge	A diverse microbial community selected for high-rate nitrification; can be further adapted to tolerate high salinity [21].
Microbial Inocula	Bradyrhizobium japonicum	Symbiotic bacteria used to study potential synergies between biological N₂ fixation and urea fertilization in legumes [22].
Analytical Methods	Ion Chromatography / Spectrophotometry	For quantitative measurement of key nitrogen species (NH₄⁺, NO₂⁻, NO₃⁻) in liquid samples [21] [10].
Analytical Methods	Free Ammonia Calculation	Derived from measured NH₄⁺-N concentration, pH, and temperature using established equilibrium equations [21].

Physicochemical and Biological Methodologies for Nitrogen Recovery

In the context of Bioregenerative Life Support Systems (BLSS) for long-duration space missions, the recovery of nitrogen from urine is paramount for achieving system closure. Nitrogen is a crucial nutrient for plant growth, which in turn provides food, oxygen, and water recycling for the crew. With urine containing 85% of the recoverable nitrogen in a BLSS, efficient physico-chemical processing technologies are essential to convert this waste into valuable fertilizers [3] [1]. This document details the application notes and experimental protocols for three key physico-chemical processes: Distillation, Membrane Separation, and Electrodialysis.

The following table summarizes the key performance characteristics of the primary nitrogen recovery processes discussed in this document.

Table 1: Performance Comparison of Nitrogen Recovery Processes

Process	Typical Configuration	Nitrogen Recovery Efficiency	Key Advantages	Key Challenges
Distillation	Reduced Pressure Distillation with Alkaline Conditions	20.5% to 52.2% [10]	High water recovery; Simplicity	High energy demand; Moderate N recovery
Membrane Separation	Hollow Fibre Membrane Contactor (HFMC)	Up to 99% [23]	High selectivity; Modularity	Membrane fouling; NaOH consumption for pH control
Electrodialysis	Cascading ED + Hollow Fibre Membrane Contactor	~90% Ammonia Recovery [24]	High concentration factor (~200x); Lower energy vs. Haber-Bosch	System complexity; Scaling potential

Detailed Methodologies & Experimental Protocols

Distillation-Based Nitrogen Recovery

Distillation, particularly under reduced pressure, is employed to separate volatile ammonia from hydrolyzed urine after the urea has been converted to ammonium carbonate.

Application Notes

In the "Lunar Palace 1" BLSS experiment, a two-step process was used: urine was first pre-hydrolyzed, followed by reduced pressure distillation under alkaline conditions to collect ammonia gas [10]. Two pretreatment methods were investigated: High-Temperature Acidification Method (HTAM) and Immobilized Urease Catalysis Method (IUCM). The IUCM method demonstrated superior nitrogen recovery efficiency and operational stability, making it more suitable for a controlled BLSS environment [10].

Experimental Protocol: Immobilized Urease Catalysis Method (IUCM) with Distillation

Research Reagent Solutions & Essential Materials Table 2: Key Reagents for Distillation-Based Recovery

Item	Function
Immobilized Urease	Biological catalyst that hydrolyzes urea to ammonium carbonate.
Sulfuric Acid (H₂SO₄) Solution	Acid trap to capture and stabilize volatilized ammonia as ammonium sulfate.
Sodium Hydroxide (NaOH)	Adjusts pH to alkaline conditions (>10) to shift NH₄⁺ to volatile NH₃ for distillation.
Real Human Urine	Feedstock containing urea and inorganic salts.

Procedure:

• Urine Hydrolysis: Place real human urine in a reactor. Add the immobilized urease. Maintain the temperature at 60°C and pH at 7.0 for 40 minutes with continuous mixing to ensure complete hydrolysis of urea [10].
• Distillation Setup: Transfer the hydrolyzed urine to a distillation apparatus. Connect the vapor outlet to an acid trap containing a known concentration and volume of sulfuric acid.
• Ammonia Stripping: Initiate reduced-pressure distillation. The alkaline conditions (naturally resulting from hydrolysis) will promote the conversion of NH₄⁺ to NH₃ gas, which will be carried over and captured in the acid trap, forming an ammonium sulfate solution [10].
• Product Collection: The process is complete when the temperature of the distillate drops. The ammonium sulfate in the acid trap is the final nitrogen product. The residual liquid in the distillation flask is desalinated water.

Membrane Separation for Nitrogen Recovery

Hollow Fibre Membrane Contactors (HFMCs) are gas-permeable membranes that enable selective recovery of ammonia from a nitrogen-rich feed solution.

Application Notes

HFMCs provide an interface for ammonia gas (NH₃) to transfer from a pressurized feed stream to an acid collector stream. The driving force is the concentration gradient of free ammonia across the membrane [23]. A critical operational requirement is maintaining the feed solution pH above 8.6 to shift the ammonium-ammonia equilibrium towards gaseous NH₃ [23]. This technology is notable for its high efficiency, modularity, and selectivity.

Experimental Protocol: pH-Controlled Batch Operation of HFMC

Research Reagent Solutions & Essential Materials Table 3: Key Reagents for Membrane Separation

Item	Function
Polypropylene (PP) or Polytetrafluoroethylene (PTFE) HFMC	Gas-permeable membrane providing interface for NH₃ transfer.
Sulfuric Acid (H₂SO₄) Solution	Acid collector that reacts with NH₃ to form stable ammonium sulfate fertilizer.
Sodium Hydroxide (NaOH) Solution	Maintains high pH in the feed solution to ensure a constant driving force.
Synthetic Nitrogen-Rich Feed Solution	Simulated wastewater or pre-hydrolyzed urine.

Procedure:

• System Setup: Pump the nitrogen-rich feed solution (e.g., hydrolyzed urine, pH pre-adjusted to >8.6) through the shell side of the HFMC module. Pump the sulfuric acid solution counter-currently through the lumen side. Both streams are recycled to their respective reservoirs [23].
• Batch Process Control: Monitor the pH of the feed solution in real-time.
  • The transfer of NH₃ across the membrane consumes protons (H⁺), causing the feed pH to rise.
  • Use a peristaltic pump to add NaOH automatically to maintain a constant set-point pH (e.g., 9.5) [23].
• Process Termination via pH Slope: The end of a batch cycle is determined by a drop in the rate of pH increase (pH slope), which correlates with a low residual ammonia concentration. A pre-determined threshold pH slope value signals the system to stop [23].
• Product Harvesting: Replace the exhausted feed solution with a fresh batch. The ammonium sulfate solution in the acid tank is harvested as a liquid fertilizer when nearly saturated.

Electrodialysis for Nitrogen Recovery

Electrodialysis (ED) uses an electric field to selectively separate and concentrate ions, such as ammonium (NH₄⁺), from a waste stream.

Application Notes

For nitrogen recovery in BLSS, ED is particularly effective as a preconcentration step. Recent research demonstrates a cascading ED system coupled with a Hollow Fibre Membrane Contactor (ED+HFMC) to achieve very high concentration factors (~200x), producing a fertilizer-grade solution with ~10 wt% NH₄⁺-N [24]. The energy consumption of this integrated system (1.89–6.14 kWh/kg NH₄⁺-N) is significantly lower than the Haber-Bosch process (8.9–19.3 kWh/kg N) [24].

Experimental Protocol: Cascading Electrodialysis with Integrated Stripping

Research Reagent Solutions & Essential Materials Table 4: Key Reagents for Electrodialysis

Item	Function
Cation Exchange Membrane (CEM)	Allows selective passage of cations (e.g., NH₄⁺).
Anion Exchange Membrane (AEM)	Allows selective passage of anions (e.g., Cl⁻).
Synthetic CAFO Wastewater	Model urine wastewater with ~500 mg/L NH₄⁺-N.
Sodium Sulfate (Na₂SO₄) Electrode Rinse	Conducting solution to protect electrodes.
Sulfuric Acid (H₂SO₄) Solution	Acid trap for final HFMC concentration step.

Procedure:

• ED System Configuration: Set up a bench-scale electrodialyzer with multiple cell pairs, each consisting of a CEM and an AEM. Use a potentiostat to operate in chronopotentiometry mode (constant current) [24].
• Staged Concentration:
  • Stage 1 (ED): Pump the synthetic wastewater (diluate) and a receiver stream (concentrate) through alternating compartments. Apply a current density to drive NH₄⁺ ions into the concentrate stream. This first stage can achieve a ~40x concentration factor [24].
  • The specific energy consumption (SEC) is calculated as: ( SEC = \frac{\int i \cdot A{ED} \cdot U(t) \, dt}{(c{NH4^+}(t) \cdot V(t) - c{NH4^+}(0) \cdot V(0))} ) where (i) is current density, (A{ED}) is membrane area, and (U(t)) is voltage [24].
• Final Concentration (HFMC): The concentrated stream from the ED stage is then fed to a Hollow Fibre Membrane Contactor, as described in Section 3.2.2, for final polishing and concentration, achieving an additional ~5x concentration factor and yielding a final ~10 wt% NH₄⁺-N product [24].

Process Integration and Visualization

The logical workflow for selecting and integrating these technologies, particularly for achieving high-value fertilizer products, is depicted below.

In the context of Bioregenerative Life Support Systems (BLSS) for long-distance space exploration, the efficient recovery of nitrogen from human waste streams is a critical technological challenge. The inability of current physicochemical systems on the International Space Station (ISS) to recover nitrogen for food production creates a dependency on resupply missions from Earth, which is logistically and economically prohibitive for deep space missions [3]. With human urine containing 85% of the potentially recoverable nitrogen (7-16g per crew member daily) in a BLSS, primarily in the form of urea, it represents the most significant nutrient source for reconstitution into fertilizers for food production [3]. Biological pretreatment through immobilized urease and urea-hydrolyzing biofilms serves as the foundational process for converting this urea into forms more readily assimilated by plants or other biological components in the BLSS, thereby closing the nitrogen loop [25] [3].

This Application Note provides detailed protocols for establishing and optimizing two complementary biological pretreatment strategies: enzyme-based systems using immobilized urease and microbial-based systems utilizing ureolytic biofilms. Both approaches aim to enhance the efficiency, stability, and operational control of urea hydrolysis, which is the rate-limiting step for subsequent nitrogen recovery processes such as nitrification or direct application as liquid fertilizer [26] [3].

Theoretical Background and Literature Review

Urea Hydrolysis Chemistry and Enzyme Fundamentals

Urease (urea amidohydrolase, EC 3.5.1.5) catalyzes the hydrolysis of urea into ammonia and carbamate, with the latter spontaneously decomposing to yield a second molecule of ammonia and carbon dioxide [27] [28]. In aqueous solution, this reaction results in a significant pH increase due to ammonia formation. The enzyme is a protein with precise three-dimensional structure maintained by its amino acid sequence and stabilized by specific environmental conditions [27]. Traditional free enzyme applications face limitations in BLSS due to enzyme instability, single-use nature, and difficulty in separation from reaction mixtures [27].

Immobilization Rationale and Benefits

Enzyme immobilization addresses several key limitations for space-based applications by enhancing operational stability, enabling enzyme reuse, facilitating product separation, and allowing continuous process operation [27]. The European Space Agency's MELiSSA initiative represents one of the most advanced BLSS programs implementing such engineered biological systems for over 30 years, with nitrogen recovery being a central focus of compartment III [3].

Table 1: Comparative Advantages of Immobilized Enzyme Systems in BLSS

Characteristic	Free Enzyme	Immobilized Enzyme	Relevance to BLSS
Operational Stability	Low, single-use	High, reusable	Reduces resupply mass
Process Control	Batch only	Continuous possible	Stable operation
Product Separation	Difficult	Straightforward	Downstream processing
Environmental Tolerance	Narrow pH/temperature	Broadened	Flexible operation
Space Requirements	Larger bioreactors	Compact systems	Limited spacecraft volume

Materials and Reagents

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Reagents and Materials for Immobilized Urease and Biofilm Research

Item	Function/Application	Examples/Specifications
Urease Enzyme	Biological catalyst for urea hydrolysis	Jack bean urease, microbial urease (e.g., from Sporosarcina pasteurii)
Carrier Materials	Solid supports for immobilization	Alginate, chitosan, polyacrylamide, silica gel, ion-exchange resins
Cross-linking Agents	Enzyme fixation to supports	Glutaraldehyde, dimethyl suberimidate
Activity Assay Reagents	Enzyme activity measurement	Phenol-red indicator, urea standard solutions, buffer solutions
Biofilm Support Matrix	Surface for microbial attachment	Ceramic rings, porous glass beads, activated carbon, polymer membranes
Nutrient Media	Cultivation of ureolytic bacteria	Yeast extract, ammonium salts, trace elements, urea
Analytical Instruments	Process monitoring	pH meter, NH4+-selective electrode, spectrophotometer

Application Notes: Experimental Protocols

Protocol 1: Preparation of Immobilized Urease via Covalent Binding

Principle: This method involves forming stable covalent bonds between enzyme functional groups (-NH₂, -COOH, -SH, -OH) and reactive groups on an activated carrier surface, producing a highly stable immobilized enzyme preparation [27].

Materials:

• Urease enzyme (lyophilized powder, ≥10 U/mg)
• Carrier material (silica gel, 100-200 mesh, with surface amino groups)
• Glutaraldehyde solution (2.5% v/v in phosphate buffer, pH 7.0)
• Phosphate buffer (0.1 M, pH 7.0)
• Ethanolamine solution (1.0 M, pH 8.0)
• Vacuum filtration setup
• Orbital shaker incubator

Procedure:

• Carrier Activation: Wash 10g of silica gel carrier with 100mL of deionized water followed by 50mL of phosphate buffer (0.1M, pH 7.0). Add 50mL of 2.5% glutaraldehyde solution to the washed carrier and incubate with gentle shaking (100rpm) for 2 hours at 4°C [27].
• Washing: Remove excess glutaraldehyde by vacuum filtration and wash the activated carrier with 200mL of cold phosphate buffer until the wash shows no absorbance at 280nm.
• Enzyme Binding: Dissolve 500mg of urease in 50mL of phosphate buffer (0.1M, pH 7.0). Add this solution to the activated carrier and incubate with shaking (50rpm) for 12 hours at 4°C [27].
• Blocking: Recover the immobilized enzyme by filtration and treat with 50mL of 1.0M ethanolamine (pH 8.0) for 1 hour to block remaining active sites.
• Final Wash: Wash thoroughly with 200mL of phosphate buffer followed by 100mL of deionized water. Store the prepared immobilized urease at 4°C in phosphate buffer until use.

Quality Control:

• Determine enzyme loading by measuring initial and final protein concentration in the binding solution (Bradford method).
• Assess activity by measuring ammonia production from urea hydrolysis per unit time.
• Typical immobilization efficiency: 60-80% protein binding with 30-50% retention of initial activity [27].

Protocol 2: Entrapment of Urease in Calcium Alginate Beads

Principle: Enzyme molecules are physically confined within the interstitial spaces of a polymer network, providing a gentle immobilization method suitable for whole cells or delicate enzymes [27].

Materials:

• Sodium alginate (2-4% w/v in deionized water)
• Calcium chloride solution (0.1M)
• Urease enzyme (lyophilized powder)
• Syringe with needle (22G) or peristaltic pump with droplet generator

Procedure:

• Preparation of Enzyme-Alginate Mixture: Dissolve 200mg of urease in 10mL of sodium alginate solution (2% w/v). Mix thoroughly without forming bubbles.
• Bead Formation: Using a syringe or droplet generator, add the enzyme-alginate mixture dropwise into 100mL of gently stirred 0.1M CaCl₂ solution. Maintain beads in the CaCl₂ solution for 1 hour for complete gelation [27].
• Curing and Washing: Collect the beads by filtration and wash with 50mL of phosphate buffer (0.1M, pH 7.0) to remove surface-bound enzyme and excess Ca²⁺.
• Storage: Store the beads in phosphate buffer at 4°C. Beads remain stable for several weeks.

