Assessing Bioregenerative Life Support Systems (BLSS): A TRL Analysis for Deep Space Exploration

Abstract

This article provides a comprehensive analysis of the Technology Readiness Levels (TRLs) of Bioregenerative Life Support Systems (BLSS) for long-duration deep space missions. Tailored for researchers, scientists, and technology development professionals in the space sector, it explores the foundational principles of BLSS, applies the TRL framework to assess current subsystem maturity, identifies critical challenges and optimization strategies, and offers a comparative validation against existing physicochemical systems. The analysis synthesizes current research and ground demonstrations to outline a strategic pathway for advancing BLSS from experimental concepts to mission-critical infrastructure for lunar and Martian outposts.

The Principles and Imperatives of BLSS for Deep Space

Bioregenerative Life Support Systems (BLSS) represent the most advanced class of life support technology for long-duration space missions, creating artificial ecosystems comprising complex symbiotic relationships among higher plants, microorganisms, and humans [1]. These systems aim to regenerate air, water, and food through biological processes rather than solely relying on physical/chemical systems or resupply from Earth. The evolution from earlier concepts like Closed Ecological Life Support Systems (CELSS) to modern BLSS architectures reflects a growing understanding of the need for robust, self-sustaining ecosystems capable of supporting human life in deep space environments beyond low Earth orbit [2].

The fundamental principle underlying BLSS is the creation of a closed-loop system that mimics Earth's natural biogeochemical cycles, particularly for essential elements like carbon, nitrogen, and phosphorus [3]. Unlike open-loop systems where all consumables are supplied from Earth, or hybrid systems that combine physical/chemical recycling with some biological components, a fully developed BLSS achieves high degrees of material closure through integrated biological processes [3]. This approach becomes increasingly necessary as mission durations extend and distances from Earth increase, making resupply impractical or prohibitively expensive.

Historical Development and Key Concepts

From CELSS to BLSS: Conceptual Evolution

The conceptual foundation for bioregenerative life support began with NASA's Controlled Ecological Life Support Systems (CELSS) program in the latter part of the 20th century, which focused on developing controlled environment agriculture for logistically sustainable space exploration [2]. This evolved into more integrated testing facilities such as the Bioregenerative Planetary Life Support Systems Test Complex (BIO-PLEX), which was designed as a habitat demonstration program before being discontinued in 2004 [2]. Parallel development occurred internationally, with the Soviet/Russian programs making significant early contributions to space-based life support, followed by the European Space Agency's Micro-Ecological Life Support System Alternative (MELiSSA) program, which has focused on BLSS component technology development [2].

The most extensive terrestrial demonstration of closed ecosystem principles was Biosphere 2, a 12,700 m² glass-enclosed facility that housed eight crew members for two years with nearly complete material closure [3]. This massive experiment provided invaluable data on the challenges of maintaining balanced ecological systems, including managing atmospheric composition, nutrient cycling, and ecosystem stability. Despite its scale making it impractical for direct space application, Biosphere 2 demonstrated that 100% closure is theoretically feasible for extended periods, though precise control mechanisms require further development [3].

International BLSS Initiatives

Table: Major International BLSS Development Programs

Program/Agency	Key Focus	Notable Achievements	Status
NASA CELSS/BIO-PLEX (US)	Controlled environment agriculture, integrated system testing	Early research on plant growth, water recycling, air revitalization	Discontinued (2004) [2]
Lunar Palace (CNSA)	Closed-system bioregenerative life support	Sustained 4 crew for 1 year with atmosphere, water, and nutrition closure [2]	Active, expanding [2]
MELiSSA (ESA)	Compartmentalized microbial ecosystems	Component technology development, pilot plant testing	Active, ongoing research [2]
Biosphere 2	Large-scale terrestrial ecological closure	8 crew for 2 years with near-complete material closure [3]	Completed (1991-1993)

China's Beijing Lunar Palace represents the current state-of-the-art in BLSS implementation, building upon earlier NASA research while incorporating domestic innovation [2]. The CNSA has demonstrated closed-system operations for atmosphere, water, and nutrition while sustaining a crew of four analog taikonauts for a full year, establishing China as the current leader in operational BLSS capability [2]. This achievement marks a significant milestone in the transition from theoretical concepts and component testing to integrated system implementation.

System Architectures and Functional Components

Core Subsystems and Their Interactions

A fully integrated BLSS comprises multiple interdependent subsystems that collectively maintain human life. The atmosphere revitalization subsystem typically employs photosynthetic organisms (plants and algae) to convert carbon dioxide exhaled by crew members back into oxygen while fixing carbon into biomass [1]. The water recovery subsystem processes various waste streams (urine, gray water, humidity condensate) through a combination of physical, chemical, and biological treatment processes to produce potable water [3]. The food production subsystem generates edible biomass through controlled plant cultivation, while the waste management subsystem processes solid wastes (inedible plant material, human metabolic waste) to recover nutrients and stabilize the system [1].

BLSS Component Interactions

Element Cycling and Stoichiometric Balances

The core challenge in BLSS design lies in maintaining balanced element cycles, particularly for the critical elements that comprise biological macromolecules. Humans require continuous input of specific macronutrients (C, H, O, N, P, S, K) and micronutrients (including various metals) while producing metabolic wastes that must be broken down and reassimilated into useful forms [3]. On the Moon, water and oxygen can potentially be sourced from local resources, but elements like carbon, nitrogen, and phosphorus are scarce in readily accessible forms, creating a premium on their efficient recycling within the BLSS [3].

Table: Essential Elements for BLSS and Their Recycling Challenges

Element	Human Daily Requirement	Primary BLSS Function	Lunar Availability	Recycling Criticality
Oxygen	~840 g (as O₂)	Respiration, water composition	High (in minerals)	Moderate (local sourcing possible)
Carbon	~300 g (as CO₂)	Biomass structure, energy storage	Low (scarce volatiles)	High (must be recycled)
Hydrogen	~300 g (as H₂O)	Water, organic compounds	Medium (in water ice)	Medium (partial local sourcing)
Nitrogen	~15 g	Proteins, nucleic acids	Very Low	Very High (tight cycling essential)
Phosphorus	~1 g	ATP, nucleic acids, bones	Low (in minerals)	High (must be recycled)
Potassium	~3.5 g	Nerve function, osmosis	Medium (in minerals)	High (must be recycled)

Technology Readiness Levels in BLSS Development

TRL Framework and BLSS Applications

Technology Readiness Levels (TRL) provide a systematic metric for assessing the maturity of particular technologies, with TRL 1 representing basic principles observed and TRL 9 representing systems proven in operational environments [4]. This framework is essential for evaluating BLSS components and integrated systems, as it provides a common language for researchers, funding agencies, and program managers to discuss technical maturity and transition points [5]. The table below maps representative BLSS technologies to their current TRL status based on published literature and program milestones.

Table: BLSS Component Technology Readiness Levels

BLSS Technology	Current TRL	Key Demonstrations	Major Challenges
Physical/Chemical ECLSS	9 (ISS operations)	Water recycling (~98%), CO₂ removal [6]	Limited closure for carbon & nutrients
Higher Plant Cultivation	6-7 (ISS testing)	50+ species grown in space including tomatoes, lettuce [6]	Automated cultivation, nutrient delivery
Algal Bioreactors	5-6	Air revitalization, water processing in ground tests	System stability, contamination control
Integrated BLSS	5-6 (terrestrial analogs)	Lunar Palace (1-year crew closure) [2]	System integration, closure of all elements
Waste Processing	4-5	Composting, bacterial processing in Biosphere 2 [3]	Pathogen control, nutrient recovery efficiency

BLSS Development Pathway

BLSS Technology Development Pathway

Experimental Methodologies and Testing Protocols

Integrated System Testing Protocols

Closed-Chamber Testing Methodology for BLSS involves housing human crews or analog crews within sealed facilities for extended durations while monitoring system parameters and crew health. The Beijing Lunar Palace employed a standardized protocol in which a four-person crew completed a 365-day closed habitation experiment, achieving 100% regeneration of atmosphere and water, and 55% of food [2]. Key measurements included atmospheric O₂ and CO₂ concentrations, water quality parameters, food production biomass yields, waste processing efficiency, and crew physiological and psychological parameters. Similar methodologies were employed in the Biosphere 2 experiment, though at a much larger scale and with less technological control over biological processes [3].