Performance Characteristics:

• Bead diameter: 1-3mm
• Operational stability: >20 batches with <50% activity loss
• Diffusion limitations may reduce apparent activity by 20-40% compared to free enzyme

Protocol 3: Establishment of Urea-Hydrolyzing Biofilms

Principle: This method cultivates urease-producing microorganisms (e.g., Sporosarcina pasteurii, Proteus vulgaris) as biofilms on support matrices, creating self-regenerating catalytic systems with potential for long-term operation in BLSS [28].

Materials:

• Ureolytic bacterial strain (e.g., Sporosarcina pasteurii ATCC 11859)
• Nutrient broth with urea (20g/L)
• Biofilm support matrix (ceramic rings, porous glass beads, or polymer carriers)
• Continuous-flow reactor system with aeration capability
• Artificial urine formulation

Procedure:

• Inoculum Preparation: Culture the selected ureolytic strain in nutrient broth with 20g/L urea for 24-48 hours at 30°C with shaking (150rpm) to reach mid-log phase (OD₆₀₀ ≈ 0.8-1.0).
• Biofilm Inoculation: Pack the bioreactor with selected support material. Recirculate the bacterial inoculum through the reactor for 24 hours without flow to allow initial attachment.
• Biofilm Development: Initiate continuous flow (hydraulic retention time 4-6 hours) with nutrient medium containing urea (10g/L) and essential minerals. Maintain for 7-14 days to establish mature biofilm [28].
• Process Optimization: Monitor urea hydrolysis efficiency by measuring residual urea and ammonium production. Adjust flow rate, urea concentration, and aeration to maximize conversion efficiency.

Performance Metrics:

• Target urea hydrolysis rate: >90% conversion at steady state
• Biofilm thickness: 100-500µm (visualized by microscopy)
• Operational longevity: >60 days with stable performance

Data Presentation and Analysis

Quantitative Performance Comparison

Table 3: Performance Characteristics of Different Urea Hydrolysis Systems

Parameter	Free Urease	Covalently Immobilized Urease	Alginate-Entrapped Urease	Ureolytic Biofilm
Optimal pH	7.0-7.5	6.5-8.0	6.5-7.5	7.0-8.5
Optimal Temperature (°C)	35-40	40-50	35-45	25-37
Operational Half-life	<24 hours	15-30 days	10-20 days	30-60 days (continuous)
Urea Conversion Efficiency	>95% (initial)	70-85%	60-80%	85-95% (steady state)
Reusability	Not reusable	>20 cycles	15-25 cycles	Self-regenerating
Inhibition by NH4+	Moderate	Reduced	Moderate	Variable
Activation Energy (kJ/mol)	~30	~35	~38	N/A

Process Integration and Workflow

The following diagram illustrates the experimental workflow for developing and evaluating immobilized urease systems and ureolytic biofilms for BLSS applications:

Process Integration in BLSS Context

The following diagram illustrates how urea hydrolysis integrates with broader nitrogen recovery processes in a BLSS:

Troubleshooting and Optimization

Low Immobilization Efficiency:

• Increase activation time or cross-linker concentration
• Try alternative carrier materials with different surface properties
• Verify enzyme activity prior to immobilization

Reduced Operational Stability:

• Incorporate enzyme stabilizers (e.g., polyols, sugars) during immobilization
• Optimize operational parameters (flow rate, substrate concentration)
• Implement protective pretreatment for feed streams

Suboptimal Biofilm Performance:

• Ensure adequate nutrient supply during biofilm development
• Monitor and control shear forces in the reactor
• Verify absence of inhibitory compounds in feed

Diffusion Limitations:

• Reduce carrier particle size or biofilm thickness
• Increase porosity of immobilization matrix
• Optimize reactor mixing or flow patterns

The implementation of immobilized urease and urea-hydrolyzing biofilms represents a crucial enabling technology for sustainable nitrogen management in BLSS. These biological pretreatment strategies transform unstable, single-use processes into robust, continuous operations essential for long-duration space missions. The protocols detailed in this Application Note provide researchers with methodologies to develop, optimize, and integrate these systems within the broader context of resource recovery in closed-loop life support systems. Future research directions should focus on enhancing stability under space conditions (radiation, microgravity), integrating with downstream nitrogen processing, and validating performance in ground and space demonstration trials [26] [3].

The Micro-Ecological Life Support System Alternative (MELiSSA) is a bioregenerative life support system (BLSS) project developed by the European Space Agency (ESA) to sustain human life during long-term space missions. Designed as a closed-loop artificial ecosystem, it aims to recycle waste into oxygen, water, and food through a series of interconnected biological compartments. The system is structured as an assembly of unit processes, or compartments, to simplify the behavior of an artificial ecosystem and allow a deterministic engineering approach [29]. The inability of current physicochemical-based systems on the International Space Station (ISS) to produce food and recover resources at sufficiently high efficiencies creates a dependency on resupply missions from Earth, which is logistically challenging and cost-prohibitive for missions to Mars or beyond [1]. MELiSSA represents a paradigm shift towards bioregenerative life support, combining biological and physicochemical processes to enable in situ production of vital resources through highly efficient recovery of minerals from waste streams [1].

Compartmental Framework of MELiSSA

The MELiSSA loop is engineered as a five-compartment system, with each compartment performing specific functions in the degradation and conversion of waste products. The flow of material between these compartments is designed to mimic a lake ecosystem [1]. The system's compartments, their key functions, and the primary microorganisms involved are summarized in the table below.

Table 1: Functional Overview of the MELiSSA Compartments

Compartment	Primary Function	Key Microorganisms/Components	Key Processes
CI	Organic waste degradation & solubilisation [29]	Thermophilic anoxygenic bacteria [29]	Anaerobic fermentation producing CO₂, volatile fatty acids (VFAs), and ammonia (NH₃) [29]
CII	Removal of carbon compounds [29]	Photoheterotrophic bacteria [29]	Conversion of VFAs into inorganic carbon (e.g., CO₂) and bacterial biomass [29]
CIII	Nitrification of ammonia to nitrate [29]	Nitrosomonas europaea, Nitrobacter winogradskyi [29]	Oxidation of ammonium (NH₄⁺) to nitrite (NO₂⁻) and subsequently to nitrate (NO₃⁻) [29]
CIV	Food and oxygen production, CO₂ removal, water recovery [29]	(CIVa) Arthrospira platensis (cyanobacteria); (CIVb) Higher plants (32 candidate crops) [29]	Photosynthesis using CO₂ and nutrients (e.g., NO₃⁻) to produce edible biomass, O₂, and clean water [29]
CV	Consumption and waste production	Human crew [29]	Respiration (CO₂ production), urination, defecation, and consumption of food, O₂, and water [29]

The logical relationships and primary mass flows between these compartments are visualized in the following diagram:

Nitrogen Recovery: The Core Challenge

In a BLSS, nitrogen recovery is a critical process because it is an essential nutrient for plant growth, which provides food and oxygen for the crew. With an average daily excretion of 7–16g of nitrogen per crew member, urine accounts for approximately 85% of the total potentially recoverable nitrogen in a BLSS, making it the primary target for nutrient recycling [1]. The nitrogen in urine is mostly in the form of urea, which rapidly hydrolyzes into ammonia and carbonate [1]. The challenge lies in converting this ammonia into a non-toxic and bioavailable form, primarily nitrate, for higher plants in Compartment IVb [29] [30]. This conversion is vital for closing the nitrogen loop and ensuring the system's self-sufficiency. A full nitrogen balance at the habitat level is also necessary; sufficient N₂ must be maintained for atmospheric pressure, while enough mineral nitrogen must be provided to plants for biomass production [30].

Experimental Protocols for Key Processes

Protocol: Urine Collection and Stabilization

Objective: To collect and chemically stabilize crew urine to prevent scaling (mineral precipitation) and microbial activity, enabling safe downstream processing [1].

Materials:

• Urine collection device with storage tank
• Phosphoric acid (H₃PO₄) solution
• Chromium (Cr⁶⁺) solution (Note: Due to toxicity, alternative stabilizers are an active area of research for terrestrial and space applications)

Procedure:

• Collect urine and urine flush water from the crew. The expected volume is approximately 1.80 L per crew member per day [1].
• Transfer the collected urine to a dedicated Wastewater Storage Tank Assembly (WSTA).
• Acidify the urine to a target pH of approximately 2.0 by adding a pre-determined volume of H₃PO₄ solution. This step converts volatile ammonia (NH₃) to non-volatile ammonium (NH₄⁺) and helps prevent mineral scaling [1].
• Add a chromium (Cr⁶⁺) solution to the acidified urine to maintain chemical stability and sterility during storage [1].
• The stabilized urine is now ready for processing in Compartment I.

Protocol: Nitrification in a Fixed-Bed Reactor (Compartment III)

Objective: To convert ammonium (NH₄⁺) into nitrate (NO₃⁻) using a co-culture of nitrifying bacteria in a biofilm reactor for downstream use in plant cultivation [29].

Materials:

• Fixed-bed reactor (e.g., packed column with high-surface-area media)
• Feed solution containing stabilized ammonium (from CII)
• Air supply system with fine bubble diffuser
• Nitrosomonas europaea (ammonia-oxidizing bacteria) culture
• Nitrobacter winogradskyi (nitrite-oxidizing bacteria) culture
• pH and temperature probes and controllers
• In-line sensors for dissolved oxygen (DO), NH₄⁺, NO₂⁻, and NO₃⁻

Procedure:

• Biofilm Establishment: Inoculate the fixed-bed reactor with pure cultures of N. europaea and N. winogradskyi. Continuously circulate a nutrient medium containing ammonium to allow for biofilm formation on the fixed-bed media. This process may take several weeks.
• Reactor Operation: Once a stable biofilm is established, begin continuous operation.
  • Pump the feed solution (effluent from CII, rich in NH₄⁺) into the bottom of the reactor at a controlled flow rate.
  • Simultaneously, supply compressed air to maintain a high dissolved oxygen concentration (>2 mg/L), as nitrification is an aerobic process.
  • Maintain a neutral pH (7.0-8.0) and a constant temperature optimal for the bacteria (e.g., 28-30°C). Narrow pH control bands enhance process stability [31].
• Process Monitoring: Continuously monitor the effluent for NH₄⁺, NO₂⁻, and NO₃⁻ concentrations to assess conversion efficiency. The target is complete conversion of NH₄⁺ to NO₃⁻ with negligible NO₂⁻ accumulation.
• Product Harvest: The effluent, now rich in nitrate, is directed to the hydroponic system of Compartment IVb (Higher Plant Compartment) to serve as a primary nitrogen fertilizer [29].

Table 2: Research Reagent Solutions for MELiSSA Nitrogen Recovery Research

Reagent/Material	Function in Experimentation	Example Application in MELiSSA
Nitrifying Bacteria Consortia	To convert toxic ammonia into plant-available nitrate.	Pure cultures of Nitrosomonas europaea and Nitrobacter winogradskyi are used in Compartment III fixed-bed reactors [29].
Stabilization Acids	To acidify urine, preventing urea hydrolysis and mineral scaling in collection systems.	Phosphoric acid (H₃PO₄) is used for urine stabilization in the ISS UPA and is a reference for BLSS design [1].
Hydroponic Nutrient Solution	To deliver recovered minerals, including nitrate, to plants in a controlled manner.	The nitrate-rich effluent from Compartment III is a key component of the nutrient solution for higher plants in Compartment IVb [29] [30].
Fixed-Bed Biofilm Support Media	To provide a surface for slow-growing nitrifying bacteria to attach and form a stable, concentrated biofilm.	Used in Compartment III reactors to retain high bacterial biomass, ensuring efficient nitrification despite slow growth rates [29].

Visualization of the Nitrogen Recovery Pathway

The core biochemical pathway for nitrogen recovery within the MELiSSA loop, particularly focusing on the actions of Compartment III, is detailed below. This pathway transforms ammonia from waste into a usable plant nutrient.

Nitrification is a crucial aerobic process in the nitrogen cycle, converting ammonia (NH₃–NH₄⁺) into nitrite (NO₂⁻) and then to nitrate (NO₃⁻) [32]. In the context of a Bioregenerative Life Support System (BLSS), efficient nitrogen recovery from human urine is paramount for creating a closed-loop system for plant fertilization. As a concentrated source of nitrogen, phosphorus, and potassium, urine represents a valuable resource that can sustain plant growth in a BLSS, reducing reliance on external inputs [33]. This document details the application and protocols for studying nitrification processes, with a specific focus on optimizing the conversion of urine-derived ammonia into plant-available nitrate for BLSS research.

Quantitative Data on Nitrification

The following tables consolidate key quantitative data on nitrification from field observations and engineered systems, providing benchmarks for BLSS process design and evaluation.

Table 1: Key Environmental Regulators of Nitrification [32]

Environmental Factor	Effect on Nitrification
Light / Solar Radiation	Generally inhibits nitrifier growth and activity.
pH	Lower pH (ocean acidification) decreases ammonia oxidation rates. The substrate for ammonia oxidation (NH₃) becomes less available at lower pH.
Temperature	Warming enhances enzyme activity and shifts the NH₄⁺–NH₃ equilibrium towards NH₃.
Oxygen	Process requires oxygen (aerobic).
Substrate Concentration	Plays an important role in controlling rates and nitrifier abundance.

Table 2: Representative Nitrification Rates and Organism Abundances from Environmental Systems [32]

Parameter	Number of Measurements	Observed Range	Notable Extremes
Ammonia Oxidation Rate	2393	Wide range, typically low	Up to 4900 nmol L⁻¹ d⁻¹ in the Peruvian oxygen-minimum zone.
Nitrite Oxidation Rate	1006	Wide range, typically low	High rates reported from anoxic waters.
Ammonia Oxidizer Abundance	2242	Varies by orders of magnitude	-
Nitrite Oxidizer Abundance	631	Varies by orders of magnitude	-

Experimental Protocols

Protocol: Tracking Nitrification Dynamics in a Bioreactor

This protocol outlines the methodology for monitoring the nitrification process in a closed-system bioreactor, simulating BLSS conditions for urine nitrogen recovery [34].

Materials and Reagents

• Bioreactor system with anoxic and oxic tanks
• Synthetic wastewater or real urine feedstock
• In-line sensors for pH, temperature, and dissolved oxygen
• Sampling apparatus (sterile syringes, vials)

Procedure

• System Inoculation and Operation: Inoculate the bioreactor with a nitrifying microbial community. Operate the system as an anoxic/oxic (AO) process.
• Feedstock Introduction: Introduce the feedstock (e.g., synthetic urine with ammonium as the primary nitrogen source) to the anoxic tank. Maintain a chemical oxygen demand (COD) of approximately 2,500 mg/L and a total nitrogen (TN) concentration exceeding 100 mg-N/L [34].
• Daily Monitoring: Collect daily samples from key points (influent, anoxic tank, oxic tank effluent).
• Parameter Analysis:
  • Inorganic Nitrogen Speciation: Measure concentrations of ammonium (NH₄⁺), nitrite (NO₂⁻), and nitrate (NO₃⁻) using standard methods (e.g., colorimetric assays, ion chromatography).
  • Carbon and Solids: Analyze total nitrogen (TN), inorganic carbon (IC), and mixed liquor suspended solids (MLSS).
• Rate Calculation: Calculate the nitrification rate based on the disappearance of ammonium and the appearance of nitrate over time in the oxic tank.

Protocol: Investigating Comammox Activity

This protocol describes methods for detecting and confirming the activity of complete ammonia-oxidizing (comammox) bacteria, which can be relevant for efficient, single-organism nitrification in simplified BLSS microbial communities [35].

Materials and Reagents

• Inhibitors: Allylthiourea (ATU), a potent inhibitor of canonical bacterial ammonia oxidation [35].
• Isotopic Tracer: ¹⁵N-labelled ammonium.
• Fixative: Fluorescein thiocarbamoylpropargylamine (FTCP), a fluorescently labelled acetylene analogue that binds to the AMO enzyme [35].
• Molecular Biology Reagents: PCR reagents, primers for amoA genes, FISH probes.

Procedure

• Batch Incubation: Set up batch incubations with biomass from an enrichment culture or reactor.
• Inhibition Assay:
  • Add ATU to parallel incubations.
  • Monitor the oxidation of ammonium and nitrite to nitrate.
  • Interpretation: If ammonia oxidation is inhibited by ATU but nitrite oxidation remains unaffected, it suggests the presence of a bacterial AMO enzyme. However, comammox bacteria are also sensitive to ATU. Further genetic analysis is required for confirmation.
• Stable Isotope Probing: Supply ¹⁵N-labelled ammonium to the culture and track the production of ¹⁵N-labelled nitrite and ¹⁵N-labelled N₂ gas (if anammox bacteria are present) using mass spectrometry [35].
• Genetic Detection:
  • Extract total DNA from biomass.
  • Perform metagenomic sequencing and analysis to identify organisms encoding genes for both AMO/hydroxylamine dehydrogenase (HAO) and nitrite oxidoreductase (NXR) [35].
• Enzyme Labelling (Advanced): Incubate biomass with FTCP. Use fluorescence microscopy in conjunction with Nitrospira-specific FISH probes to confirm the presence of the AMO enzyme within comammox Nitrospira cells [35].

Protocol: Urine Stabilization and Nutrient Reclamation

This protocol covers the initial processing of source-separated urine for subsequent nitrification, based on practical field applications [33].

Materials

• Source-separating toilets or urinals
• Collection containers (plastic jugs) or large storage tanks
• Dilution water

Procedure

• Collection: Collect human urine separately from other wastewater using dedicated plumbing.
• Stabilization: Store urine in sealed containers for a period (e.g., two months) to reduce pathogen load [33].
• Application/Nutrient Extraction:
  • Direct Use: Dilute stored urine with water (e.g., 1:3 to 1:10 ratio) and apply directly as a fertilizer [33].
  • Nutrient Concentration: Process urine via distillation to concentrate nitrogen compounds. Precipitate phosphorus as struvite (magnesium ammonium phosphate) for a solid, slow-release fertilizer [33].

Process Visualization

Nitrification Pathways in Nitrogen Recovery

Experimental Workflow for Nitrification Analysis

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents and Materials for Nitrification Research

Research Reagent / Material	Function and Application
Allylthiourea (ATU)	A specific inhibitor of the bacterial ammonia monooxygenase (AMO) enzyme. Used in inhibition assays to distinguish bacterial ammonia oxidation from archaeal activity and to probe comammox function [35].
¹⁵N-labelled Ammonium	A stable isotopic tracer. Used to track the pathway of ammonia through the nitrification process (e.g., to ¹⁵N-nitrite/ nitrate) or linked processes like anammox (to ²⁹N₂/³⁰N₂), allowing for precise rate measurements [35].
Fluorescein Thiocarbamoylpropargylamine (FTCP)	A fluorescent probe that acts as a suicide substrate for the AMO enzyme. Used for in situ labelling and visualization of cells possessing active AMO, typically via microscopy [35].
Specific PCR Primers	Oligonucleotides designed to target functional genes such as amoA (for AOA, AOB, comammox) and 16S rRNA genes of nitrifiers. Used for detecting and quantifying nitrifying populations in microbial communities [32] [35].
FISH Probes	Fluorescently labelled oligonucleotide probes that bind to specific ribosomal RNA sequences. Used for the microscopic identification, enumeration, and spatial localization of nitrifying microorganisms within samples [35].
Urea	Serves as an alternative ammonium source for nitrification studies. Used to test an organism's ability to produce ammonium via urease activity and subsequently nitrify it [35].

The integration of advanced biotechnologies within Bioregenerative Life Support Systems (BLSS) is paramount for enabling long-duration human space exploration. These systems create an Earth-like microenvironment where oxygen, water, and food are recycled using biological and engineering control technologies, representing the most advanced life support capability for missions beyond low-Earth orbit [36]. A critical challenge in maintaining these closed-loop systems is the efficient management of nitrogen, an essential element for plant growth and protein synthesis. Nitrogen recovery from human waste, particularly urine, presents a significant opportunity to enhance system closure and sustainability. Emerging bioelectrochemical and microbial technologies offer promising pathways for converting this waste stream into valuable, nitrogen-rich fertilizers, thereby supporting crop production and reducing reliance on external supplies [37] [38].

The paradigm is shifting from viewing wastewater as a problem to be treated to recognizing it as a source of valuable resources. Reactive nitrogen in wastewater can be recovered and utilized, challenging the conventional approach of converting it to inert nitrogen gas (N₂), which requires significant energy input and results in the loss of a bioavailable nutrient [38]. This is particularly relevant in space habitats, where the nitrogen in human urine is equivalent to a substantial portion of annual fertilizer demand; one study estimates it could fulfill approximately 14% of global fertilizer needs, highlighting its immense potential value within a BLSS [37].

Core Biotechnologies for Nitrogen Recovery and Valorization

Bioelectrochemical Systems (BES) for Nitrogen Recovery

Bioelectrochemical Systems represent a cutting-edge platform for simultaneous wastewater treatment and resource recovery. These systems utilize the unique extracellular electron transfer capabilities of electroactive bacteria (e.g., Geobacter species) to convert the chemical energy of organic pollutants directly into electrical energy or valuable products [38]. For nitrogen recovery, BESs leverage the electric field between anode and cathode to drive the directional migration of charged ions, such as ammonium (NH₄⁺), across ion exchange membranes, effectively concentrating nitrogen from a waste stream.

A key microbial process enabled by BES is Dissimilatory Nitrate Reduction to Ammonium (DNRA). Certain electroactive bacteria can reduce nitrate (NO₃⁻) and nitrite (NO₂⁻) present in wastewater to ammonium, rather than to nitrogen gas. This pathway is crucial because it conserves bioavailable nitrogen in a form readily usable by plants, overcoming a major limitation of traditional nitrogen removal processes [38]. The concentrated ammonium stream can then be directly used as a fertilizer or further processed into more stable products like ammonium sulfate.

Table 1: Comparison of Carbon Sources for Microbial Biomanufacturing of High-Value Products

Characteristic	Ethanol (C₂)	Glucose	Acetate (C₂)	Methanol (C₁)
Molecular Weight	46	180	60	32
Carbon Content (%)	52.1	40	40.7	37.5
State at Room Temp.	Liquid	Solid	Liquid	Liquid
ATP Yield (mol/g)	0.326	0.178	0.133	-
Carbon to Acetyl-CoA Recovery	100%	66.67%	100%	-
NADH Generated	2	4	0	-
Key Advantage	High energy density, simple metabolism	Standard, well-understood	Non-corrosive, simple pathway	Abundant and cheap

Source: Adapted from [39]

Engineered Microbial Biosynthesis

Beyond nutrient recovery, engineered microbes can transform recovered nitrogen compounds into a diverse array of high-value products. This biomanufacturing relies on using microorganisms as cellular factories. The choice of carbon source is critical in these processes, as it significantly impacts both the efficiency and the cost of production [39].

As shown in Table 1, ethanol is emerging as a superior carbon source for producing acetyl-CoA-derived compounds. Its advantages include a higher energy density than glucose, 100% carbon recovery during catabolism to acetyl-CoA, and the generation of reducing power (NADH) without an additional energy input [39]. Furthermore, ethanol is less toxic to microorganisms than methanol and is easier to handle than gaseous C1 sources like CO₂. Microbial metabolism of ethanol primarily occurs through the alcohol dehydrogenase (ADH) pathway, where ethanol is first converted to acetaldehyde and then to acetate, which is subsequently activated to acetyl-CoA to feed the central metabolism [39].

Application Note: A Prototype for Integrated Urine Processing and Fertilizer Generation

A Stanford-led research team has developed an integrated prototype that exemplifies the convergence of these biotechnologies for BLSS applications. The system is designed to recover nitrogen from urine as ammonium sulfate, a valuable fertilizer, using a solar-powered, electrochemical process [37].

The following diagram illustrates the core workflow and technologies involved in this integrated nutrient recovery system:

Key Performance Data and Economic Outlook

The Stanford prototype demonstrated significant efficiency improvements through system integration. The use of waste heat from solar panels to warm the liquid increased power generation by nearly 60% and improved ammonia recovery efficiency by more than 20% compared to non-integrated designs [37]. This synergistic use of energy is critical for optimizing performance in resource-limited environments like a lunar or Martian base.

Economic modeling for this technology indicates strong potential, particularly in regions with high fertilizer costs and limited energy infrastructure. The model projected that the system could generate up to $4.13 per kilogram of nitrogen recovered in a context like Uganda, more than double the potential earnings in the U.S. [37]. This underscores the economic viability and relevance of such decentralized systems.

Table 2: Quantitative Performance and Economic Metrics of the Urine Processing Prototype

Parameter	Value	Context / Impact
Nitrogen in Human Urine	~14% of annual fertilizer demand	Highlights global potential of resource recovery [37]
Power Generation Increase	~60%	Achieved by integrating PV waste heat into the process [37]
Ammonia Recovery Efficiency	>20% improvement	Result of optimized heat and electrical current management [37]
Economic Potential (Uganda)	\$4.13 per kg N recovered	Demonstrates high value in resource-limited settings [37]
System Closure Degree (BLSS)	Up to 98.2%	Achieved in the 370-day "Lunar Palace 365" mission [36]

Experimental Protocols

Protocol 1: Operation of a Solar-Powered Nitrogen Recovery Reactor

This protocol details the setup and operation of an electrochemical system for recovering nitrogen from a synthetic urine stream, simulating a BLSS-compatible process.

Research Reagent Solutions & Materials:

• Ion Exchange Membranes: Cation-exchange membrane (e.g., Nafion) to selectively allow ammonium ion (NH₄⁺) passage.
• Electrolyte Solution: Phosphate buffer or sodium sulfate solution for the cathode chamber.
• Synthetic Urine Feedstock: Prepared with urea, salts, creatinine, and other constituents to mimic human urine.
• Absorption Solution: Dilute sulfuric acid (H₂SO₄) to trap recovered ammonia as ammonium sulfate.
• Solar Photovoltaic (PV) Module: With a connected copper tube cold plate for waste heat collection.
• Data Acquisition System: To monitor voltage, current, temperature, and pH.

Procedure:

• Reactor Assembly: Construct a two-chamber electrochemical reactor separated by the cation-exchange membrane. Integrate the copper tube cold plate from the PV module into the urine feedstock line to pre-warm the solution.
• System Initialization: Fill the anode chamber with the synthetic urine feedstock. Fill the cathode chamber with the electrolyte solution and the absorption sulfuric acid trap.
• Power Connection: Connect the electrodes to the solar PV module, ensuring the electrical configuration allows for control of the current supplied to the electrochemical system.
• Process Monitoring: Initiate the feed flow and apply power. Monitor and record the following parameters every 30 minutes for the first 4 hours, then hourly:
  • Temperature of the urine feedstock pre- and post-heat exchange.
  • Current (mA) and Voltage (V) supplied to the reactor.
  • pH in the anode and cathode chambers.
• Product Collection: After 6-8 hours of operation, or when the absorption solution pH rises significantly, collect the ammonium sulfate solution from the cathode trap for analysis.
• Analysis:
  • Quantify ammonium concentration in the product solution using standard colorimetric methods (e.g., Nessler's reagent or indophenol blue method).
  • Calculate the nitrogen recovery efficiency based on the initial nitrogen content in the urine feedstock.

Protocol 2: Cultivation of an Engineered Microbial Strain for Acetyl-CoA Derived Product Synthesis Using Ethanol

This protocol describes the cultivation of a microorganism (e.g., Escherichia coli) engineered to produce a high-value, acetyl-CoA-derived product (e.g., a biopolymer) using ethanol as the primary carbon source.

Research Reagent Solutions & Materials:

• Engineered Microbial Strain: E. coli with metabolic pathway optimized for ethanol assimilation and product synthesis.
• Minimal Salt Medium (MSM): Contains essential salts, nitrogen source (e.g., recovered ammonium sulfate), trace elements, but no carbon source.
• Sterile Ethanol Feed: 100% pure, filter-sterilized.
• Antifoam Agent: To control foaming during fermentation.
• Bioreactor: Bench-top fermenter with controls for dissolved oxygen (DO), temperature, and pH.
• Product-Specific Analysis Kits: e.g., for GC-MS, HPLC, or enzymatic assays.

Procedure:

• Pre-culture Preparation: Inoculate a single colony of the engineered strain into a small volume of MSM supplemented with a low concentration of ethanol (e.g., 0.5% v/v). Incubate overnight with shaking.
• Bioreactor Inoculation: Transfer the pre-culture to the bioreactor containing sterile MSM. The initial working volume should be 60-70% of the total vessel volume.
• Fermentation Parameters: Set and maintain the following conditions:
  • Temperature: 37°C
  • pH: 7.0 (controlled with NH₄OH, linking to the nitrogen recovery theme)
  • Dissolved Oxygen (DO): Maintain at >30% saturation via agitation and aeration.
• Fed-Batch Operation: Once the initial ethanol is consumed (indicated by a DO spike), initiate a fed-batch mode. Feed sterile ethanol at a controlled rate to maintain a low, non-inhibitory concentration in the broth (e.g., <0.1% v/v). This strategy helps mitigate ethanol stress on the microbes [39].
• Process Monitoring: Take samples every 2-4 hours to measure:
  • Optical Density (OD₆₀₀) for cell growth.
  • Residual ethanol concentration.
  • Extracellular product concentration.
• Harvest and Analysis: Terminate the fermentation after 24-48 hours or when growth plateaus. Centrifuge the culture to separate cells from the supernatant.
  • Analyze the supernatant for the target high-value product using appropriate analytical methods (HPLC, GC-MS).
  • Calculate the product yield from ethanol and the specific productivity.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagent Solutions for Microbial Bioprocessing and Nitrogen Recovery

Reagent / Material	Function / Application	Example & Notes
Cation Exchange Membrane	Selectively transports ammonium ions (NH₄⁺) in electrochemical cells.	Nafion membrane; critical for separating and concentrating nitrogen in recovery systems [37] [38].
Electroactive Bacteria	Catalyze redox reactions and electron transfer in Bioelectrochemical Systems.	Geobacter sulfurreducens; capable of Dissimilatory Nitrate Reduction to Ammonium (DNRA) [38].
Ethanol (C₂ Carbon Source)	High-energy, efficient substrate for microbial production of acetyl-CoA-derived products.	Preferred over glucose for higher ATP yield and 100% carbon recovery to acetyl-CoA [39].
Minimal Salt Medium (MSM)	Defined medium for cultivating engineered microbes, forcing them to use the provided target substrates.	Used with recovered ammonium sulfate as the nitrogen source and ethanol as the carbon source [39].
Dilute Sulfuric Acid (H₂SO₄)	Traps recovered ammonia gas to form a stable fertilizer product.	Forms ammonium sulfate ((NH₄)₂SO₄) upon contact with NH₃ [37].
CRISPR/Cas9 Systems	Enables precise genome editing for metabolic engineering of microbial cell factories.	Used to optimize strains for ethanol utilization and high-value product synthesis [40].

Overcoming Operational Challenges and Enhancing Process Efficiency

In the context of Bioregenerative Life Support Systems (BLSS) for long-duration space missions, the efficient recovery of nitrogen from crew urine is paramount for closing the nutrient loop and enabling in-situ production of food [3] [41]. Urine is the largest source of recoverable nitrogen, with 85% of the total potentially recoverable nitrogen found in this waste stream, primarily in the form of urea [3]. Hydrolysis, the process of breaking down urea into ammonia and carbon dioxide, is a critical first step for making this nitrogen available for subsequent biological processing and conversion into fertilizers for plant growth [10] [41]. The optimization of hydrolysis parameters—specifically temperature, pH, and Hydraulic Retention Time (HRT)—is therefore a fundamental research focus for advancing the technological maturity of BLSS. This Application Note provides a detailed comparative analysis of two primary hydrolysis methods and standardized protocols for their optimization, framed within the overarching goal of developing robust nitrogen recovery strategies for space exploration.