Component-Level Testing Protocols focus on individual BLSS subsystems under controlled laboratory conditions. For plant growth systems, standard metrics include Biomass Accumulation Rate, Edible Yield Percentage, Gas Exchange Rates (photosynthesis and respiration), Water Transpiration Efficiency, and Nutrient Uptake Profiles [3]. For waste processing systems, critical parameters include Volume Reduction Ratio, Pathogen Inactivation Efficiency, Nutrient Recovery Percentage, and Process Stability under variable loading conditions. These component-level tests typically precede integrated system testing and continue throughout BLSS development to refine individual technologies.

BLSS Research Reagent Solutions

Table: Essential Research Reagents for BLSS Experimentation

Reagent/Category	Function in BLSS Research	Example Applications
Nutrient Solutions	Provide essential elements for plant growth	Hydroponic systems, algal bioreactors [3]
DNA Sequencing Kits	Microbial community analysis	Monitoring microbiome stability in closed systems [6]
Gas Standards	Instrument calibration for atmospheric monitoring	O₂, CO₂ sensors in closed environments [3]
Water Quality Test Kits	Monitoring recycled water safety	pH, conductivity, microbial contamination testing [3]
Plant Growth Regulators	Optimize crop yields in controlled environments	Timing of harvest, stress response modification

Comparative Performance Analysis

System-Level Performance Metrics

The table below provides a comparative analysis of different life support system architectures based on key performance parameters derived from published experimental data and mission demonstrations. This comparison highlights the relative advantages and limitations of each approach and illustrates the performance gains offered by increasingly bioregenerative systems.

Table: Life Support System Architecture Comparison

Parameter	Open Loop (Resupply)	Physical/Chemical (ECLSS)	Hybrid BLSS	Fully Bioregenerative BLSS
Water Closure (%)	0	~98 (ISS demonstration) [6]	>99	~100 (theoretical)
Oxygen Closure (%)	0	~100 (ISS operations)	100	100
Food Closure (%)	0	0	55-80 (Lunar Palace: 55%) [2]	>90 (theoretical)
Mass Initial (kg/crew-day)	~38.5 (including hygiene) [3]	~20 (with resupply of filters)	~10 (with partial resupply)	~5 (with minimal resupply)
Crew Time (%)	<5	5-10	15-30 (Biosphere 2 experience) [3]	20-40 (estimated)
System Complexity	Low	Medium-High	High	Very High
TRL	9 (Apollo, Shuttle)	9 (ISS operations)	5-6 (Lunar Palace) [2]	4-5 (experimental)

Crop-Specific Performance Data

Plant cultivation systems form the foundation of BLSS food production and air revitalization capabilities. Research has identified several plant species with favorable characteristics for BLSS implementation, including high edible biomass ratio, favorable growth characteristics, and complementary nutrient profiles. Performance data collected from ground-based tests and limited space-based experiments provide critical parameters for system design.

Table: Crop Performance in Controlled Environments

Crop Species	Edible Biomass Yield (g/m²/day)	Light Use Efficiency (g/MJ)	Cultivation Duration (days)	Oxygen Production (g/m²/day)	Water Transpiration Ratio
Wheat	25-50	1.5-3.5	60-80	15-30	450-650
Potato	40-70	2.5-4.5	90-120	20-40	350-550
Lettuce	50-100	1.0-2.0	28-35	10-20	200-350
Tomato	20-40	1.0-2.5	90-120	15-25	400-600
Sweet Potato	30-60	2.0-4.0	100-140	18-35	300-500

Current Challenges and Research Frontiers

Technical and Biological Limitations

Despite significant progress, BLSS implementation faces several persistent challenges. System Stability and Reliability remains a concern, as demonstrated by the atmospheric fluctuations experienced in Biosphere 2 that required external intervention [3]. The complex, nonlinear dynamics of ecological systems make predictive control difficult, particularly at smaller scales where buffer capacities are limited. Crop Diversity and Nutritional Completeness presents another challenge, as current BLSS food production systems provide limited variety and may not meet all human nutritional requirements without supplementation [2].

Microbiome Management is crucial for both system function and human health, yet controlling microbial communities in closed systems remains imperfect. The rare biosphere phenomenon—where low-abundance microorganisms can rapidly proliferate under changing conditions—presents a particular challenge for system stability and crew health [3]. Additionally, elemental balancing across all essential nutrients, particularly trace metals, requires sophisticated monitoring and control to prevent depletion or toxic accumulation over time [3].

Path Forward: Integration and Scaling

The most immediate research priorities for advancing BLSS include developing Advanced Monitoring and Control Systems capable of maintaining system homeostasis with minimal crew intervention. This includes sensor networks for real-time assessment of microbial communities, nutrient flows, and system health [6]. Waste Processing Integration needs particular attention, as efficient conversion of waste streams to plant-available nutrients remains a bottleneck in closing element cycles [3]. Crop Optimization through both conventional breeding and genetic engineering offers potential for improving yields, resource use efficiency, and nutritional quality in space-appropriate cultivars.

As noted in recent assessments, the strategic gap in U.S. BLSS capabilities relative to Chinese programs necessitates urgent investment in integrated testing facilities and programmatic efforts to mature these technologies for deployment in the coming decade [2]. The success of endurance-class human space exploration missions to Mars and beyond will depend on resolving these challenges and demonstrating reliable, robust BLSS operation in space environments.

For long-duration human missions to the Moon and Mars, Bioregenerative Life Support Systems (BLSS) represent an indispensable technology for enabling crew survival beyond Earth's supply lines. Unlike physical/chemical-based systems that merely recycle water and air, BLSS creates an artificial closed ecosystem composed of humans, plants, animals, and microorganisms that can regenerate oxygen, water, and food through biological processes [7]. These systems minimize resupply needs from Earth by performing in-situ recycling of vital resources while preventing pollution of extraterrestrial environments [7]. As space agencies plan for sustained lunar presence and eventual Mars missions, BLSS technology transitions from a scientific concept to a critical mission driver for achieving long-term human presence in space.

The development path for extraterrestrial BLSS follows a three-stage strategy beginning with hydroponic plant cultivation combined with processed local resources, advancing to systems utilizing transformed local soils, and ultimately achieving high closure with transformed local soils and comprehensive waste recycling [7]. This progression mirrors historical efforts like NASA's Controlled Ecological Life Support Systems (CELSS) program and Bioregenerative Planetary Life Support Systems Test Complex (BIO-PLEX) habitat demonstration program, though many early U.S. initiatives were discontinued and subsequently advanced by other international partners [2].

BLSS vs. Physical/Chemical Systems: A Quantitative Comparison

Current human spaceflight operations in Low Earth Orbit rely primarily on Physical/Chemical Life Support Systems (PCLSS) that focus on recycling water and regenerating oxygen through mechanical and chemical processes. These systems are limited by their inability to produce food and their eventual consumable depletion. In contrast, BLSS incorporates biological components that can regenerate multiple resources simultaneously, including food production through plant cultivation and bioconversion processes [7].

Table 1: Performance Comparison Between BLSS and Physical/Chemical Life Support Systems

System Parameter	Physical/Chemical Systems	BLSS Approach	Mission Impact
Food Production	None (100% resupply)	In-situ production via higher plants & crops	Eliminates food resupply; provides fresh nutrition
O2 Regeneration	Electrolysis of water	Plant photosynthesis	Renewable O2; consumes CO2
Water Recovery	Mechanical & chemical processing (~85-90%)	Biological processing + physico-chemical	Potentially higher recovery rates; multiple pathways
Waste Processing	Limited storage/processing	Bioconversion (e.g., yellow mealworms, microorganisms)	Converts waste to resources (feed, fertilizer)
Closure Duration	Limited by consumables & system degradation	Theoretical long-term sustainability (years)	Enables multi-year missions without resupply
Closure Degree	Partial closure of air & water loops	Higher closure potential for O2, H2O, food	Reduced mass & volume of stored consumables

Table 2: BLSS Closure Capabilities Demonstrated in Terrestrial Analog Missions

Mission/Facility	Duration	Crew Size	Closure Achievements	Key Limitations
Lunar Palace 1 (China)	370 days	4	Food production, O2 regeneration, water recycling, waste processing	Earth-based; planetary protection unverified
BIOS-3 (Russia)	Multiple up to 180 days	3	100% O2, 95% H2O recycled, ~50% food	Limited food variety; small crew size
Biosphere 2 (US)	2 years	8	Complex ecosystem with multiple habitats	System instability; O2 depletion issues
CELSS/NASA Tests	Various (days-months)	Varies	Component validation (plant growth, etc.)	Never fully integrated with crew

Technology Readiness Assessment: Where BLSS Stands Today

The Technology Readiness Level (TRL) framework, originally developed by NASA, provides a systematic method for assessing the maturity of BLSS technologies [8]. According to this scale, technologies progress from TRL 1 (basic principles observed) to TRL 9 (actual system proven in operational environment) through sequential development and testing phases [8]. Most current BLSS components exist at TRL 4-6, with some integrated systems reaching TRL 6-7 through analog testing [2].

The most significant challenge in BLSS development is bridging the "Valley of Death" between TRL 6 (system/subsystem model demonstrated in relevant environment) and TRL 7 (system prototype demonstration in operational environment) [8]. This transition requires moving from Earth-based analog testing to actual space deployment, representing a substantial increase in both cost and technical risk [8]. Currently, only China's Lunar Palace 1 has achieved integrated BLSS testing at a scale and duration (370 days with 4 crewmembers) that approaches TRL 6, while other international efforts remain at lower maturity levels [9] [2].

BLSS Technology Readiness Journey

Experimental Protocols and Methodologies in BLSS Research

Reliability Testing Protocol - Lunar Palace 1

The 370-day closed human experiment conducted in Lunar Palace 1 provides the most comprehensive dataset for BLSS reliability analysis [9]. The experimental methodology followed a rigorous protocol:

• System Configuration: LP1 consisted of nine integrated units: temperature and humidity control unit (THCU), water treatment unit (WTU), LED light source unit (LLSU), solid waste treatment and yellow mealworm feeding unit (SWT-YMFU), two plant cabins (PC1, PC2), plant cultivation substrate unit (PCSU), mineral element supply unit (MESU), and atmosphere management unit (AMU) [9].

• Failure Data Collection: Researchers precisely recorded the number and time of each unit failure throughout the 370-day experiment, creating detailed time-series failure data for statistical analysis [9].

• Stochastic Modeling: Using maximum likelihood estimates, researchers calculated the failure rate (λ) for each unit. For example, the SWT-YMFU failure stochastic process had λ = 0.0108 d⁻¹ with a 95% confidence interval of [0.0029, 0.0277] d⁻¹ [9].

• Monte Carlo Simulation: Researchers generated 10,000 simulated BLSS life cycles based on the failure distribution parameters to estimate system reliability and mean time between failures [9].

• Lifetime Estimation: Through statistical analysis of simulation results, the team calculated the average BLSS lifespan as 19,112.37 days (approximately 52.4 years) with a 95% confidence interval of [17,367.11, 20,672.68] days [9].

Biological Component Integration Protocol

BLSS research employs standardized protocols for integrating biological components:

• Plant Cultivation: Selection of appropriate plant species (typically 5 food crops, 29 vegetables, and 1 fruit as in LP1) with optimized growth conditions including LED lighting spectra, nutrient delivery systems, and atmospheric composition control [9].

• Animal Protein Production: Implementation of yellow mealworm (Tenebrio molitor L.) cultivation systems that convert inedible plant biomass into animal protein for human consumption, creating an additional trophic level in the ecosystem [9].

• Waste Processing: Development of fermentation processes that convert inedible plant biomass mixed with human feces and food residues into soil-like substrate for plant growth, completing nutrient cycling loops [9].

• Gas Exchange Monitoring: Continuous measurement of O₂ production, CO₂ consumption, and trace gas concentrations to ensure atmospheric stability and detect system imbalances [9].

BLSS Material Flow and Integration

The Scientist's Toolkit: Essential BLSS Research Components

Table 3: Key Research Reagents and Materials for BLSS Experimentation

Research Component	Function	Example Application
Higher Plant Species	Food production, O₂ regeneration, CO₂ consumption	Food crops (wheat, potato), vegetables (29 types), fruits [9]
Yellow Mealworms (Tenebrio molitor L.)	Bioconversion of inedible biomass to animal protein	Conversion of plant waste to human-edible protein [9]
Microbial Consortia	Waste processing, nutrient recycling	Solid waste fermentation, water purification [9]
LED Lighting Systems	Optimized plant growth with energy efficiency	Specific light spectra for different crop types [9]
Hydroponic/Nutrient Delivery Systems	Plant cultivation without soil	Mineral element supply to plant roots [9]
Atmospheric Monitoring Sensors	Trace gas detection, O₂/CO₂ balance	Real-time atmospheric management [9]
Water Recycling Systems	Water purification and recovery	Integration of biological and physico-chemical processing [9]

Critical Gaps and Strategic Recommendations

Despite significant progress, BLSS development faces several critical research gaps that must be addressed to achieve mission-ready status:

• Radiation Effects: Limited understanding of deep space radiation effects on biological systems, including plant growth, microbial communities, and overall ecosystem stability [2].

• Microgravity Adaptation: Incomplete data on how partial gravity (Moon: 0.16g, Mars: 0.38g) affects biological processes including plant growth, gas exchange, and nutrient uptake [7].

• System Closure: Current systems demonstrate high but incomplete closure, with Lunar Palace 1 achieving significant but not total resource recycling [9].

• Technology Readiness: Most BLSS components remain at TRL 4-6, requiring substantial development to reach TRL 7-9 for operational missions [2] [8].

Strategic investments are needed in ground-based testing facilities that can simulate space environments, flight demonstration missions to validate technologies in actual space conditions, and international collaboration frameworks to leverage global expertise [2]. The successful 370-day Lunar Palace 1 mission demonstrates that with sufficient commitment, these technical barriers can be overcome to enable the next era of human space exploration [9].

Bioregenerative Life Support Systems (BLSS) represent a critical technological frontier for enabling long-duration human space missions beyond low Earth orbit, where resupply from Earth becomes increasingly impractical [10] [11]. These systems aim to create sustainable, closed-loop environments that regenerate essential resources by integrating biological processes. Unlike current physical-chemical life support systems, BLSS leverage the metabolic capabilities of living organisms to recycle waste, revitalize atmosphere, produce food, and purify water [12]. The core concept mimics Earth's ecological principles, where the waste products of one compartment become the resources for another, thereby establishing a circular economy within the isolated environment of a spacecraft or planetary habitat [10]. As space agencies worldwide plan for crewed missions to the Moon and Mars within the next decade, developing reliable BLSS has become paramount for reducing dependency on Earth-based supplies and ensuring mission success [12] [11].

The three fundamental biological components that form the backbone of any BLSS are higher plants, microalgae, and microbial recyclers, each playing distinct yet interconnected roles. These organisms function as producers, oxygen generators, and waste processors within the closed system [10]. This review provides a comparative analysis of these core biological components, examining their specific functions, current technological readiness, experimental supporting data, and integration challenges within the context of BLSS development for deep space exploration.