Hydrolysis Methodologies and Comparative Analysis

For BLSS applications, two main methodological approaches for urine hydrolysis have been studied extensively: High-Temperature Acidification Method (HTAM), a physicochemical process, and the Immobilized Urease Catalysis Method (IUCM), a biological process [10]. The choice between these methods involves trade-offs between reaction efficiency, energy consumption, and system stability.

Table 1: Comparison of Hydrolysis Methods for Urine Treatment in BLSS

Parameter	High-Temperature Acidification Method (HTAM)	Immobilized Urease Catalysis Method (IUCM)
Principle	Chemical hydrolysis under high temperature and low pH [10].	Enzymatic hydrolysis catalyzed by immobilized urease [10].
Optimal Temperature	99 °C [10]	60 °C [10]
Optimal pH	Low pH (e.g., [H⁺] = 2 mol/L) [10]	Neutral pH (pH = 7) [10]
Reaction Time	7 hours [10]	40 minutes [10]
Nitrogen Recovery Efficiency	39.7% [10]	52.2% [10]
Key Advantages	Effective hydrolysis without biological catalysts.	Higher efficiency, lower energy demand, superior reaction stability [10].
Key Challenges	High energy input, prolonged processing time, lower efficiency [10].	Potential enzyme cost and immobilization support requirements.

The experimental data, derived from real human urine tests conducted for the "Lunar Palace 1" BLSS project, clearly demonstrates the performance differential between these methods [10]. IUCM achieves a significantly higher nitrogen recovery efficiency (52.2%) than HTAM (39.7%) under its respective optimal conditions. Furthermore, IUCM operates at a lower temperature and requires a fraction of the reaction time, making it a more energy-efficient and rapid process [10]. The study also reported that the immobilized urease showed excellent reaction stability, which is a critical factor for the long-term, reliable operation required in a BLSS [10].

Experimental Protocols for Hydrolysis Optimization

Protocol A: Immobilized Urease Catalysis Method (IUCM)

Principle: This protocol utilizes the enzyme urease, immobilized on a solid support, to catalytically hydrolyze urea in urine into ammonia and carbon dioxide at neutral pH and moderate temperatures [10].

Materials:

• Real Human Urine: Collect and store frozen. Thaw and bring to room temperature before use.
• Immobilized Urease: Urease enzyme fixed on a chosen solid support (e.g., chitosan beads).
• Reaction Vessel: Jacketed glass bioreactor with temperature control.
• pH Meter and Titrants: For monitoring and maintaining pH.
• Heating Circulator: For precise temperature control of the reactor jacket.
• Ammonia Probe or Spectrophotometer: For quantifying ammonia concentration and tracking hydrolysis efficiency.

Procedure:

• Reactor Setup: Place the immobilized urease into the bioreactor.
• Urine Introduction: Add the pre-treated urine to the reactor.
• Parameter Control: Adjust the heating circulator to maintain the reaction temperature at 60 °C. Use a pH stat or manual addition of a mild acid/base to maintain a constant pH of 7.0.
• Reaction Monitoring: Allow the reaction to proceed for 40 minutes, periodically measuring the ammonia concentration.
• Efficiency Calculation: After the reaction, calculate the Nitrogen Recovery Efficiency using the formula provided in Section 3.3.

Protocol B: High-Temperature Acidification Method (HTAM)

Principle: This protocol employs high temperature and low pH to chemically hydrolyze urea in urine without a biological catalyst [10].

Materials:

• Real Human Urine
• Strong Acid: e.g., Hydrochloric Acid (HCl) or Sulfuric Acid (H₂SO₄).
• Reflux System: A sealed reaction vessel equipped with a condenser to prevent volume loss.
• Hot Plate or Oven: Capable of maintaining temperatures up to 100 °C.
• Ammonia Trapping System: e.g., an acid trap containing a boric acid solution.
• Titration Setup or Ammonia Probe.

Procedure:

• Urine Acidification: Transfer urine to the reaction vessel and add a strong acid to achieve a proton concentration of [H⁺] = 2 mol/L.
• System Assembly: Securely attach the condenser and ammonia trapping system to the vessel.
• Heating and Hydrolysis: Place the vessel in a temperature-controlled oven or on a hot plate set to 99 °C for a duration of 7 hours.
• Ammonia Collection: The released ammonia gas is carried via the condenser and captured in the acid trap.
• Analysis: Quantify the amount of nitrogen captured in the trap via titration or direct measurement to determine the Nitrogen Recovery Efficiency.

Data Analysis and Calculation

Nitrogen Recovery Efficiency: This is the primary metric for evaluating hydrolysis performance. It is calculated as follows [10]: Nitrogen Recovery Efficiency (%) = (Mass of Nrecovered / Mass of N in initial urine) × 100%

Table 2: Key Parameter Ranges for Hydrolysis Optimization Studies

Parameter	Low Range	High Range	Analytical Method
Temperature	20 °C	99 °C	Thermocouple / Heater [10] [42]
pH	2 (acidic)	11 (alkaline)	pH Meter / pH Stat [10]
Hydraulic Retention Time (HRT)	5 minutes	24 hours	Controlled batch time or flow rate [43] [10]
Ammonia Concentration	-	-	Spectrophotometry, Ion-Selective Electrode, or Acid Titration [10]
Urea Concentration	-	-	HPLC or Urease-Based Diagnostic Kits [10]

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagent Solutions for Hydrolysis Optimization

Item	Function/Benefit	Application Context
Recombinant β-Glucuronidase (e.g., BGTurbo)	Efficiently cleaves drug glucuronides in urine; fast hydrolysis (as low as 5 min) across a range of temperatures (20–55 °C) [43] [42].	Forensic toxicology; studying the stability of pharmaceutical metabolites in recycled water streams [43].
Immobilized Urease	Biological catalyst for urea hydrolysis; enables high nitrogen recovery at lower temperatures and neutral pH, improving energy efficiency [10].	Core component of the IUCM protocol for nitrogen recovery in BLSS [10].
Polytetrafluoroethylene (PTFE) Membranes	Hydrophobic membranes used to recover volatilized ammonia from hydrolyzed urine; high recovery rates (e.g., ~72% from real digestate) [44].	Downstream ammonia capture and concentration following the hydrolysis step [44].
Sulfuric Acid (H₂SO₄) Solution	Serves as both a pH-lowering agent for HTAM and a capture solution for ammonia, converting it into stable ammonium sulfate fertilizer [44].	Essential chemical for HTAM and for ammonia absorption in membrane contactors [10] [44].

Workflow and Pathway Visualization

The following diagram illustrates the logical decision workflow for selecting and optimizing a hydrolysis strategy within a BLSS framework, integrating the two primary methods discussed in this note.

Managing Microbial Contamination and Pathogen Inactivation

In the context of Bioregenerative Life Support Systems (BLSS) for long-duration space missions, the recovery of nitrogen from human urine is paramount for closing the nutrient loop and enabling in-situ food production [1] [3]. A crew member excretes an average of 7–16 grams of nitrogen per day, with urine accounting for approximately 85% of the total potentially recoverable nitrogen within the system [1] [3]. The efficient recycling of this nitrogen into forms usable by plants or edible microorganisms is critical for producing dietary protein and sustaining crewed missions beyond Earth's orbit [30].

However, the management of microbial contamination and the implementation of reliable pathogen inactivation protocols present significant challenges. Untreated urine waste streams can harbor pathogens that threaten system stability and crew health. Furthermore, contamination can interfere with the biological processes, such as nitrification, that are central to nitrogen recovery in systems like the European Space Agency's MELiSSA (Micro-Ecological Life Support System Alternative) loop [1] [3]. This application note details practical strategies and protocols for ensuring microbial safety within the urine processing components of a BLSS, directly supporting the broader thesis of robust nitrogen recovery.

Pathogen Inactivation Technologies: Mechanisms and Applications

Pathogen Inactivation (PI) technologies function by irreversibly damaging the nucleic acids of pathogens (viruses, bacteria, fungi, protozoa), thereby blocking their replication [45] [46]. While several PI methods have been developed for terrestrial biomedical applications, such as treating blood products, their principles and workflows are highly relevant to processing liquid waste streams like urine in a BLSS.

The following table summarizes the core mechanisms of several key PI technologies.

Table 1: Comparison of Pathogen Inactivation Technologies

Technology Name	Core Agent	Mechanism of Action	Key Consideration for BLSS
INTERCEPT System [46]	Amotosalen HCl (S-59) & UVA Light	Photoactive compound cross-links nucleic acids (DNA/RNA) upon UVA activation.	Requires post-illumination compound removal.
MIRASOL PRT System [46]	Riboflavin (Vitamin B2) & UV Light	Mediates oxygen-independent electron transfer, causing irreversible nucleic acid damage upon UV exposure.	Riboflavin and its byproducts are non-toxic; no removal needed.
THERAFLEX UV-Platelets [46]	UVC Light (254 nm)	Shortwave UVC light directly damages nucleic acids, forming pyrimidine dimers.	No photoactive compounds required; simple and rapid process.
Solvent/Detergent Treatment [45]	Solvent & Detergent (e.g., Triton X-100)	Dissolves the lipid envelope of pathogens, compromising membrane integrity.	Effective primarily against enveloped pathogens.
Heat Inactivation [47]	Elevated Temperature	Denatures proteins and nucleic acids.	A simple, physical method; parameters are pathogen-specific.

The selection of a PI technology for a BLSS must consider factors such as compatibility with downstream nitrifying bacteria, the potential for toxic byproduct formation, energy requirements, and operational complexity in a microgravity environment.

Experimental Protocol: UVC-Based Pathogen Inactivation for Liquid Waste Streams

The following protocol is adapted from the THERAFLEX UVC principle for application in a BLSS urine processing unit [46].

Principle: Shortwave UVC light (254 nm) directly interacts with microbial nucleic acids, forming pyrimidine dimers that block replication, effectively inactivating viruses, bacteria, fungi, and protozoa [46].

Materials:

• Source Material: Hydrolyzed and pre-filtered urine stream.
• UVC Illumination Device: A system capable of delivering UVC light at 254 nm.
• Irradiation Bag/Container: UVC-transparent, sterile container (e.g., specially designed plastic bag).
• Agitation Platform: A device to ensure continuous, uniform mixing of the liquid during illumination.

Procedure:

• Sample Preparation: Pre-filter the hydrolyzed urine to remove large particulate matter that could shield microorganisms from UVC light.
• Loading: Aseptically transfer a defined volume of the pre-filtered liquid into the UVC-transparent irradiation bag. The bag thickness and liquid path length are critical design parameters for ensuring uniform light penetration.
• Illumination & Agitation:
  • Place the bag on the agitation platform within the UVC illumination device.
  • Initiate intense agitation to ensure homogeneous exposure.
  • Illuminate the sample with UVC light (254 nm) for a predetermined duration (e.g., <1 minute to several minutes, based on dose-response validation). The required UVC dose (J/m²) must be validated to achieve the target log-reduction in the specific matrix.
• Product Handling: The treated liquid is now pathogen-reduced and can be safely directed to downstream nitrogen recovery compartments, such as a nitrifying bioreactor.

Validation: The success of inactivation must be confirmed by culture-based methods, attempting to grow microbes from the treated sample under optimal conditions, and by molecular methods like qPCR to quantify the reduction in viable pathogen load [47].

Preventing Contamination in Urine Sample Collection and Handling

Pre-analytical contamination during urine acquisition is a well-documented problem in clinical medicine and offers critical lessons for BLSS [48] [49] [50]. In a closed-loop habitat, introducing contaminants can disrupt the finely tuned microbial ecology of the recycling system.

Protocol: Aseptic Midstream Urine Collection for BLSS

Principle: The "midstream" collection technique minimizes the introduction of dermal and urogenital commensals into the sample by using the initial urine flow to flush the urethra [48] [49].

Materials:

• Sterile Urine Collection Container: Airtight, leak-proof container produced and assembled under cleanroom conditions. Individual, tamper-evident packaging is essential to maintain sterility until use [49].
• Sterile Cleansing Wipes: Single-use wipes with appropriate, skin-safe disinfectant.
• Personal Protective Equipment (PPE): Sterile, disposable gloves.

Procedure:

• Hygiene: The crew member and assistant don sterile gloves. The crew member performs hand hygiene.
• Cleansing: The crew member cleanses the peri-urethral area thoroughly with a sterile wipe.
• Voiding Sequence:
  • Initiate urination into the habitat's waste collection toilet.
  • Without stopping the urine flow, pass the sterile collection container into the stream and collect a volume of 50-100 mL.
  • Remove the container before the flow stops.
• Securing the Sample: Immediately close the container with its sterile lid, ensuring an airtight seal. Label the container with a unique identifier.

Contamination Control: Studies have shown that devices designed to automate midstream collection (e.g., Peezy, Whiz Midstream) do not necessarily reduce contamination rates compared to well-executed standard verbal instructions [48]. Therefore, comprehensive crew training in this protocol is more critical than relying on specialized collection devices.

The Scientist's Toolkit: Key Research Reagents and Materials

The following table lists essential materials for research and development in microbial management for BLSS, drawing from the cited protocols and general laboratory practice.

Table 2: Research Reagent Solutions for Contamination and Inactivation Studies

Item	Function/Description	Application in BLSS Research
Sucrose Phosphate Glutamine (SPG) Buffer [47]	Stabilizing buffer for preserving viability of delicate microorganisms during storage.	Used in maintaining stock cultures of nitrifying bacteria or specific pathogens for challenge studies.
Riboflavin (Vitamin B2) [45] [46]	A natural photosensitizer used in the MIRASOL PRT system for pathogen inactivation.	Investigating PI in nutrient streams without introducing toxic chemicals; its byproducts are safe.
Amotosalen HCl (S-59) [45] [46]	A synthetic psoralen that cross-links nucleic acids upon UVA activation.	A reference compound for evaluating high-efficacy PI in process development.
L929 Cell Line [47]	A mouse fibroblast cell line susceptible to infection.	Used as a bioassay to validate the infectivity of obligate intracellular bacteria (e.g., Rickettsia) after PI treatment.
HotSHOT DNA Extraction Reagents [47]	A rapid, alkaline lysis-based method for nucleic acid extraction.	For quick molecular monitoring of microbial community dynamics and pathogen load in waste streams.
Sterile, Gamma-Irradiated Collection Containers [49]	Specimen containers sterilized by ionizing radiation to prevent initial contamination.	Essential for aseptic sampling of urine and other liquid wastes within the habitat to preserve process integrity.

Integrating robust microbial contamination control and pathogen inactivation strategies is a non-negotiable prerequisite for safe and efficient nitrogen recovery from urine in a BLSS. The protocols and technologies outlined here—from UVC inactivation to aseptic collection—provide a foundational framework. Future work must focus on adapting and validating these methods for the unique constraints of the space environment, including microgravity, resource limitations, and the need for fully autonomous operation. By proactively addressing these challenges, the goal of a sustainable, closed-loop life support system for deep space exploration becomes increasingly attainable.

In the context of Bioregenerative Life Support Systems (BLSS) for long-duration space missions, the recovery of water and nutrients from waste streams is paramount for system closure. Nitrogen recovery from urine, a resource-rich in water and essential nutrients, represents a critical process [3]. However, human urine also contains pharmaceuticals and hormones, a category of organic micropollutants, which can pose a significant threat to the stability of the biological components within a BLSS and the health of the crew if not adequately addressed [51] [52]. These micropollutants are characterized by their persistence and potential biological activity even at very low concentrations (nanograms to micrograms per liter) [53]. In terrestrial wastewater, these compounds have been shown to adsorb onto other pollutants, like microplastics, forming a 'plastisphere' that can protect harmful microorganisms and viruses, thereby increasing their stability and potential for dissemination [51]. This application note details protocols for monitoring and analyzing these micropollutants within the specific framework of BLSS nitrogen recovery research, ensuring the safety and sustainability of closed-loop life support.