Comparative Analysis of Core Biological Components

The following table summarizes the primary functions, advantages, and challenges of the three core biological components in BLSS:

Table 1: Comparative Analysis of Core BLSS Components

Component	Primary Functions in BLSS	Key Advantages	Technical Challenges
Higher Plants	Food production, O₂ generation, CO₂ removal, water transpiration, psychological benefits [10]	Provides diverse nutrition, high edible biomass, familiar food sources, enhances crew well-being [10]	Requires large growth area, long growth cycles, energy-intensive lighting, susceptible to gravity effects [10]
Microalgae & Cyanobacteria	Rapid O₂ production, CO₂ sequestration, biofuel production, nutrient recycling, water purification [13] [14]	High growth rates, efficient photosynthesis, utilizes waste streams, adaptable to various conditions [13] [14]	Requires processing for consumption, potential toxin production, culture stability, sensitive to contamination [15] [14]
Microbial Recyclers	Waste processing (solid & liquid), nutrient recovery, soil fertility enhancement, pharmaceutical synthesis [12] [11] [16]	Versatile metabolic capabilities, efficient decomposition, enables resource loop-closure, can produce edible biomass [12] [11] [16]	Pathogen risk management, process control optimization, integration complexity, variable efficiency [12] [16]

Technology Readiness Levels (TRL) Assessment

The current developmental status of these biological components varies significantly, with microalgae-based systems generally demonstrating higher technology readiness for specific functions like atmosphere revitalization, while integrated plant cultivation systems for staple crop production remain at earlier development stages for space applications [10] [12]. Microbial recycling technologies have demonstrated promise in ground-based prototypes but require further testing for space implementation [16]. The MELiSSA (Micro-Ecological Life Support System Alternative) project represents one of the most advanced BLSS development programs, incorporating multiple biological compartments in an integrated loop [12]. Current research focuses on increasing the TRL of these systems through ground-based demonstrators and limited spaceflight experiments, with the goal of deploying functional BLSS for lunar missions within this decade and Martian missions in the 2030s [12] [11].

Higher Plants in BLSS

Functional Roles and Species Selection

Higher plants serve as multifunctional components in BLSS, providing nutritional, atmospheric, hydrological, and psychological benefits [10]. Their photosynthetic capability enables simultaneous carbon dioxide absorption and oxygen release, while their transpiration process contributes to water purification. The selection of plant species for BLSS depends heavily on mission parameters including duration, destination, and available resources [10]. For short-duration missions in low Earth orbit, fast-growing species such as leafy greens (lettuce, kale), microgreens, and dwarf cultivars of horticultural crops are prioritized for their rapid harvest cycles and minimal spatial requirements [10]. These plants primarily serve as dietary supplements to augment prepackaged food, providing fresh produce rich in phytochemicals that may help counteract physiological stresses associated with spaceflight [10].

For long-duration missions and planetary outposts, staple crops capable of providing substantial carbohydrates, proteins, and fats become essential, including wheat, potato, rice, and soy [10]. These species are selected based on multiple criteria including nutritional content, resource efficiency (water, nutrients, light), edible-to-waste biomass ratio, and compatibility with controlled environment agriculture [10]. The inclusion of higher plants also provides non-nutritional benefits through "horticultural therapy," potentially mitigating psychological challenges associated with confinement and isolation during extended missions [10].

Experimental Data and Cultivation Protocols

Table 2: Experimental Performance Data of Selected Plant Species for BLSS

Plant Species	Growth Cycle (days)	Edible Biomass Yield	O₂ Production Rate	Cultural System	Reference Studies
Red Romaine Lettuce	30-45	100-200 g/m²/day	Moderate	Veggie System (ISS)	[12]
Mizuna Mustard	35-50	80-150 g/m²/day	Moderate	Veggie System (ISS)	[12]
Wheat (Apogee)	60-70	300-500 g/m²/cycle	High	Advanced Plant Habitat	[10]
Potato (SPACETATER)	90-120	500-800 g/m²/cycle	Moderate-High	Hydroponic System	[10]
Tomato (Micro-Tom)	80-100	200-400 g/m²/cycle	Moderate	Hydroponic System	[10]

Detailed Experimental Protocol for Plant Cultivation in BLSS:

• Growth Platform Setup: Utilize specialized systems such as NASA's Vegetable Production System (Veggie) or Advanced Plant Habitat (APH), which provide controlled lighting, nutrient delivery, and environmental monitoring [12].
• Planting Methodology: Implement seed pillows or rooting substrates containing controlled-release fertilizers, with surface-sterilized seeds to minimize microbial contamination [12].
• Environmental Parameters: Maintain temperature at 22-26°C, relative humidity at 60-70%, CO₂ concentration at 1000-2000 ppm, and light intensity at 200-300 μmol/m²/s for optimal photosynthesis [10] [12].
• Nutrient Delivery: Employ hydroponic or aeroponic systems with recirculating nutrient solutions containing essential macro and micronutrients, with regular monitoring and adjustment of pH (5.8-6.2) and electrical conductivity (1.5-2.5 mS/cm) [10].
• Harvest and Processing: Conduct aseptic harvest procedures, with microbial safety verification (pathogen screening) prior to human consumption [12].

Integration Challenges and Future Research

Significant challenges persist in integrating higher plants into functional BLSS, including their substantial mass, volume, and energy requirements [10]. Plant growth facilities demand significant power for lighting systems and thermal control, creating competing resource demands within the spacecraft habitat [10]. The effects of altered gravity environments (microgravity or partial gravity) on plant growth, development, and physiological processes require further investigation, as these factors influence critical functions such as root nutrient uptake, gas exchange, and structural support [10]. Future research priorities include optimizing growth systems for increased energy efficiency, developing automated monitoring and maintenance systems, and breeding or engineering plant varieties specifically adapted to space environment conditions [10].

Microalgae and Cyanobacteria in BLSS

Functional Roles and Species Selection

Microalgae and cyanobacteria serve as highly efficient biological components in BLSS, primarily functioning in atmospheric revitalization through rapid CO₂ sequestration and O₂ generation [13] [14]. These photosynthetic microorganisms offer several advantages over higher plants, including significantly higher growth rates, more efficient space utilization, and ability to thrive on waste streams and inedible biomass [14]. Selected species also demonstrate capability for water purification through nutrient uptake from wastewater, simultaneously producing valuable biomass that can be utilized for food, feed, or biofuel production [17] [14].

Species selection for BLSS applications prioritizes organisms with robust growth characteristics, high nutritional value, and resilience to space environment factors. The cyanobacterium Anabaena sp. PCC 7938 demonstrates particular promise due to its dual capability for carbon fixation (photosynthetic) and nitrogen fixation (diazotrophic), potentially enabling it to utilize atmospheric nitrogen directly [11]. Spirulina (Arthrospira platensis) is widely investigated for its high protein content (60-70% by dry weight) and excellent nutritional profile, while Chlorella vulgaris is valued for its rapid growth and efficient nutrient recycling capabilities [13] [14]. These microorganisms can be cultivated in various wastewater streams, simultaneously treating water while producing valuable biomass, thereby closing resource loops within the BLSS [17].

Experimental Data and Cultivation Protocols

Table 3: Experimental Performance Data of Microalgae and Cyanobacteria for BLSS

Species	Growth Rate (g/L/day)	Protein Content (% DW)	O₂ Production Rate	CO₂ Sequestration	Cultivation System
Chlorella sorokiniana	0.2-0.4 [18]	45-55% [14]	0.1-0.3 g/L/day [18]	1.7-2.0 kg CO₂/kg biomass [14]	Recycled Harvesting Water System [18]
Spirulina platensis	0.3-0.5 [13]	60-70% [13]	0.2-0.4 g/L/day [13]	1.5-1.8 kg CO₂/kg biomass [14]	Photobioreactor [13]
Anabaena sp. PCC 7938	0.15-0.3 [11]	50-60% [11]	0.1-0.25 g/L/day [11]	1.2-1.6 kg CO₂/kg biomass [11]	Photobioreactor [11]
Methylococcus capsulatus	0.2-0.35 [16]	52% [16]	N/A	N/A	Methane Bioreactor [16]

Detailed Experimental Protocol for Microalgae Cultivation in BLSS:

• Culture System Setup: Utilize photobioreactors (closed systems) or open pond systems, with photobioreactors generally preferred for space applications due to better contamination control and process optimization [15] [14].
• Inoculation and Medium Preparation: Inoculate with axenic cultures in optimized growth media such as BG-11 for cyanobacteria or Zarrouk's medium for Spirulina, with potential adaptation to use nutrients derived from waste streams [18] [13].
• Environmental Control: Maintain temperature at 25±1°C, light intensity at 200 μmol/(m²·s) with light/dark cycles of 12h:12h, and continuous CO₂ supplementation at 1-5% v/v [18] [14].
• Process Optimization: Implement recirculating cultivation strategies with harvesting water recycling, which has demonstrated 65-85% water savings and 55% nutrient reduction while maintaining productivity through multiple cycles [18].
• Harvesting and Processing: Employ centrifugation, filtration, or flocculation for biomass harvesting, followed by processing for product extraction (lipids, proteins, carbohydrates) or direct utilization as nutritional biomass [15].

Diagram 1: Microalgae cultivation workflow in BLSS

Integration Challenges and Future Research

Despite their promise, microalgae systems face challenges including the potential accumulation of growth-inhibiting substances in recirculated media, such as soluble algal products, organic metabolites, and increased chemical oxygen demand, which can reduce productivity over multiple cultivation cycles [18]. Contamination risk management remains a significant concern, particularly in semi-open systems [14]. Future research directions include developing more robust cultivation systems with integrated monitoring and control, optimizing nutrient recycling from waste streams, and genetic engineering of strains to enhance desired characteristics such as nutritional content, growth efficiency, and resilience to space environmental factors [13] [14].

Microbial Recyclers in BLSS

Functional Roles and Species Selection

Microbial recyclers serve as the fundamental decomposers in BLSS, enabling resource recovery from various waste streams including human metabolic waste, inedible plant biomass, and food waste [12] [11]. These microorganisms perform essential nutrient cycling functions, converting waste materials into forms usable by other biological components within the system, thereby closing ecological loops that would otherwise require external inputs or result in resource loss [11]. Their roles extend beyond waste processing to include soil fertility enhancement in plant growth systems, biological air remediation, and even direct production of edible microbial biomass [11] [16].

Diverse microbial species with specialized metabolic capabilities are employed for different recycling functions. Anaerobic digesters such as methanogenic archaea process solid waste to produce methane, which can subsequently be utilized by methylotrophic bacteria like Methylococcus capsulatus to generate microbial biomass with high protein content (52%) suitable for consumption [16]. Nitrifying bacteria convert ammonia from urine into nitrate, a more readily usable nitrogen source for plants [11]. Siderophilic cyanobacteria such as Leptolyngbya JSC-1 demonstrate capability in bioweathering of regolith, releasing trapped minerals and making them bioavailable for other organisms [13]. This function is particularly valuable for in situ resource utilization on lunar or Martian surfaces, where local materials can be processed to support BLSS operations [13].

Experimental Data and Processing Protocols

Table 4: Performance Data of Microbial Recycling Systems for BLSS

Process Type	Microbial Species/Consortium	Feedstock	Product Output	Efficiency/Conversion Rate	System Parameters
Anaerobic Digestion	Mixed anaerobic consortium	Solid human waste	Methane, volatile fatty acids	49-59% solid removal in 13 hours [16]	Mesophilic (35°C), pH 7-8 [16]
Single-Cell Protein Production	Methylococcus capsulatus	Methane from digestion	Microbial biomass	52% protein, 36% fats [16]	Aerobic, 35-45°C [16]
Alkaline Waste Processing	Halomonas desiderata	Liquid waste	Microbial biomass	15% protein, 7% fats [16]	pH 11, mesophilic [16]
Thermophilic Processing	Thermus aquaticus	Liquid waste	Microbial biomass	61% protein, 16% fats [16]	70°C (158°F) [16]
Regolith Bioweathering	Leptolyngbya JSC-1	Martian regolith analog	Bioavailable minerals	Production of 2-ketoglutaric acid [13]	Minimal medium, phototrophic [13]

Detailed Experimental Protocol for Microbial Waste Recycling:

• Reactor System Configuration: Establish enclosed, cylindrical fixed-film reactors with high-surface-area materials to support microbial biofilm development, enabling efficient waste conversion with minimal reactor volume [16].
• Process Inoculation: Introduce specialized microbial consortia adapted to target waste streams, with potential for extremophilic organisms tolerant to elevated temperature (thermophiles) or alkaline conditions to suppress pathogen growth [16].
• Waste Feed Preparation: Process waste streams to appropriate consistency, with solid waste potentially requiring size reduction and liquid waste filtration to prevent reactor clogging [16].
• Process Optimization: Maintain optimal environmental conditions for target microorganisms, including temperature control (varies by species), pH regulation, and mixing to ensure uniform contact between microbes and substrate [16].
• Product Recovery and Utilization: Separate microbial biomass through settling, centrifugation, or filtration for direct consumption or as feed for other BLSS components; capture gaseous products (methane, CO₂) for reuse in other system components [16].

Diagram 2: Microbial waste recycling pathways in BLSS

Integration Challenges and Future Research

Key challenges for microbial recyclers in BLSS include ensuring process reliability and preventing pathogen establishment in the recycled waste streams [16]. System stability must be maintained despite variations in waste composition and loading rates, which can occur with crew changes or unusual mission events [16]. Future research priorities include developing integrated systems that combine multiple microbial processes for comprehensive waste management, optimizing reactor designs for space and energy efficiency, and exploring genetic engineering approaches to enhance the capabilities of microbial recyclers for specific BLSS applications [11].

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 5: Essential Research Reagents and Materials for BLSS Experimentation

Reagent/Material	Function in BLSS Research	Application Examples	Special Considerations
BG-11 Medium	Standardized nutrient medium for cyanobacteria cultivation [18]	Cultivation of Anabaena sp., Nostoc, and other cyanobacteria [18]	Can be adapted with nutrients from waste streams or regolith processing [13]
Zarrouk's Medium	Optimized growth medium for Spirulina cultivation [13]	Production of Spirulina biomass for nutritional applications [13]	Elements can be sourced from regolith processed by siderophilic cyanobacteria [13]
Simulated Regolith	Martian and lunar soil analogs for ISRU studies [13]	Bioweathering experiments, plant growth trials in extraterrestrial soils [13]	Must match mineralogical composition of target extraterrestrial environment [13]
Fixed-Film Filter Materials	High-surface-area supports for microbial biofilm development [16]	Waste processing reactors, aquatic filtration systems [16]	Commercial aquarium materials can be adapted for methane production systems [16]
Ionic Liquids	Green solvents for extraction of bioactive compounds [15]	Extraction of lipids, proteins, carbohydrates from microalgae biomass [15]	Tunable properties for selective compound extraction; alternative to conventional solvents [15]

The successful development of BLSS for deep space exploration depends on the effective integration of higher plants, microalgae, and microbial recyclers, with each component playing complementary roles in creating sustainable closed-loop systems [10] [12] [11]. Current research demonstrates significant progress in individual component development, with the MELiSSA project representing one of the most advanced integrated approaches [12]. However, substantial challenges remain in scaling these systems for space applications, optimizing their resource efficiency, and ensuring long-term reliability in the harsh space environment [10] [11].

Future research directions should prioritize system integration studies, examining how these biological components interact within closed systems and respond to perturbations [10] [11]. Advanced monitoring and control technologies will be essential for maintaining system stability with minimal crew intervention [11]. Synthetic biology approaches offer promising avenues for enhancing the capabilities of all three biological components, potentially engineering plants for higher yields and reduced resource requirements, microalgae for improved productivity and valuable compound production, and microbes for more efficient waste processing and nutrient recycling [13] [19]. As research advances, BLSS technology will not only enable human exploration of deep space but may also provide sustainable solutions for resource management here on Earth, supporting the United Nations Sustainable Development Goals through applications in closed-loop agriculture, waste management, and environmental protection [11].

Bioregenerative Life Support Systems (BLSS) are artificial ecosystems designed to sustain human life during long-duration space missions by recycling waste into oxygen, water, and food. These systems break down human waste materials through biological processes, transforming them into nutrients and CO₂ for plants and other edible organisms, which in turn provide essential life support for astronauts [20]. By creating a materially closed loop, BLSS can significantly reduce mission mass and volume compared to systems relying entirely on physical/chemical processes and Earth resupply [21] [20]. This capability is particularly vital for future autonomous long-duration missions to the Moon and Mars where resupply from Earth is impractical or impossible [20].