Background and Significance

Micropollutants in the Context of BLSS

In a BLSS, the goal is to achieve a high degree of closure by recycling all vital resources. The European Space Agency's MELiSSA (Micro-Ecological Life Support System Alternative) project is a prime example, designed as a five-compartment bioengineered system to produce food, oxygen, and recycle water by recovering minerals from waste streams [3]. Urine is the primary source of recoverable nitrogen in such systems, with each crew member excreting 7–16g of nitrogen per day, predominantly in the form of urea [3]. Current physicochemical systems on the International Space Station, such as the Urine Processor Assembly (UPA), are highly effective at water recovery but are not designed to recover nitrogen or handle complex organic micropollutants [3]. Biological treatment methods, such as those explored in the "Lunar Palace 1" experiment, aim to recover both water and nitrogen but face challenges with removal efficiency and potential interference from micropollutants [10].

Risks Posed by Pharmaceuticals and Hormones

Pharmaceuticals and hormones are pseudo-persistent contaminants—they are continuously introduced into the recycled waste stream. Their presence in a BLSS can lead to several risks:

• Disruption of Microbial Communities: The nitrifying and other beneficial bacteria essential for nitrogen recovery in compartments like MELiSSA's Compartment III can be inhibited by these bioactive compounds [3] [54].
• Uptake by Plants: If treated water containing micropollutants is used for irrigation, plants can accumulate these substances, potentially introducing them into the food chain [51].
• Threat to Crew Health: The potential for chronic, low-level exposure to a mixture of pharmaceuticals and hormones through reclaimed water or food poses an unknown risk to crew health over long-duration missions [51].

Analytical Methods for Micropollutant Assessment

Effect-based methods and chemical analysis are recommended for a comprehensive assessment of micropollutant presence and activity.

Solid-Phase Extraction (SPE) for Micropollutant Enrichment

Given the low concentrations of micropollutants, a pre-concentration step is essential. Solid-phase extraction is the most common technique for enriching micropollutants from aqueous samples, such as processed urine or reclaimed water streams in a BLSS [53].

Table 1: Comparison of Solid-Phase Extraction Sorbents for Micropollutant Enrichment

Sorbent Type	Best Suited For	Key Advantages	Optimal pH	Typical Recovery of Estrogenic Activity
HRX	Generic extraction of a wide range of micropollutants	Best overall recovery for diverse chemicals and bioactivity [53]	pH 7 [53]	Good
HLB	Selective extraction of estrogenic compounds; suitable as a universal sorbent	Excellent for estrogenic chemicals; reduces matrix cytotoxicity [53]	pH 3 [53]	~100% after 112 days storage [53]

Protocol: Standardized Solid-Phase Extraction for Bioanalysis

This protocol is adapted for the enrichment of micropollutants from BLSS-processed water streams prior to bioanalytical or chemical analysis [53].

I. Materials and Reagents

• SPE Cartridges: Oasis HLB or equivalent hydrophilic-lipophilic balanced sorbent (e.g., 60 mg, 3 mL).
• Solvents: Methanol (LC-MS grade), Acetone (for cleaning), and Water (LC-MS grade).
• Sample: Processed water or urine distillate from BLSS. Filter through a 0.45 μm glass fiber filter to remove particulate matter.
• Equipment: Vacuum manifold, glass test tubes, calibrated pipettes.

II. Procedure

• Conditioning: Sequentially pass 3 mL of Methanol and 3 mL of Water through the SPE cartridge. Do not let the sorbent bed run dry.
• Loading: Load the acidified sample (adjusted to pH 3 with H₃PO₄ for estrogenic analysis, or unadjusted for generic screening) onto the cartridge at a steady flow rate of 5-10 mL/min.
• Washing: After sample loading, wash the cartridge with 3 mL of Water to remove salts and other polar interferences.
• Drying: Apply a strong vacuum (e.g., 15-20 in. Hg) for 10-15 minutes to dry the sorbent completely.
• Elution: Elute the captured micropollutants into a clean glass test tube using 2 x 2.5 mL of Methanol. Gently push air through the cartridge to collect the entire eluent.
• Reconstitution: Evaporate the eluent to complete dryness under a gentle stream of nitrogen. Reconstitute the dry extract in 100 μL of a solvent compatible with the downstream bioassay or chemical analysis (e.g., DMSO for bioassays, methanol/water for LC-MS).

Effect-Based Bioassays

Bioassays are critical for detecting the cumulative biological effect of all bioactive micropollutants in a sample, including unknown compounds and transformation products.

• YES (Yeast Estrogen Screen): Used to detect estrogenic activity by measuring the activation of the human estrogen receptor in a genetically modified yeast strain [53].
• Fish Embryo Toxicity Test: An in vivo assay used to detect sublethal and lethal toxicity in water samples [53].

Quantitative Data on Micropollutant Removal

Understanding the removal efficiency of micropollutants across different biological treatments is vital for designing a BLSS wastewater processing unit.

Table 2: Removal Efficiencies of Selected Micropollutants in Biological Treatment Processes Data derived from studies on municipal wastewater treatment, indicating potential performance in optimized BLSS conditions [54].

Micropollutant	Category	Activated Sludge (Aerobic)	Oxic Biofilm	Anaerobic Treatment
Bezafibrate	Lipid regulator	Significant removal [54]	-	-
Atenolol	Beta-blocker	Significant removal [54]	-	-
Diclofenac	Anti-inflammatory	Persistent	Complementary removal [54]	-
Venlafaxine	Antidepressant	Persistent	-	Removed [54]
Tramadol	Analgesic	Persistent	-	Removed [54]
Carbamazepine	Anticonvulsant	Persistent	Persistent	Persistent

The data illustrates that a single biological process is insufficient for comprehensive micropollutant removal. A combination of aerobic and anaerobic treatments may be necessary to target a broader spectrum of these persistent compounds [54].

Integration into BLSS Nitrogen Recovery

The following diagram illustrates a proposed workflow for integrating micropollutant monitoring and management into a BLSS nitrogen recovery process, such as the MELiSSA loop.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents and Materials for Micropollutant Research in BLSS

Item	Function/Application	Example/Citation
Oasis HLB Sorbent	Broad-spectrum SPE enrichment of micropollutants with different physicochemical properties [53].	Waters Oasis HLB Cartridges
17β-Estradiol (E2)	Reference standard for calibrating estrogenic activity in bioassays and chemical analysis [53].	Sigma-Aldridge, >98% purity
Internal Standards (Isotope-Labeled)	Correct for analyte loss during sample preparation in quantitative LC-MS analysis [55].	e.g., Carbamazepine-d₁₀, Venlafaxine-d6
Synthetic Wastewater	For controlled laboratory experiments to study micropollutant degradation without complex matrix interference [54].	Contains acetate, peptone, NH₄Cl, salts [54]
Accelerated Solvent Extractor (ASE)	Automated, efficient extraction of micropollutants and their transformation products from solid matrices (e.g., soil, bio-solids) [55].	Dionex ASE 350
LC-HRMS System	Non-targeted screening and identification of unknown micropollutants and their transformation products [55].	e.g., Thermo Scientific Q-Exactive Orbitrap

The successful implementation of a long-duration BLSS hinges on the safe and efficient recycling of nitrogen and water from urine. The presence of pharmaceuticals and hormones in these streams represents a significant challenge that must be proactively addressed. By integrating robust analytical protocols, such as solid-phase extraction coupled with effect-based bioassays, and designing treatment trains that combine multiple biological processes, researchers can monitor, understand, and mitigate the risks posed by these micropollutants. This proactive approach is essential to ensure the health and stability of both the biological components of the life support system and the crew it sustains.

Improving Process Stability under Simulated Space Conditions

In the context of Bioregenerative Life Support Systems (BLSS) for long-duration space missions, the efficient recovery of nitrogen from human urine is paramount for achieving system closure. Nitrogen is a crucial nutrient required for the cultivation of higher plants, which provide food, oxygen, and water recycling for the crew [30] [56]. With the daily human excretion of 7–16g of nitrogen, 85% of which is found in urine, it represents the most significant recoverable nitrogen source within a BLSS [3]. However, the space environment, characterized by microgravity and increased radiation, poses significant challenges to the process stability of biological systems. This application note details protocols for two key urine pretreatment methods investigated to enhance the stability and efficiency of nitrogen recovery under constraints relevant to space missions [10].

Research Reagent Solutions

The table below catalogues the essential materials and reagents used in the featured nitrogen recovery experiments.

Table 1: Key Research Reagents and Materials for Nitrogen Recovery Experiments

Item Name	Function/Description	Application Context
Immobilized Urease	Biological catalyst hydrolyzing urea into ammonia and carbon dioxide [10].	Urine pretreatment in IUCM [10].
Urease Activity	Critical performance parameter for IUCM; loss of 6.7% after 10 uses [10].	Stability assessment of immobilized urease [10].
Phosphoric Acid (H₃PO₄)	Acidifying agent for urine chemical stabilization; prevents scaling vs. H₂SO₄ [3].	Urine stabilization in storage [3].
Hexavalent Chromium (Cr⁶⁺)	Oxidizing agent in urine stabilization; prevents urea hydrolysis and NH₄⁺ formation [3].	Urine stabilization in storage [3].
Reduced Pressure Distillation	Method for collecting water vapor and ammonia gas from pretreated urine [10].	Nitrogen and water recovery post-pretreatment [10].

The following table summarizes the performance and optimal conditions for two primary urine pretreatment methods, as investigated for application in BLSS.

Table 2: Comparison of Urine Pretreatment Methods for Nitrogen Recovery

Parameter	High-Temperature Acidification Method (HTAM)	Immobilized Urease Catalysis Method (IUCM)
Principle	Chemical hydrolysis of urea under high temperature and acidic conditions [10].	Enzymatic hydrolysis of urea catalyzed by immobilized urease [10].
Optimal Temperature	99 °C [10]	60 °C [10]
Optimal pH	[H⁺] = 2 mol/L (Highly acidic) [10]	pH = 7 (Neutral) [10]
Processing Time	7 hours [10]	40 minutes [10]
Maximum Nitrogen Recycle Efficiency	39.7% [10]	52.2% [10]
Key Advantages	-	Higher efficiency, milder conditions, reagent reusability [10].

Figure 1: Workflow for nitrogen and water recovery from urine, featuring two pretreatment pathways.

Detailed Experimental Protocols

Protocol A: High-Temperature Acidification Method (HTAM)

This protocol describes the chemical hydrolysis of urea in urine under high-temperature and acidic conditions.

Materials and Reagents

• Urine Sample: Fresh or stabilized real human urine [10].
• Acid Solution: e.g., Sulfuric acid (H₂SO₄) or Phosphoric acid (H₃PO₄) for pH adjustment [3] [10].
• Reaction Vessel: Heat-resistant glassware (e.g., three-necked flask).
• Heating Mantle: With precise temperature control.
• pH Meter.
• Condenser: To prevent volume loss during heating.

Step-by-Step Procedure

• Urine Preparation: Collect and filter urine if necessary. Test and record initial urea and total nitrogen concentrations [10].
• Acidification: Transfer a measured volume of urine (e.g., 100 mL) to the reaction vessel. Under constant stirring, carefully add the acid solution to adjust the hydrogen ion concentration to the target level (e.g., [H⁺] = 2 mol/L) [10].
• Heating and Hydrolysis: Assemble the condenser and heat the urine-acid mixture to the target temperature (e.g., 99 °C). Maintain this temperature for the duration of the hydrolysis reaction (e.g., 7 hours) [10].
• Termination and Analysis: After the reaction time, cool the mixture to room temperature. The pretreated urine is now ready for the subsequent reduced pressure distillation step to recover ammonia [10].

Protocol B: Immobilized Urease Catalysis Method (IUCM)

This protocol outlines the enzymatic hydrolysis of urea using immobilized urease, a more efficient and milder alternative to HTAM.

Materials and Reagents

• Urine Sample: Fresh or stabilized real human urine [10].
• Immobilized Urease: Urease enzyme fixed onto a solid support [10].
• Buffer Solution: To maintain neutral pH (e.g., phosphate buffer).
• Temperature-Controlled Incubator or Water Bath.
• Filtration Setup or Fixed-Bed Bioreactor.

Step-by-Step Procedure

• Urine Preparation: Collect and filter urine. Adjust the pH to the optimal value for the enzyme (e.g., pH 7) using a buffer solution [10].
• Reaction Setup:
  • Batch Method: Combine the pH-adjusted urine with a measured quantity of immobilized urease in a sealed container [10].
  • Continuous Flow Method: Pack the immobilized urease into a column and pass the pH-adjusted urine through it.
• Incubation/Reaction: Place the batch mixture or the entire column in a temperature-controlled environment set to the optimal temperature (e.g., 60 °C). Allow the reaction to proceed for the determined time (e.g., 40 minutes) [10].
• Separation: If using the batch method, separate the pretreated urine from the immobilized urease by filtration. The immobilized urease can be reused for subsequent cycles [10].
• Downstream Processing: The hydrolyzed urine, now rich in ammonia, is transferred to the reduced pressure distillation unit for recovery [10].

Figure 2: Step-by-step workflow for Protocol A (HTAM) and Protocol B (IUCM).

The Immobilized Urease Catalysis Method (IUCM) presents a superior nitrogen recovery pathway for BLSS applications compared to the High-Temperature Acidification Method (HTAM), achieving a higher nitrogen recovery efficiency of 52.2% under milder and less energy-intensive conditions [10]. Its reusability—demonstrating only a 6.7% activity loss after 10 uses—is a critical attribute for long-duration missions where resource conservation and waste minimization are imperative [10]. Future research must focus on validating the stability and performance of these biological systems, particularly the urease enzyme and nitrifying bacteria, under real simulated space conditions such as microgravity and increased ionizing radiation [3]. Integrating this efficient nitrogen recovery loop with hydroponic systems for higher plant cultivation is the essential next step to closing the mass balance and achieving a truly sustainable bioregenerative life support system for the exploration of deep space [30] [56].

Energy Efficiency and Resource Consumption of Different Recovery Technologies

In the context of Bioregenerative Life Support Systems (BLSS) for long-duration space missions, the efficient recovery of nitrogen from human urine is a critical technological challenge. On Earth, shipping fertilizers is costly, but in space, resupply is often impossible, making the in-situ recycling of nutrients from waste streams an absolute necessity for food production [30]. Nitrogen is a paramount nutrient for plant growth, and human urine is a primary source of it within a closed habitat, containing approximately 80% of the recoverable nitrogen and contributing the majority of the nitrogen load in wastewater [1] [14]. The development of energy- and resource-efficient nitrogen recovery technologies is therefore a cornerstone for achieving the self-sufficiency required for future lunar bases or Martian exploration [36]. This application note provides a detailed comparison of prevailing nitrogen recovery technologies and standardized protocols for their assessment within BLSS-oriented research.

Various technologies have been developed to recover nitrogen from urine, each with distinct operational principles, efficiency, and resource demands. They can be broadly categorized into biological, physical-chemical, and electrochemical processes.

• Biological Nitrification relies on ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) to convert ammonia into nitrate, stabilizing nitrogen in a form readily available for plant uptake [9]. Its main advantage is high nutrient recovery efficiency with low chemical demand, though it can be sensitive to environmental conditions and requires longer start-up times [9] [57].
• Membrane Gas Separation is a physical-chemical process where urine is alkalized to convert ammonium ions (NH₄⁺) into free ammonia (NH₃). This ammonia gas passes through a hydrophobic membrane and is captured in an acid solution to produce a liquid fertilizer like ammonium sulfate [58]. It is noted for its compactness and rapid nutrient recovery.
• Struvite Precipitation involves the addition of a magnesium source to urine to precipitate magnesium ammonium phosphate (MgNH₄PO₄·6H₂O), simultaneously recovering nitrogen and phosphorus [58]. While prized for phosphorus recovery, its nitrogen recovery efficiency is typically lower than other methods.
• Electrochemical Processes represent an emerging field that uses electrical currents to directly recover nitrogen or to drive other reactions, such as membrane-based separation. These processes show promise for on-site applications and potential energy recovery, though they are not yet as mature as other technologies [57].

The following table summarizes the performance and resource consumption of these key technologies based on current research.

Table 1: Performance and Resource Consumption of Nitrogen Recovery Technologies

Technology	Nitrogen Recovery Efficiency	Energy Consumption	Chemical Consumption	Key Challenges
Biological Nitrification [9] [57]	High; maintains nearly all nutrients	Low energy demand	Low chemical demand	Long start-up time, process instability, sensitivity to high salinity & pH
Membrane Gas Separation [58]	~85% harvesting efficiency	Moderate (pumping, temp. control)	Acid for absorption (e.g., H₂SO₄)	Membrane fouling, chemical requirement for pH elevation
Struvite Precipitation [58]	Lower than other methods (focus on P)	Low to Moderate	Magnesium source (e.g., MgO, MgCl₂)	Low nitrogen recovery efficiency, produces solid waste
Electrochemical Processes [57]	Varies; rapidly developing	Can be high; potential for energy production	Varies by process	Early stage of development, can be capital intensive

Experimental Protocols for Technology Assessment

To ensure comparable results across different BLSS research initiatives, standardized experimental protocols are essential. The following sections provide detailed methodologies for assessing two prominent nitrogen recovery pathways.

Protocol for Biological Nitrification in a Sequencing Batch Reactor (SBR)

1. Principle: This protocol uses a Sequencing Batch Reactor (SBR) to biologically stabilize nitrogen in source-separated urine by converting ammonia to nitrate through aerobic microbial activity. This process prevents nitrogen loss and produces a nitrate-rich liquid fertilizer [9].

2. Reagents and Materials:

• Source-separated human urine
• Inoculum of nitrifying bacteria (e.g., from an active nitrifying reactor)
• Mineral salts medium (for micronutrients, if required)
• Sodium hydroxide (NaOH) and Hydrochloric acid (HCl) for pH control
• Aerated SBR vessel with temperature control
• Dissolved oxygen (DO) meter and pH meter
• Peristaltic pumps for feeding and effluent removal

3. Procedure:

• Step 1: Reactor Startup and Inoculation. Fill the SBR with activated nitrifying sludge. Gradually acclimate the biomass to urine by progressively increasing the urine concentration in the feed over several weeks while monitoring ammonia and nitrite levels [9].
• Step 2: SBR Operation. Operate the reactor in sequential cycles. A typical 12-hour cycle includes:
  • Feed Phase (15 min): Add a predetermined volume of urine.
  • Reaction Phase (10-10.5 hours): Maintain aerobic conditions (DO > 2 mg/L) with continuous aeration. Monitor pH and adjust to ~7.5-8.0 using NaOH or HCl as needed to optimize nitrifier activity [9].
  • Settle Phase (1 hour): Stop aeration to allow biomass to settle.
  • Draw Phase (15 min): Withdraw a volume of clarified, nitrified effluent.
• Step 3: Monitoring and Analysis. Regularly analyze the influent and effluent for key parameters: Total Ammonia Nitrogen (TAN), nitrite (NO₂⁻-N), nitrate (NO₃⁻-N), and pH. Monitor mixed liquor suspended solids (MLSS) to track biomass concentration.

4. Data Analysis:

• Nitrogen Conversion Efficiency: Calculate as (1 - [TAN]_effluent / [TAN]_influent) * 100%.
• Process Stability: Monitor the ratio of nitrite to nitrate; a sudden increase may indicate inhibition of nitrite-oxidizing bacteria (NOB).

The workflow for this SBR process is outlined below.

Protocol for Nitrogen Recovery via Hydrophobic Gas-Permeable Membrane

1. Principle: This method exploits the pH-dependent equilibrium between ammonium (NH₄⁺) and free ammonia (NH₃). Under alkaline conditions, NH₃ is formed, diffuses through a hydrophobic membrane, and is trapped in an acid solution on the permeate side, producing a concentrated ammonium salt solution [58].

2. Reagents and Materials:

• Source-separated human urine
• Hydrophobic gas-permeable membrane module (e.g., ePTFE)
• Sulfuric acid (H₂SO₄, 1-2 N) or other absorbent acid
• Peristaltic pumps for urine and acid recirculation
• pH meter and controller
• Heating bath with temperature control

3. Procedure:

• Step 1: Urine Alkalization. Adjust the pH of the urine feed to a value between 9 and 10 using a strong base like sodium hydroxide (NaOH). This shifts the ammonium-ammonia equilibrium towards gaseous NH₃ [58].
• Step 2: Membrane System Setup. Configure a continuous or semi-batch membrane reactor. The alkalized urine is pumped along one side of the membrane. A diluted acid stream is pumped counter-currently on the permeate side.
• Step 3: Process Optimization. Key parameters to control and optimize include:
  • Feed Temperature: Maintain at 35-40°C to enhance NH₃ stripping [58].
  • Acid Flux: Optimize flow rate to ensure complete capture of NH₃ (e.g., 350 L/m²h) [58].
  • Hydraulic Retention Time (HRT): An HRT of ~8 hours can achieve up to 85% ammonia harvesting efficiency [58].
• Step 4: Product Collection. The acid stream, now rich in ammonium sulfate [(NH₄)₂SO₄], is collected as the final fertilizer product.

4. Data Analysis:

• Ammonia Harvesting Efficiency: (1 - [TAN]_treated_urine / [TAN]_feed_urine) * 100%.
• Acid Consumption: Moles of acid consumed per mole of nitrogen recovered.

The logical relationship of the membrane separation process is as follows.

The Scientist's Toolkit: Research Reagent Solutions

A standardized set of reagents and materials is crucial for ensuring reproducibility in nitrogen recovery research. The following table details essential items for the experiments described in this note.

Table 2: Essential Research Reagents and Materials

Item Name	Specification / Example	Primary Function in Experiment
Source-Separated Urine	Collected via non-mixing toilets/urinals	The primary feedstock containing urea and nitrogen for recovery [9].
Nitrifying Inoculum	Activated sludge from a nitrifying wastewater plant	Provides the microbial consortium (AOB & NOB) for biological nitrification [9].
Hydrophobic Membrane	Zeus Aeos ePTFE or equivalent	Serves as the physical barrier for selective ammonia gas transfer in membrane processes [58].
Absorbent Acid	1-2 N Sulfuric Acid (H₂SO₄)	Traps permeated ammonia gas to form a stable ammonium salt fertilizer [58].
pH Adjusters	NaOH pellets, HCl solution	Controls the pH to optimize microbial activity (nitrification) or shift NH₄⁺/NH₃ equilibrium (membrane stripping) [9] [58].
Magnesium Source	MgO, MgCl₂	Added as a precipitant for simultaneous nitrogen and phosphorus recovery via struvite formation [58].

The choice of nitrogen recovery technology for a BLSS involves critical trade-offs between energy efficiency, resource consumption, reliability, and integration complexity. Biological nitrification offers high efficiency with low energy and chemical inputs but requires careful control of operational stability. In contrast, membrane gas separation provides a more rapid and compact solution with high recovery rates, albeit with greater energy and chemical demands. As BLSS research advances, the integration of these technologies, alongside robust protocols for monitoring and control, will be paramount for closing the nitrogen loop and enabling sustainable, long-duration human presence in space. Future work should focus on hybrid systems that leverage the strengths of different processes to maximize overall system resilience and efficiency.

Comparative Technology Assessment and Validation in Integrated Systems

In the context of Bioregenerative Life Support Systems (BLSS) for long-duration space missions, the efficient recovery of nitrogen from human urine is paramount. Nitrogen is a critical nutrient for plant growth, which in turn provides food and oxygen for crew members. With the high cost of resupply from Earth, achieving a high degree of closure in the nitrogen cycle is essential for mission sustainability [3]. This document outlines application notes and protocols for evaluating key performance metrics—Nitrogen Recovery Efficiency, Energy Cost, and System Stability—for nitrogen recovery processes within BLSS research.

Performance Metrics and Data Comparison

Evaluating nitrogen recovery technologies requires a standard set of quantitative metrics. The following parameters are crucial for inter-protocol comparison and system-level integration.

Table 1: Key Performance Metrics for Nitrogen Recovery in BLSS

Metric	Definition	Formula/Unit	Target for BLSS
Nitrogen Recovery Efficiency (NRE)	Mass of nitrogen in usable product per mass of nitrogen in feed urine.	NRE (%) = (N_product / N_urine) * 100	>85% [3]
Energy Cost	Electrical and thermal energy consumed per mass of nitrogen recovered.	Energy (kWh/kg N)	Minimized; a primary driver for shifting from physicochemical to biological systems [3]
Process Stability	Ability to maintain target NRE under variable loads and potential inhibitory conditions.	Operational duration without failure or significant (>10%) efficiency drop.	Robust to FA (≥84 g N/m³) and salinity [21]
Nitrification Rate	Speed of ammonium conversion to nitrate in biological systems.	Rate (mg N/L·d)	Suspended growth: >1000 mg N/L·d; Attached growth: ~400-800 mg N/L·d [21]
Hydraulic Retention Time (HRT)	Average time urine remains in the bioreactor.	HRT (days)	Lower HRT enables smaller system volume [21]

Data from various nitrogen recovery techniques reveals a performance spectrum. Biological nitrification can achieve high NRE, producing nitrate-rich fertilizer ideal for plant growth [21]. In one suspended-growth system, a Nitrogen Conversion Efficiency (NCE) of 94.7% was reported, demonstrating high proficiency in converting ammonium to nitrate [21].

Table 2: Comparative Analysis of Nitrogen Recovery Techniques

Technique	Mechanism	Final Product	Key Advantages	Key Challenges for BLSS
Nitrification (Suspended Growth)	Biological oxidation of ammonia to nitrate by microbes (AOB/NOB)	Nitrate solution	High nitrification rates (>1000 mg N/L·d), low sludge production, high NCE [21]	Sensitivity to extreme Free Ammonia (FA), requires sludge separation [21]
Nitrification (Attached Growth)	Biofilm-mediated oxidation of ammonia to nitrate	Nitrate solution	Biomass retention, system resilience [21]	Lower process rates (e.g., 380-800 mg N/L·d), carrier transport/replacement [21]
Ammonia Stripping	pH adjustment and air/steam stripping of gaseous NH₃, absorbed in acid	Ammonium sulfate (solution/crystal)	Well-established, produces a familiar fertilizer [59]	High energy demand, handling of corrosive chemicals [59]
Chemical Precipitation	Precipitation with Mg and PO₄ salts	Struvite (MgNH₄PO₄)	Simultaneous N and P recovery, slow-release fertilizer [60]	N co-precipitation is a side-benefit to P recovery, product market [60]

Experimental Protocols

Protocol: Robustness Testing of Urine Nitrification

Objective: To evaluate the stability and inhibition response of a suspended-growth nitrification system to high salinity and extreme free ammonia (FA) concentrations, simulating potential system failures [21].

Materials:

• Sequencing Batch Reactor (SBR), 150 L pilot scale.
• pH, temperature, and dissolved oxygen (DO) probes and controllers.
• Internal heater and aeration system.
• Activated sludge inoculum, adapted to urine.
• Synthetic or real human urine, concentrated as required.
• Sodium chloride (NaCl).
• Sodium hydroxide (NaOH) for pH control.

Workflow:

• Reactor Startup & Acclimatization: Inoculate the SBR with activated sludge. Feed with diluted urine, gradually increasing the nitrogen loading rate over 20 days to acclimate the biomass. Maintain DO >2 mg/L, pH at 6.8-7.2, and temperature at 28-30°C [21].
• Baseline Performance: Establish baseline nitrification rates and NCE with a stable urine load.
• Salinity Stress Test: Introduce a high-salinity shock by adding NaCl to increase conductivity to a target level (e.g., 30-90 mS/cm). Monitor nitrification efficiency (ammonium and nitrite levels) for 24-48 hours to assess inhibition of Ammonia-Oxidizing Bacteria (AOB) and Nitrite-Oxidizing Bacteria (NOB) [21].
• Free Ammonia Inhibition Test: Induce a high FA event by allowing pH to rise (e.g., to 8.5-9.0) under high ammonium concentration. This can generate extreme FA concentrations (>80 g N/m³). Monitor the response of AOB and NOB and the time to full inhibition [21].
• System Recovery: Following inhibition, restore optimal operational conditions (pH ~7.0, normal loading). Document the time required for the system to recover >90% of its baseline nitrification performance.

Diagram: Urine Nitrification Robustness Testing Workflow

Protocol: Nitrogen Recovery via Vacuum Thermal Stripping-Acid Absorption

Objective: To determine the efficiency and energy cost of recovering nitrogen from urine as ammonium sulfate using a combined thermal stripping and acid absorption process.

Materials:

• Thermostatically controlled stripping column.
• Vacuum pump.
• Condensation and collection system.
• Acid absorption column filled with sulfuric acid (H₂SO₄).
• pH meter and temperature sensors.
• Concentrated urine feedstock.

Workflow:

• Urine Hydrolysis: Pre-treat urine to convert urea to ammonium. This can be achieved via high-temperature acidification or enzymatic (urease) processing [15].
• Stripping Unit Setup: Feed the hydrolyzed urine into the stripping column. Apply vacuum and heat (e.g., 60-80°C) to volatilize ammonia (NH₃) [60].
• Ammonia Capture: Direct the vapor stream, containing water and ammonia, to the condensation unit to recover water. The non-condensable gas (NH₃) is then passed through the absorption column, where it reacts with H₂SO₄ to form ammonium sulfate solution [(NH₄)₂SO₄] [60] [15].
• Product Crystallization (Optional): Evaporate the ammonium sulfate solution to crystallize the solid fertilizer product.
• Metrics Calculation: Analyze the total nitrogen content in the initial urine and the final ammonium sulfate product to calculate NRE. Precisely monitor the electrical energy for heating/vacuum and thermal energy input to calculate total Energy Cost (kWh/kg N).

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagent Solutions for Nitrogen Recovery Experiments

Reagent/Material	Function/Application	Example Use in Protocol
Activated Sludge	Mixed microbial consortium for biological nitrification.	Inoculum for sequencing batch reactors (SBRs) in nitrification robustness tests [21].
Urease Enzyme	Hydrolyzes urea in urine to ammonium and carbonate.	Pre-treatment of urine to stabilize nitrogen and make it available for recovery processes [15].
Synthetic Urine	Standardized, reproducible feedstock for controlled experiments.	Allows for comparison of different technologies without the variability of real urine.
Zeolite/Clinoptilolite	Ammonium-selective ion-exchange medium.	Capturing and concentrating ammonium from urine for later recovery or direct use as a slow-release fertilizer [60].
Sulfuric Acid (H₂SO₄)	Acidifying agent for pH control and reactant for ammonium sulfate production.	Used in the acid absorption column to capture stripped ammonia gas [60] [59].
Magnesium and Phosphate Salts	Precipitating agents for struvite formation.	Simultaneous recovery of nitrogen and phosphorus as struvite (MgNH₄PO₄) [60].

Achieving high nitrogen recovery efficiency with minimal energy cost and robust stability is a cornerstone of sustainable BLSS. Biological nitrification offers high efficiency and lower energy demands but requires careful management of inhibitory factors like free ammonia and salinity. Physicochemical methods like stripping provide alternative pathways with different product profiles. The protocols and metrics detailed herein provide a framework for researchers to quantitatively assess and compare technologies, ultimately guiding the development of closed-loop life support systems for the future of space exploration.