The successful implementation of BLSS technology represents a critical strategic capability for deep space exploration. As space agencies plan for sustained lunar presence and eventual Mars missions, biological life support has reemerged as an area of intense international interest and competition [21]. This review systematically compares three pioneering ground demonstrators—BIO-PLEX, MELiSSA, and Lunar Palace 1—that have significantly advanced the technological readiness of BLSS components and systems.

Comparative Analysis of Major BLSS Demonstrators

Historical Development and Geopolitical Context

The development of BLSS technology has been shaped by shifting geopolitical priorities and funding decisions over several decades. NASA's Controlled Ecological Life Support Systems (CELSS) program, initiated in the 1980s, eventually led to the Bioregenerative Planetary Life Support Systems Test Complex (BIO-PLEX) habitat demonstration program [21]. This ambitious project aimed to integrate biological and physical/chemical systems for closed-loop life support. However, following the release of the Exploration Systems Architecture Study (ESAS) in 2004, NASA discontinued BIO-PLEX and physically demolished the facilities, redirecting focus toward shorter-term lunar missions relying on resupply [21].

During this period of reduced NASA investment, other space agencies continued BLSS development. The European Space Agency (ESA) established the Micro-Ecological Life Support System Alternative (MELiSSA) program in 1989, pursuing a more gradual, systematic approach to BLSS component development [21] [20]. Most notably, China's space program analyzed and incorporated research from canceled NASA programs into their own development efforts, resulting in the Beijing Lunar Palace (Lunar Palace 1) initiative [21]. This strategic adoption of previous research enabled China to rapidly advance its BLSS capabilities, successfully demonstrating closed-system operations that sustain crews of four analog taikonauts for up to a full year [21].

System Architectures and Design Philosophies

Table 1: Comparison of BLSS Demonstrator System Architectures

Demonstrator	Lead Agency/Country	Primary Architecture	Key Organisms/Components	Closure Goals
BIO-PLEX	NASA, USA	Integrated biological-physical/chemical system	Higher plants, bacteria, physical/chemical processors	Full closure of air, water, and portion of food
MELiSSA	ESA, Europe	Compartmentalized artificial ecosystem with five distinct loops	Cyanobacteria, nitrifying bacteria, higher plants (e.g., lettuce), microalgae	Progressive closure targeting nearly complete recycling
Lunar Palace 1	CNSA/Beihang University, China	Single integrated closed ecosystem	Higher plants (wheat), microalgae, insects (silkworm), microorganisms	>98% material closure demonstrated

The three BLSS demonstrators employed distinct architectural approaches reflecting their respective program philosophies. BIO-PLEX envisioned a comprehensive integrated system combining biological and physical/chemical technologies [21]. MELiSSA adopted a rigorously compartmentalized architecture inspired by aquatic ecosystems, dividing biological processes into five interconnected compartments, each with specific metabolic functions [20]. This modular approach allowed for targeted research on individual subsystems while working toward full integration.

Lunar Palace 1 implemented a single integrated ecosystem supporting multiple trophic levels, including higher plants, insects, and microorganisms [22] [23]. This design emphasized practical closure metrics, successfully achieving greater than 98% material closure during the 105-day "Lunar Palace 365" experiment [24]. The system's ability to maintain gases concentrations at stable levels through biological regulation represented a significant advancement in BLSS operational reliability [23].

Key Experimental Results and Performance Metrics

Table 2: Experimental Results and Performance Metrics from BLSS Demonstrators

Performance Metric	BIO-PLEX	MELiSSA	Lunar Palace 1
Maximum Crew Size Tested	Not reached operational testing	Small-scale component testing	4 crew members
Longest Duration Test	Program canceled before full testing	Continuous laboratory-scale operation	365 days (Lunar Palace 365)
Atmospheric Closure	Target: Full O₂ and CO₂ balance	High closure in experimental compartments	Demonstrated robust stabilization
Water Recovery Rate	Target: >95%	Component development ongoing	>95% in 105-day experiment
Food Production	Target: Partial food provision	100% food concept modeled	Significant portion of diet
Waste Processing	Integrated biological-physicochemical	Nitrogen recovery via nitrifying bacteria	Complete nutrient recycling

The experimental outcomes from these demonstrators reveal both capabilities and remaining challenges. MELiSSA has developed sophisticated stoichiometric models describing the cycling of elements (C, H, O, N) through its compartments, enabling predictive control of mass flow fluctuations [20]. The program's operational pilot plant at Universitat Autònoma de Barcelona has demonstrated continuous operation of connected compartments implementing part of the full metabolic loop [20].

Lunar Palace 1 has achieved the most impressive operational milestones, supporting crewed testing for extended durations with high closure rates [24]. Their ground-based experiments have provided crucial data on system stability, crew psychology, and microbiological dynamics during long-term isolation [22]. The 105-day experiment demonstrated effective water treatment and recycling systems capable of maintaining potable water standards throughout the testing period [23].

Experimental Protocols and Methodologies

Stoichiometric Modeling and Mass Balance Analysis

A foundational methodology common to advanced BLSS programs is the development of detailed stoichiometric models describing elemental flows through the system. The MELiSSA program has pioneered this approach through a compact set of chemical equations with fixed coefficients to simulate the flow of all relevant compounds for a crew of six [20]. This modeling strategy enables researchers to balance the dimensions of different compartments to achieve high degrees of closure at steady state, with optimal models demonstrating zero loss for 12 out of 14 compounds, with only minor losses for oxygen and CO₂ between iterations [20].

The stoichiometric modeling process typically involves several methodical steps. First, researchers conduct a comprehensive literature review to identify all relevant metabolic processes and their chemical equations. For MELiSSA, this review covered publicly available literature from 1989 to 2022 [20]. Next, mathematical models are developed to describe the cycling of key elements (C, H, O, N) through each compartment. These models are then validated against experimental data from individual compartment operations. Finally, integrated models simulate the entire closed-loop system, identifying critical bottlenecks and optimization opportunities [20].

Gas Stabilization Protocols and Control Systems

Maintaining gaseous equilibrium (particularly O₂ and CO₂ concentrations) at stable levels is critically important for crew safety in closed environments. Lunar Palace 1 researchers developed sophisticated protocols employing microalgae as biological gas regulators [23]. Their experimental methodology involved constructing a closed integrative system (CIS) containing lettuce, silkworms, and microalgae (Spirulina platensis) as representative components of a BLSS [23].

The experimental protocol for gas stabilization involves several key steps. First, researchers develop mathematical models of gas dynamics using system dynamics and artificial neural networks (ANN) based on mechanistic understanding and experimental data [23]. These models capture the complex nonlinear relationships between biological processes and gas concentrations. Next, closed-loop control systems with Linear-Quadratic Gaussian (LQG) servo controllers are designed to regulate microalgae growth, indirectly affecting gas concentrations through biological activity [23]. The system is then validated through real-time simulation, enabling rapid prototyping of controllers and optimization of response parameters. This methodology demonstrated that silkworms, lettuce, and microalgae could grow robustly within the controlled system while maintaining gas concentrations at target levels [23].

Long-Duration Crewed Testing Protocols

The most comprehensive BLSS validation comes from long-duration crewed tests, which evaluate both technical performance and human factors. Lunar Palace 1 established rigorous protocols for their landmark 365-day test, which set a world record for closed life support system duration [24]. The experimental design typically includes multiple overlapping monitoring systems tracking physiological, psychological, and system performance metrics.

Crewed testing protocols involve several methodical phases. First, extensive pre-mission baseline data collection establishes normal parameters for both crew and systems. During the mission, continuous monitoring tracks air composition, water quality, food production, waste processing efficiency, and microbial dynamics [22]. Crew members regularly complete standardized assessments of cognitive function, emotional state, and interpersonal dynamics to identify potential issues related to confinement [22]. Material flow is meticulously quantified at all system interfaces to calculate closure rates. Post-mission analysis compares all parameters with baseline data to identify adaptive responses and system evolution over time [24].

Diagram 1: BLSS Mass Flow and Control Logic. This diagram illustrates the compartmentalized architecture of advanced BLSS like MELiSSA and Lunar Palace 1, showing material flows (solid arrows) and control feedback loops (dashed arrows) that maintain system equilibrium.