The recovery of nitrogen from human urine is a critical challenge for achieving closure of Bioregenerative Life Support Systems (BLSS) in long-duration space missions. With water recovery rates exceeding 90% already feasible in current environmental control and life support systems (ECLSS), nitrogen recovery efficiency remains a limiting factor, with some methods achieving as little as 20.5% efficiency [10]. This application note provides a detailed technical comparison of three promising nitrogen recovery technologies—Biological Activated Carbon (BAC), Immobilized Urease Catalysis, and Distillation—evaluating their performance characteristics, operational requirements, and implementation protocols for BLSS applications.

Technology Comparisons

Performance Metrics

Table 1: Comparative performance of nitrogen recovery technologies for urine processing in BLSS

Parameter	Biological Activated Carbon (BAC)	Immobilized Urease Catalysis	Distillation-Based Methods
Nitrogen Recovery Efficiency	79.33% (synthetic urine) [61]	52.2% (optimal conditions) [10]	20.5-39.7% (depending on pretreatment) [10]
Urea Hydrolysis Rate	High (52.9% in first 2 days) [61]	Very High (85% with free enzyme in 1 day) [61]	Dependent on pretreatment method [10]
Optimal Temperature	Ambient (25°C) [61]	60°C [10]	99°C (HTAM) [10]
Optimal pH	Neutral [61]	7 [10]	Acidic (HTAM: [H+]=2 mol/L) [10]
Processing Time	5 days HRT [61]	40 minutes [10]	7 hours (HTAM) [10]
Water Recovery	Integrated with membrane filtration [61]	Not primary function	70-97.75% [61] [62]
Technology Readiness	Ground demonstrated [61]	Ground demonstrated [10]	Space flight experienced (ISS) [1]

Operational Considerations

Table 2: Operational requirements and constraints

Consideration	Biological Activated Carbon	Immobilized Urease	Distillation
Key Advantages	Continuous urease production by immobilized microorganisms; stable biofilm community [61]	Rapid reaction; enhanced stability against pH/temperature changes [61]	High water recovery; flight-proven technology [61] [1]
Limitations	Requires 35 days for biofilm stabilization [61]	Enzyme source and short lifetime may limit large-scale application [61]	Limited nitrogen recovery without pretreatment; scaling issues [10] [1]
Energy Requirements	Moderate (aeration, circulation) [61]	Low to moderate (temperature control) [10]	High (heating, vacuum systems) [10]
Microbial Ecology	Diverse community (Bacillus, Sporosarcina, Pseudomonas, Paracoccus) [61]	Minimal (enzyme-based)	Minimal (physical/chemical process)
Downstream Compatibility	Compatible with plant growth systems [61]	Provides ammonium for direct plant uptake [61]	Requires additional nutrient processing for plant use [10]

Experimental Protocols

Biological Activated Carbon System

Principle: BAC utilizes urease-producing microorganisms immobilized on activated carbon to continuously hydrolyze urea to ammonium and carbon dioxide [61].

Materials:

• Coconut shell granulated activated carbon (specific surface: 800-900 m²/g)
• Bacterial inoculants extracted from soil
• Synthetic urine composition: urea (24 g/L), NaCl (5.85 g/L), KH₂PO₄ (3.40 g/L), KCl (2.00 g/L), NH₄Cl (0.50 g/L), CaCl₂ (0.34 g/L), MgSO₄·7H₂O (0.34 g/L), creatinine (0.60 g/L), acetic acid (0.34 g/L)
• Membrane bioreactor configuration

Procedure:

• BAC Cultivation: Mix bacterial inoculants with powder activated carbon (optimal dosage: 100 g/L) and cultivate for 35 days with daily synthetic urine replacement until stable urea hydrolysis is achieved [61].
• Reactor Operation: Configure membrane-BAC reactor with 5-day hydraulic retention time (HRT).
• Monitoring: Sample daily for urea concentration (diacetylmonoxime method), ammonium concentration (Nessler's method), and pH.
• Biofilm Characterization: Analyze biofilm development via SEM and CLSM; identify microbial community through 16S rRNA high-throughput sequencing [61].

Immobilized Urease Catalysis Method

Principle: Urease enzyme tethered to support matrix catalyzes urea hydrolysis to ammonium and carbon dioxide under optimized conditions [10].

Materials:

• Immobilized urease (coconut shell AC-tethered, loading capacity: 78.8 mg/g)
• Urease activity assay reagents
• Real human urine samples
• Temperature-controlled reaction vessel

Procedure:

• Condition Optimization: Conduct batch experiments to determine optimal temperature (60°C), pH (7.0), and reaction time (40 minutes) [10].
• Enzyme Activity Assay: Monitor urease activity using standard protocols.
• Nitrogen Recovery: Process urine samples under optimal conditions, measuring initial and final urea and ammonium concentrations.
• Stability Testing: Evaluate remaining activity after 50 hours of operation to assess operational lifespan [61].

Distillation with Pretreatment

Principle: Thermal distillation with pretreatment converts urea to volatile ammonia, which is recovered through condensation [10].

Materials:

• Reduced pressure distillation apparatus
• Acid reagents for High Temperature Acidification Method (HTAM: H₂SO₄ or HCl)
• Immobilized urease for alternative pretreatment
• pH adjustment chemicals

Procedure:

• Urine Pretreatment (HTAM): Acidify urine to [H+]=2 mol/L and heat to 99°C for 7 hours [10].
• Alternative Pretreatment (IUCM): Hydrolyze urine with immobilized urease at 60°C, pH 7 for 40 minutes [10].
• Distillation: Transfer pretreated urine to reduced pressure distillation system.
• Collection: Collect distillate and measure water volume and ammonium concentration in both distillate and residue.
• Calculation: Determine nitrogen recovery efficiency using mass balance approaches.

Integration Pathways for BLSS

The Scientist's Toolkit

Table 3: Essential research reagents and materials for nitrogen recovery experiments

Reagent/Material	Function/Application	Technical Specifications	Reference
Coconut Shell Activated Carbon	BAC support matrix providing high surface area for microbial immobilization	Specific surface: 800-900 m²/g; mechanical hardness	[61]
Urease Enzyme	Hydrolyzes urea to ammonium and carbon dioxide	Immobilized loading capacity: 78.8 mg/g (urease/AC)	[61]
PTFE-PP Membrane	Hydrophobic membrane for distillation processes	0.45 µm pore size; PTFE active layer with PP support	[62]
Soil Bacterial Inoculants	Source of urease-producing microorganisms for BAC	Extract from soil with deionized water (2:3 soil to water ratio)	[61]
Synthetic Urine Formulation	Standardized testing medium	Urea (24 g/L), NaCl (5.85 g/L), electrolytes, organics	[61]
16S rRNA Sequencing Reagents	Microbial community analysis in BAC systems	High-throughput sequencing of biofilm communities	[61]

The selection of nitrogen recovery technology for BLSS applications involves critical trade-offs between efficiency, resource requirements, and system complexity. Biological Activated Carbon offers the highest nitrogen recovery efficiency and continuous operation through microbial urease production but requires extended startup times and more complex system management. Immobilized Urease Catalysis provides rapid processing with good efficiency but faces potential enzyme sourcing and stability challenges. Distillation methods, while flight-proven and excellent for water recovery, demonstrate limited nitrogen recovery efficiency without energy-intensive pretreatments. Future BLSS architectures may benefit from hybrid approaches that leverage the complementary strengths of these technologies to achieve both high water and nitrogen closure rates essential for long-duration space missions.

Bioregenerative Life Support Systems (BLSS) are essential for long-term, crewed space missions, as they aim to recycle waste into oxygen, water, and food. A critical process within a BLSS is the recovery of nitrogen from human urine, which accounts for approximately 85% of the recoverable nitrogen and is vital for fertilizing plant growth systems [1]. This document details the application notes and experimental protocols for validating nitrogen recovery processes in two major ground-based analogue tests: the Lunar Palace 365 experiment and the MELiSSA (Micro-Ecological Life Support System Alternative) project. The content is structured to provide researchers and scientists with a clear framework for data comparison, experimental replication, and system analysis.

The Lunar Palace 1 (LP1) and MELiSSA projects are pioneering BLSS test beds that integrate biological and physicochemical processes to close the loop on essential element cycles.

• Chinese Lunar Palace 1 (LP1): This ground-based facility is a 160 m² system with a total volume of 500 m³. It integrates plant cultivation cabins, a comprehensive cabin for crew living, and a solid waste treatment cabin [63] [64]. The "Lunar Palace 365" project was a 370-day manned test that provided crucial data on system performance and microbial dynamics [63].
• MELiSSA (European Space Agency): This project is designed as a five-compartment, bioengineered system mimicking the ecosystem of a lake. Its goal is to recover minerals from waste streams completely, with a particular focus on nitrogen recovery from urine in its third compartment [1].

Table 1: Key Characteristics of Lunar Palace and MELiSSA

Feature	Lunar Palace 1	MELiSSA
Primary Developer	Beijing University of Aeronautics and Astronautics (China)	European Space Agency (ESA)
System Type	Multi-compartment BLSS (Plant cabins, crew cabin, waste cabin) [63]	Five-compartment, lake-ecosystem-inspired loop [1]
Key Nitrogen Source	Human metabolic waste (implied) [1]	Human urine (85% of recoverable N) [1]
Nitrogen Recovery Goal	Recycling within the closed ecosystem [63]	Highly efficient recovery for plant/algal fertilization [1]
Major Validation Mission	Lunar Palace 365 (370-day mission) [63]	Ongoing ground and future space demonstrations [1]

Quantitative Data and Performance Metrics

Validation in ground demonstrators relies on collecting robust quantitative data to assess the system's stability and the efficiency of its core processes, such as nutrient recycling and microbial management.

Data from the Lunar Palace 365 mission revealed that personnel changes significantly affected the diversity of airborne bacterial communities, underscoring the human crew's impact on the BLSS environment [64]. Furthermore, research showed that plants were the primary source of surface fungal communities, and despite crew turnover, the potential for mycotoxin production remained stable, indicating a healthy ecological balance [63].

Table 2: Key Quantitative Findings from Lunar Palace 365 Studies

Parameter Investigated	Key Finding	Implication for BLSS Validation
Airborne Bacterial Diversity	Significantly influenced by crew changeover [64]	Crew composition is a critical variable for environmental monitoring.
Surface Fungal Diversity	Higher alpha diversity than in other confined habitats (e.g., ISS); primary origin is plants [63]	Plant modules are a major determinant of the mycobiome.
Mycotoxin Gene Abundance	No significant difference despite occupant or location changes [63]	Suggests a balanced, healthy fungal community in the presence of plants.
Crew & Mission Duration	8 total crew split into 2 groups; 370-day mission duration [63] [64]	Provides context for the scale and duration of human-in-the-loop testing.

Experimental Protocols for BLSS Validation

Detailed and standardized protocols are fundamental for generating reliable and comparable data in BLSS research. The following sections outline critical methodologies.

Protocol 1: Surface Mycobiome and Mycotoxin Potential Analysis

This protocol, derived from the Lunar Palace 365 study, describes how to characterize the fungal community and its potential to produce toxins on interior surfaces [63].

1. Sample Collection

• Materials: Sterile swab tube (e.g., containing 3 mL of 0.85% NaCl solution), standard 10 cm x 10 cm sterilization specification plate.
• Procedure:
  • Place the sterilization plate on the pre-defined sampling point.
  • Use the sterile swab to thoroughly sample the surface area within the plate.
  • Immediately place the swab back into the tube, seal it, and label it.
  • Include field controls by waving a clean swab in the air at the sampling location. Process unused sampling materials as lab controls.

2. DNA Extraction

• Materials: FastDNA Spin Kit or equivalent.
• Procedure:
  • Centrifuge the sample tube at 1,400 ×g for 5 minutes to separate the fluid from the swab. Discard the swab.
  • Transfer the fluid to a 2.0 mL centrifuge tube.
  • Incubate the tube at 70°C for 10 minutes at 1000–1200 rpm in a shaking mixer to promote lysis.
  • Extract DNA from the fluid using the FastDNA Spin Kit, following the manufacturer's instructions [63].

3. Molecular Analysis

• Fungal Community (Illumina Sequencing):
  • Target: Nuclear ribosomal internal transcribed spacer 1 (ITS1) region.
  • Primers: ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) [63].
  • Process: Perform a two-step amplification prior to MiSeq Illumina sequencing.
• Mycotoxin Gene Quantification (qPCR):
  • Targets: Amplify genes specific to mycotoxin pathways (e.g., idh, ver1, nor1, tri5) and the ITS1 region for absolute quantification [63].

Protocol 2: Airborne Microbiome and Resistome Monitoring

This protocol details the method for assessing the bacterial community and antibiotic resistance genes (ARGs) in the air of a BLSS, as performed in Lunar Palace 365 [64].

1. Air Sampling

• Materials: Air purifier with High-Efficiency Particulate Air (HEPA) filter (e.g., Xiaomi Air Purifier 2).
• Procedure:
  • Place the air purifier at the designated sampling location (e.g., plant cabin, comprehensive cabin).
  • Sample air continuously over a defined period (e.g., 30 days) to accumulate sufficient biomass on the filter for analysis.

2. DNA Extraction and Downstream Analysis

• Procedure:
  • Process the collected dust from the HEPA filter for DNA extraction.
  • Perform a multi-faceted molecular analysis:
    • 16S rRNA Amplicon Sequencing: To evaluate bacterial diversity and species composition.
    • Shotgun Metagenomic Sequencing: To assess the functional potential of the microbial community.
    • Quantitative PCR (qPCR): To determine the absolute abundance of bacteria and specific Antibiotic Resistance Genes (ARGs) [64].

Workflow Visualization

The following diagram illustrates the logical sequence and decision points for the core experimental protocols described in this document.

Diagram 1: BLSS Validation Workflow

The Scientist's Toolkit: Research Reagent Solutions

The successful implementation of the protocols above depends on a set of essential reagents and materials.

Table 3: Essential Research Reagents and Materials for BLSS Validation

Item	Function / Application
Sterile Swab & Buffer	Collection and temporary preservation of surface microbial samples [63].
HEPA Filter-based Air Sampler	Collection of airborne microbial particles and dust over extended periods [64].
DNA Extraction Kit	Isolation of high-quality genomic DNA from diverse sample types (surface, air dust) for downstream molecular analysis [63].
PCR Reagents & Primers	Amplification of target genetic regions (e.g., ITS for fungi, 16S rRNA for bacteria, specific mycotoxin/ARG genes) [63].
qPCR Master Mix	Absolute quantification of specific genes and taxonomic markers [63] [64].
Illumina Sequencing Reagents	High-throughput analysis of microbial community composition and functional potential [63] [64].
FastDNA Spin Kit	Specifically cited for efficient DNA isolation from environmental samples in the LP1 study [63].
Urine Stabilization Acid	Prevents scaling and stabilizes urea in urine waste streams, a critical first step in nitrogen recovery [1].

The development of Bioregenerative Life Support Systems (BLSS) is crucial for long-term crewed space missions, aiming to sustainably produce food, recycle water, and regenerate oxygen [30]. Within these closed-loop systems, nitrogen recovery from human urine presents a pivotal strategy for providing essential nutrients for plant growth, thereby reducing reliance on external resupply from Earth [3]. The average human urine excretion contains 7–16g of nitrogen per day, accounting for approximately 85% of the potentially recoverable nitrogen in a BLSS, making it the primary source of this vital nutrient [3]. This document details application notes and protocols for the agronomic validation of fertilizers derived from urine, specifically within the context of soilless plant cultivation systems, which are integral to advanced BLSS designs like the Micro-Ecological Life Support System Alternative (MELiSSA) [30] [3].

The following table summarizes primary nitrogen recovery pathways from urine and their expected performance in soilless cultivation.