Technology Readiness Assessment

TRL Evaluation Framework

Technology Readiness Levels (TRL) provide a systematic framework for assessing the maturity of BLSS technologies, with the scale ranging from TRL 1 (basic principles observed) to TRL 9 (actual system proven in mission operations) [8]. This framework is particularly valuable for managing the complex progression of biological systems from laboratory research to flight-ready technology. The TRL scale helps quantify technological risk, with early-stage technologies (TRL 1-3) carrying high uncertainty but relatively low development costs, while intermediate levels (TRL 4-6) require increasing investment in prototyping and testing [8].

The most challenging transition in technology development often occurs between TRL 5-6 and TRL 7, known as the "Valley of Death" where technologies must move from demonstration in relevant environments to actual operational environments [8]. For space technologies, this typically means progressing from ground-based analog testing to actual spaceflight demonstration. The costs and technical challenges associated with this transition are substantial, with NASA studies noting that advancing from TRL 5 to TRL 6 can cost multiple times more than all previous development work combined [8].

Current TRL Status of BLSS Demonstrators

Table 3: Technology Readiness Levels of BLSS Components and Systems

System/Component	Demonstrator	Current TRL	Key Accomplishments	Remaining Challenges
Integrated BLSS	Lunar Palace 1	5-6 (System demonstrated in relevant environment)	365-day crewed operation with >98% closure	Space environment validation
Waste Processing	MELiSSA	5 (Component validation in relevant environment)	Compartment-level validation, predictive control	Full integration and crew testing
Food Production	BIO-PLEX (legacy)	4-5 (Laboratory/Relevant environment validation)	Conceptual design complete	System implementation and testing
Gas Stabilization	Lunar Palace 1	6 (System prototype in relevant environment)	Biological control with microalgae demonstrated	Space-grade reliability and automation
Water Recovery	Multiple	6-7 (System prototype in operational environment)	>95% recovery in Lunar Palace 105-day test	Long-term reliability with biological systems

The current TRL assessment reveals that BLSS technology has progressed significantly but still faces substantial development before being ready for deployment on deep space missions. Lunar Palace 1 currently represents the most advanced integrated system, achieving TRL 5-6 through their extended crewed demonstrations [24]. MELiSSA components vary in maturity, with some individual compartments reaching TRL 5 through extensive laboratory testing, though full integration remains at lower TRL levels [20]. The original BIO-PLEX concept never progressed beyond TRL 3-4 before program cancellation, though its design concepts continue to influence current development efforts [21].

The "Valley of Death" between TRL 6 and TRL 7 presents particular challenges for BLSS, as it requires moving from ground-based demonstrations to space-based testing [8]. No BLSS has yet achieved this critical transition, though plans for lunar surface demonstrations could provide the necessary operational environment to advance these technologies to higher readiness levels [24].

Diagram 2: TRL Progression and Valley of Death. This diagram illustrates the Technology Readiness Level framework, highlighting the challenging transition between TRL 6 and TRL 7 where many BLSS technologies currently face development barriers.

Research Reagent Solutions and Essential Materials

Table 4: Key Research Reagents and Materials for BLSS Experimentation

Reagent/Material	Function in BLSS Research	Example Applications
Limnospira indica (Spirulina)	Oxygen production, CO₂ consumption, food source	MELiSSA C4a compartment; Lunar Palace 1 gas stabilization [20] [23]
Nitrosomonas europaea	Ammonia oxidation in nitrogen cycle	MELiSSA C3 compartment for nitrification process [20]
Higher plants (wheat, lettuce)	Food production, oxygen generation, water transpiration	BIO-PLEX plant growth chambers; Lunar Palace 1 food provision [21] [23]
Silkworm (Bombyx mori L.)	Animal protein source, waste consumer	Lunar Palace 1 closed integrative system [23]
Rhodospirillum rubrum	Photoheterotrophic waste processing	MELiSSA C2 compartment for volatile fatty acid conversion [20]
Stoichiometric model compounds	Mass balance validation and prediction	MELiSSA elemental cycling calculations (C, H, O, N) [20]
Gas analysis sensors	Real-time monitoring of O₂ and CO₂ concentrations	Lunar Palace 1 gas stabilization control systems [23]

The research and development of BLSS technology relies on specialized biological reagents and analytical tools. Microalgae species, particularly Limnospira indica (formerly Spirulina) and Chlorella vulgaris, serve multiple crucial functions as oxygen generators, carbon dioxide consumers, and nutritional sources [20] [23]. These microorganisms exhibit extreme metabolic flexibility and extensive adaptability, making them ideal for gas stabilization during system perturbations [23].

Higher plants including wheat, lettuce, and other crops form the foundation of nutritional support in BLSS, with research focusing on optimizing growth conditions, nutrient delivery, and harvest cycles [20]. Insect species such as silkworms provide animal protein while contributing to waste processing loops [23]. Sophisticated sensor arrays and control systems are essential research tools for maintaining system equilibrium, with Lunar Palace 1 employing Linear-Quadratic Gaussian (LQG) servo controllers to regulate biological processes based on real-time environmental data [23].

The comparative analysis of BIO-PLEX, MELiSSA, and Lunar Palace 1 reveals both significant progress and substantial challenges in BLSS development. Lunar Palace 1 has demonstrated the highest level of integrated system performance through extended crewed tests, while MELiSSA has developed sophisticated compartmentalized architectures and control methodologies. The legacy of BIO-PLEX continues to influence current design concepts despite its premature cancellation.

Future BLSS research will necessarily focus on addressing the TRL "Valley of Death" by advancing from ground-based demonstrations to space-based validation [8] [24]. Chinese researchers have indicated that future work will emphasize "lunar probe payload carrying experiments to study mechanisms of small uncrewed closed ecosystem in space and clarify the impact of space environmental conditions on the ecosystem" [24]. This approach will enable ground-based BLSS parameters to be corrected based on actual space environment performance data.

The strategic implications of BLSS development extend beyond technical considerations to geopolitical dimensions in space exploration. As noted in recent analyses, "China has surpassed the US and its allies in both scale and preeminence of these emerging efforts and technologies" in bioregenerative life support [21]. Closing the current technology gaps will require sustained investment and international collaboration to achieve the BLSS capabilities necessary for human endurance-class missions to Mars and beyond.

Applying the TRL Scale to BLSS Components and Systems

The Technology Readiness Level (TRL) framework is a systematic metric used to assess the maturity of a particular technology across its development lifecycle. Originally developed by NASA in the 1970s, this scale has become a standardized measurement system across space agencies worldwide, including the European Space Agency (ESA) [5]. The TRL scale consists of nine discrete levels, with TRL 1 representing the lowest maturity (basic principles observed) and TRL 9 representing the highest (flight-proven success) [4].

This framework provides project managers, engineers, and stakeholders with a common language for evaluating technical maturity, enabling informed decision-making regarding funding, technology transition, and risk management [8]. For complex research domains such as Bioregenerative Life Support Systems (BLSS) essential for long-duration deep space missions, the TRL framework offers a critical assessment tool to gauge progress toward mission-ready status and identify development gaps [2]. Both NASA and ESA have institutionalized TRL assessments within their technology development processes, though with some contextual variations that reflect their distinct operational priorities and standardization approaches [25] [5].

TRL Definitions and Comparative Analysis

Detailed Breakdown of the TRL Scale

The nine-level TRL scale represents a technology's progression from fundamental research to operational deployment. The table below provides a comprehensive comparison of NASA and ESA definitions for each TRL level:

Table 1: Comparative Definitions of TRL Levels by NASA and ESA

TRL	NASA Definition [26]	ESA Definition [5]	Shared Exit Criteria
1	Basic principles observed and reported	Basic principles observed	Peer-reviewed publication of underlying research
2	Technology concept and/or application formulated	Technology concept formulated	Documented description addressing feasibility and benefit
3	Analytical and experimental critical function and/or characteristic proof-of-concept	Experimental proof of concept	Documented analytical/experimental results validating key parameters
4	Component and/or breadboard validation in laboratory environment	Technology validated in lab	Documented test performance demonstrating agreement with predictions
5	Component and/or breadboard validation in relevant environment	Technology validated in relevant environment	Documented test performance and definition of scaling requirements
6	System/subsystem model or prototype demonstration in a relevant environment	Technology demonstrated in relevant environment	Documented test performance in relevant environment
7	System prototype demonstration in a space environment	System prototype demonstration in operational environment	Documented test performance in operational environment
8	Actual system completed and "flight qualified" through test and demonstration	System complete and qualified	Documented test performance verifying analytical predictions
9	Actual system "flight proven" through successful mission operations	Actual system proven in operational environment	Documented mission operational results

Agency-Specific Implementation Frameworks

While NASA and ESA share the fundamental TRL structure, their implementation frameworks reflect distinct organizational standards and requirements.