Table 1: Agronomic Performance of Urine-Derived Nutrient Sources in Soilless Systems

Fertilizer Source	Key Nitrogen Form	Reported Agronomic Efficacy	Considerations for Soilless Systems
Stabilized Urine (via Nitrification) [65]	Nitrate (NO₃⁻)	Nitrification rate: 563.7 mg N·L⁻¹·d⁻¹ (with betaine) [65]	Stabilization prevents pH rise and nitrogen loss; suitable for closed-loop hydroponics [65].
Electrochemical Recovery (Ammonia Stripping) [37]	Ammonium Sulfate [(NH₄)₂SO₄]	Potential earnings up to \$4.13 per kg N recovered (Uganda model) [37]	Provides a concentrated, sterile fertilizer solution; ideal for precise nutrient solution formulation [37].
Combined Physicochemical Processing (e.g., ECLSS) [3]	Distillate (Water) / Concentrated Waste	Water recovery efficiency ~85%; nitrogen typically not recovered for plants [3]	Highlights the limitation of non-biological systems; used as a benchmark for BLSS improvement [3].

Detailed Experimental Protocols

Protocol 1: Ultra-Rapid Start-Up of Urine Nitrification for Nutrient Solution Production

This protocol describes a method for rapidly initiating a stable biological nitrification process to convert urine into a nitrate-rich fertilizer for hydroponic systems [65].

• Objective: To achieve a rapid start-up of nitrification for source-separated urine, producing a stabilized nitrate solution for plant nutrition.
• Principle: Ammonia nitrogen in hydrolyzed urine is oxidized to nitrate by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) under aerobic conditions. The addition of the osmoprotectant betaine mitigates microbial inhibition caused by high salinity and ammonia levels in urine [65].
• Materials:
  • Source-separated human urine
  • Aerobic nitrifying sludge (e.g., 10 ± 0.05 g MLVSS·L⁻¹)
  • Betaine
  • 2 L beakers or aerated bioreactors
  • Air pumps and diffusers
  • Water quality probes (pH, NH₄⁺-N, NO₂⁻-N, NO₃⁻-N)
• Step-by-Step Procedure:
  • Urine Collection and Hydrolysis: Collect source-separated urine and store it to allow for complete hydrolysis of urea to ammonia [65].
  • Reactor Setup: Place the nitrifying sludge in the reactor. Dilute the hydrolyzed urine with water to achieve a target NH₄⁺-N concentration (e.g., 1000 mg/L is used in studies) [65].
  • Betaine Addition: Add betaine to the reactor at a concentration of 50 mg/L. Maintain a control reactor without betaine for comparison [65].
  • Aeration and Operation: Initiate continuous aeration to maintain dissolved oxygen levels above 2 mg/L. Operate in batch or continuous mode, monitoring key parameters daily.
  • Monitoring: Track the concentrations of NH₄⁺-N, NO₂⁻-N, and NO₃⁻-N to calculate the nitrification rate. System stability is achieved when NH₄⁺-N is consistently below 5 mg/L and NO₂⁻-N accumulation is negligible [65].
• Expected Outcomes: With betaine addition, the system start-up time can be reduced from 98 days to 36 days, with a high nitrification rate of 563.7 mg N·L⁻¹·d⁻¹ achieved [65]. The resulting nitrified urine is a stabilized nutrient solution rich in nitrate.

Protocol 2: Electrochemical Recovery and Validation of Ammonium Sulfate Fertilizer

This protocol outlines a procedure for recovering nitrogen from urine as ammonium sulfate using an electrochemical system powered by solar energy, and its subsequent agronomic testing [37].

• Objective: To recover nitrogen from urine as ammonium sulfate and evaluate its efficacy as a fertilizer in a hydroponic system.
• Principle: An electrochemical cell uses solar-generated electricity to drive the separation of ammonia from urine. Ions are moved across membranes, ultimately trapping ammonia as ammonium sulfate, a common fertilizer [37].
• Materials:
  • Electrochemical nutrient recovery prototype [37]
  • Photovoltaic solar panels with waste heat collection system
  • Source-separated urine
  • Hydroponic growth system (e.g., Nutrient Film Technique - NFT)
  • Target crop seeds (e.g., lettuce, basil)
  • Standard Hoagland's nutrient solution (for control)
• Step-by-Step Procedure:
  • Fertilizer Production: Process collected urine through the electrochemical system. Use solar-derived electricity and waste heat to enhance ammonia recovery efficiency [37].
  • Nutrient Solution Formulation: Formulate a hydroponic nutrient solution where the nitrogen component is entirely provided by the recovered ammonium sulfate. A control solution should use an equivalent nitrogen concentration from a conventional source like potassium nitrate.
  • Plant Growth Trial: Sow seeds in the hydroponic system. Once established, transplant seedlings into separate channels for the test and control solutions.
  • System Management and Monitoring: Maintain pH and electrical conductivity (EC) of the nutrient solutions within optimal ranges for the chosen crop. Recirculate the solution in a closed system.
  • Data Collection: After a growth cycle (e.g., 4-5 weeks for lettuce), measure agronomic parameters: fresh and dry biomass yield, plant height, leaf area, and chlorophyll content.
• Expected Outcomes: The study should demonstrate that the ammonium sulfate fertilizer from urine can support plant growth comparable to conventional fertilizers. Nitrogen use efficiency (NUE) of the crops should be calculated and optimized [30].

Visualization of Workflows and Pathways

Nitrogen Recovery and Utilization Pathway in a BLSS

The following diagram illustrates the logical flow of nitrogen from waste streams to plant biomass within a Bioregenerative Life Support System.

Nitrogen Loop in a BLSS

Experimental Workflow for Fertilizer Validation

This diagram outlines the specific experimental workflow for validating the efficacy of a urine-derived fertilizer.

Fertilizer Validation Workflow

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Reagents and Materials for Urine-Derived Fertilizer Research

Item	Function/Application	Research Context
Betaine	Osmoprotectant to enhance salt tolerance of nitrifying bacteria in high-ammonia urine [65].	Critical for rapid start-up and stable operation of urine nitrification bioreactors [65].
Nitrifying Sludge	A mixed microbial consortium containing Ammonia-Oxidizing Bacteria (AOB) and Nitrite-Oxidizing Bacteria (NOB) [65].	Serves as the biocatalyst in biological nitrification systems for converting ammonia to nitrate [65].
Ion-Selective Membranes	Facilitate the selective transport of ions (e.g., NH₄⁺) in electrochemical nutrient recovery cells [37].	Key component in electrochemical systems for separating and concentrating ammonia from urine [37].
Inert Hydroponic Substrate (e.g., Perlite, Mineral Wool)	Provides physical support for plant roots in soilless systems without altering nutrient chemistry [66].	Used in substrate-based hydroponic systems to evaluate plant uptake of nutrients from novel fertilizers [66].
pH & EC Meters	Monitor and control the acidity and ionic strength (nutrient concentration) of the hydroponic nutrient solution [30].	Essential for maintaining optimal growing conditions and ensuring valid comparisons in fertilization trials [30].

{Techno-Economic Analysis and Scalability for Mission Deployment}

The development of Bioregenerative Life Support Systems (BLSS) is critical for long-duration space missions, aiming to create closed-loop systems that recycle water, oxygen, and nutrients. Nitrogen is a vital element for protein synthesis and plant growth within these systems. This document provides a detailed techno-economic analysis and scalability assessment of a novel Nitrogen Recovery Process from Human Urine, a technology pivotal for enhancing the sustainability and self-sufficiency of BLSS by converting a major waste stream into a valuable agricultural resource. The process is adapted from recent terrestrial research demonstrating the feasibility of urine valorization [25] [67].

Techno-Economic Analysis

A preliminary techno-economic analysis was conducted to evaluate the process's viability for mission deployment. The analysis is based on a system designed to process urine from a crew of four astronauts, with key performance and economic metrics summarized below.

Table 1: Key Performance Metrics for Nitrogen Recovery

Metric	Value	Reference / Calculation Basis
Nitrogen Recovery Efficiency	~7.5 kg N per m³ urine	[25] [67]
Estimated Daily Urine Production per Crew Member	1.5 L	Standard life support system design parameter
System Capacity for 4-Person Crew	6 L urine/day	(4 crew × 1.5 L/day)
Annual Nitrogen Output (Estimated)	~1.64 kg N	(6 L/day × 365 days × 7.5 kg N / 1000 L)
Equivalent Tomato Production Capacity (Earth Open-Field)	~525 kg annually	[25] [67]
Electrochemical Conversion Efficiency (Urea to Fertilizer)	~100%	[68]

Table 2: Scalability and Resource Projections

Scale Scenario	Required Urine Source	Estimated Land Footprint	Key Technical Consideration
Lab-scale (Producing 1 kg fertilizer/day)	6,382 households or 3,800 dairy cows	100 m²	Serves as a terrestrial analog for sizing initial BLSS modules [68].
Mission-scale (Supporting 4 crew)	Integrated crew life support	< 5 m² (estimated)	Focus on system miniaturization, automation, and integration with hydroponics.

Table 3: Economic and Operational Advantage Analysis

Parameter	Traditional Haber-Bosch N-Fixation	Proposed Urine Nitrogen Recovery	Advantage for Mission Deployment
Primary Energy/Feedstock	Natural Gas (Fossil Fuels)	Human Metabolic Waste (In-situ Resource)	Dramatic reduction in Earth-upmass; Enhanced mission closure and sustainability.
CO2 Emissions	High (~2.5 tons CO2/ton NH3)	Negligible (process is part of carbon cycle)	Significant reduction in mission lifecycle emissions [25] [67].
Water Pollution Potential	High (from fertilizer runoff)	Prevented (N is captured, not released as effluent)	Mitigates internal water purification load within the BLSS [25] [67].
Process Intensity	High-pressure, high-temperature	Ambient or mild conditions (Electrochemical/Biological)	Improved crew safety; lower energy and hardware mass requirements.

Experimental Protocols

Protocol: Nitrified Nitrogen Fertilizer Production from Urine

This protocol details the production of a nitrate-rich liquid fertilizer suitable for hydroponic systems, based on the method described by the Spanish research team [25] [67].

1. Principle: Urine is collected and stabilized. Microorganisms are used to convert urea and ammonium sequentially into nitrate (nitrification) under alkaline conditions, producing a plant-available nitrogen solution.

2. Materials:

• Source-separating, waterless urinals or collection system.
• Alkaline agent (e.g., potassium hydroxide, KOH).
• Bioreactor with aeration and temperature control (e.g., 25-30°C).
• Nitrifying bacterial inoculum (e.g., from established aquatic filters).
• Filtration unit (0.22 µm).
• pH and nitrate meters.

3. Procedure:

Diagram 1: Nitrified fertilizer production workflow.

• Step 1: Urine Collection. Collect urine using a source-separation system to minimize contamination.
• Step 2: pH Stabilization & Hydrolysis. Adjust the collected urine to a pH of >9 using KOH. This stabilizes the urine by preventing urea hydrolysis and ammonia volatilization during storage. Allow the mixture to stand for 24-48 hours to ensure pathogen reduction.
• Step 3: Microbial Nitrification. Inoculate the stabilized urine with a nitrifying bacterial consortium in a bioreactor. Dilute the urine with water if necessary (e.g., 1:1) to reduce ammonium toxicity to microbes. Maintain vigorous aeration, a temperature of 25-30°C, and a pH of 7.5-8.5. Monitor ammonium and nitrate levels. The process is complete when ammonium is undetectable and nitrate levels have plateaued (typically 2-4 weeks).
• Step 4: Filtration. Pass the nitrified solution through a 0.22 µm filter to remove bacterial biomass, producing a sterile liquid fertilizer.
• Step 5: Quality Control & Application. Analyze the final product for nitrate concentration and pH. Adjust the pH to the optimal range for the target crop (typically 5.5-6.5 for hydroponics) before introducing it into the hydroponic nutrient solution.

Protocol: Electrochemical Conversion to Solid Urea Derivative (Overcarbonamide)

This protocol describes an alternative method for converting urine urea into a solid, stable fertilizer, overcarbonamide, using an electrochemical process, as reported by the Chinese research team [68].

1. Principle: An electrochemical reactor is used to selectively oxidize urea in a wastewater stream, leading to the nearly quantitative formation and precipitation of pure overcarbonamide, a solid peroxide-containing urea derivative with applications as a fertilizer and disinfectant.

2. Materials:

• Electrochemical reactor with appropriate electrodes (e.g., Ni-based anode).
• Power supply.
• Urine or synthetic urine feed.
• Filtration or centrifugation setup.
• Drying oven.

3. Procedure:

Diagram 2: Electrochemical conversion to solid fertilizer.

• Step 1: Feed Preparation. Pre-filter urine to remove large particulates. The electrolyte composition may be adjusted to optimize reaction efficiency.
• Step 2: Electrochemical Conversion. Pump the feed into the electrochemical reactor. Apply a controlled voltage/current to the electrodes. The specific parameters (e.g., current density, reaction time) are optimized to achieve near 100% conversion of urea to overcarbonamide [68]. The product precipitates out of the solution.
• Step 3: Solid-Liquid Separation. Separate the solid overcarbonamide from the treated liquid stream using filtration or centrifugation.
• Step 4: Product Drying. Gently dry the collected solid to remove residual moisture, resulting in a stable, storable solid fertilizer.

The Scientist's Toolkit: Research Reagent Solutions

Table 4: Essential Materials and Reagents for Nitrogen Recovery Research

Item	Function/Application	Notes for BLSS Context
Potassium Hydroxide (KOH)	Alkaline agent for urine stabilization and pH control during nitrification.	Preferred over sodium hydroxide to avoid Na+ accumulation in closed-loop hydroponics.
Nitrifying Bacterial Consortium	Bioinoculant for converting ammonium to nitrate in Protocol 3.1.	Select for robust, space-compatible strains; potential for in-situ cultivation.
Electrochemical Reactor	Core unit for the direct conversion of urea to overcarbonamide (Protocol 3.2).	Requires development of compact, low-mass, and highly efficient designs.
0.22 µm Sterilization Filter	For removing microbial biomass from the liquid fertilizer product post-bioprocessing.	Critical for preventing contamination of the hydroponic subsystem.
Ion-Selective Electrodes / HPLC	For monitoring ammonium, nitrite, and nitrate levels during processes.	On-line, automated sensors are ideal for continuous mission operations.
Source-Separation Sanitation Hardware	For collecting undiluted, low-contamination urine.	Integral to system design; impacts downstream processing efficiency.

Scalability for Mission Deployment

Scalability from laboratory prototypes to integrated flight systems presents several key challenges and considerations:

• System Integration and Mass Balance: The nitrogen recovery system must be tightly coupled with the food production (hydroponic) and water recovery subsystems. Dynamic modeling is required to match the rate of fertilizer production with plant nutrient demand.
• Reliability and Redundancy: Given the criticality of food production, the system must have high reliability and built-in redundancy or alternative nutrient supply strategies.
• Crew Time and Automation: Processes must be highly automated to minimize crew time required for maintenance and operation. Protocol 3.2 (Electrochemical) may offer advantages here due to its potential for continuous operation and fewer biological stability concerns.
• Pathogen and Pharmaceutical Contaminants: Rigorous testing is needed to ensure the complete removal or degradation of pathogens and pharmaceutical residues present in urine [25] [67]. The electrochemical method may offer superior pathogen kill.
• Technology Readiness Level (TRL): Current systems are at TRL 3-4. Roadmaps should focus on integrated testing in increasingly realistic analog environments (e.g., Antarctic stations, lunar analog missions) to advance to TRL 6+.

The presented techno-economic data and protocols establish a foundation for developing nitrogen recovery processes that are technically feasible, economically advantageous, and scalable for future BLSS missions, turning a waste management challenge into a pillar of mission sustainability.

Conclusion

Nitrogen recovery from urine is a linchpin for achieving the material closure required for sustainable, long-duration space missions. A hybrid approach, integrating the robustness of physicochemical methods with the resource efficiency of biological systems, appears most promising. Future research must prioritize enhancing the resilience of biological components to space conditions, integrating multiple nutrient recovery (N, P, K) processes, and automating control systems for unprecedented operational stability. Success in this domain will not only enable deep space exploration but also yield transformative technologies for sustainable resource management on Earth.

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