NASA's TRL Framework: NASA's system provides detailed technology descriptions across hardware and software domains [26]. For instance, at TRL 4, hardware validation involves "a low fidelity system/component breadboard built and operated to demonstrate basic functionality," while software requires that "key, functionally critical software components are integrated and functionally validated" [26]. This specificity offers developers precise criteria for advancement. NASA emphasizes rigorous testing protocols at each transition, particularly between TRL 5-6 where technology must advance from laboratory to relevant environments, and at TRL 7-8 where systems must demonstrate performance in actual space environments [4] [26].

ESA's TRL Framework: ESA employs the ISO 16290 TRL Scale, adopting international standardization to facilitate collaboration across European member states and international partnerships [25]. The agency incorporates TRL within the broader European Cooperation for Space Standardization (ECSS) system, particularly following the ECSS-E-HB-11A handbook guidelines established in 2017 [27] [28]. This handbook provides detailed processes for Technology Readiness Assessment (TRA), establishes Technology Readiness Status Lists (TRSL), and links TRL management with Critical Item Lists (CIL) for project risk management [28]. ESA's framework includes specific adaptations for various disciplines including software, EEE components, and materials manufacturing processes [28].

Application to Bioregenerative Life Support Systems (BLSS)

BLSS Technology Status and Mission Criticality

Bioregenerative Life Support Systems represent a critical enabling technology for long-duration deep space missions and sustained lunar or Martian habitation [2]. These systems aim to create closed-loop ecosystems that regenerate oxygen, water, and food through biological processes while recycling waste, dramatically reducing reliance on Earth resupply [2]. Current BLSS technologies span the TRL spectrum, with most integrated systems residing at mid-TRL levels, presenting a significant strategic challenge for upcoming exploration initiatives.

The historical development of BLSS reveals a dramatic divergence in agency approaches. NASA's Bioregenerative Planetary Life Support Systems Test Complex (BIO-PLEX) was discontinued after 2004, creating substantial capacity gaps [2]. In contrast, China's CNSA has advanced aggressively in this domain, demonstrating a fully integrated system that supported a crew of four analog taikonauts for a full year—a capability that currently surpasses NASA's operational readiness [2]. The European Space Agency maintains the MELiSSA (Micro-Ecological Life Support System Alternative) program, though it "never approached closed-systems human testing" [2]. This technological disparity highlights the strategic implications of TRL advancement rates in BLSS development.

Experimental Protocols for BLSS TRL Advancement

Advancing BLSS technologies through the TRL ladder requires specialized experimental protocols tailored to biological system validation:

TRL 3-4 Transition Protocol (Proof of Concept to Laboratory Validation)

• Objective: Validate component functionality and initial integration
• Methodology: Construct bench-scale bioreactors and plant growth modules
• Testing Parameters: Measure gas exchange rates (O₂ production, CO₂ absorption), water purification efficiency, and biomass production
• Success Criteria: Documented performance metrics demonstrating agreement with predictive models for at least 90 days of continuous operation
• Environmental Controls: Precise regulation of temperature, humidity, light intensity, and nutrient delivery

TRL 5-6 Transition Protocol (Laboratory to Relevant Environment)

• Objective: Demonstrate subsystem integration in simulated operational environment
• Methodology: Integrate multiple BLSS components (waste processing, food production, air revitalization) within a closed test chamber
• Testing Parameters: System closure degree (>95%), mass and energy balances, stability under variable loads, and reliability metrics
• Success Criteria: Functional performance for 6-12 months with >98% closure of water and atmosphere loops, and >50% food production closure
• Relevant Environment Elements: Mission-analogous lighting, pressure, radiation, and thermal conditions

TRL 7-8 Transition Protocol (Relevant Environment to Space Qualification)

• Objective: Validate integrated system performance in actual space environment
• Methodology: Deploy BLSS subsystems on orbital platforms (ISS, lunar gateway) or analog habitats
• Testing Parameters: Microgravity/partial gravity effects on biological processes, radiation impacts, system autonomy, and crew interaction
• Success Criteria: Continuous operation for mission-duration equivalents with defined performance margins and contingency response protocols
• Documentation Requirements: Complete verification and validation data, maintenance protocols, and failure mode analyses

Table 2: BLSS Technology Readiness Assessment for Deep Space Missions

BLSS Subsystem	Current NASA TRL	Current ESA TRL	Target TRL for Lunar Mission	Target TRL for Mars Mission
Air Revitalization	6-7	5-6	8	9
Water Recovery	7	6	8	9
Food Production	5-6	5	7	8
Waste Processing	5	4-5	7	8
System Integration	4-5	4	6-7	8

BLSS Research Reagent Solutions and Essential Materials

Table 3: Essential Research Reagents and Materials for BLSS Technology Development

Item	Function	Application in BLSS Research
Hydroponic Nutrient Solutions	Provide essential macro/micronutrients	Plant growth optimization in controlled environments
Gas Analyzers (O₂, CO₂)	Monitor atmospheric composition	Closed-loop gas exchange measurements
DNA Sequencing Kits	Microbial community analysis	Bioreactor process monitoring and optimization
Ion-Selective Electrodes	Nutrient solution monitoring	Real-time detection of essential minerals in hydroponic systems
Biofilm Assay Kits	Microbial attachment quantification	Waste processing bioreactor performance optimization
Radiation Dosimeters	Measure ionizing radiation exposure	Biological system response assessment in space environments
Lyophilized Microbial Cultures	Waste processing bioreactor inoculation	System startup and process stability studies
Plant Tissue Culture Media	Sterile plant propagation	Genetic preservation and controlled production studies

TRL Assessment Methodologies and Tools

Technology Readiness Assessment Process

The Technology Readiness Assessment (TRA) represents a systematic process for evaluating technology maturity against standardized TRL criteria. Both NASA and ESA employ formalized TRA methodologies that involve:

• Technology Characterization: Detailed documentation of technical parameters, performance requirements, and operational environments
• Evidence Collection: Compilation of experimental data, test reports, analytical models, and demonstration results
• Gap Analysis: Identification of discrepancies between current technology status and target TRL requirements
• Independent Review: Assessment by qualified technical experts not directly involved in the technology development
• TRL Assignment: Formal determination of technology maturity level based on established criteria

For BLSS technologies, TRA requires special consideration of biological system complexities, including variability, adaptability, and ecological interactions that differ from traditional hardware systems [2]. The European Cooperation for Space Standardization (ECSS) handbook ECSS-E-HB-11A provides specific guidelines for TRL assessment within space projects, addressing the unique challenges of biological and life support technologies [27].

Visualization of TRL Assessment Workflow

The following diagram illustrates the comprehensive TRL assessment workflow for space technologies, incorporating agency-specific requirements:

TRL Assessment Workflow for Space Technologies

Risk Management and the "Valley of Death"

A critical concept in TRL progression is the technological "Valley of Death" – the gap between technology validation in laboratory settings (TRL 4-6) and operational demonstration in space environments (TRL 7-8) [8]. This transition requires substantial funding increases, with costs from TRL 5-6 potentially exceeding all previous development stages combined [8]. For BLSS technologies, this challenge is particularly acute due to the complex biological interactions and extended testing timelines required for validation.

Strategies to bridge this "Valley of Death" include:

• Incremental Testing Approaches: Utilizing analog environments (Antarctic stations, closed chambers) and partial-gravity simulation platforms
• International Collaboration: Leveraging partnerships to distribute development costs and access diverse testing facilities
• Phased Demonstration Missions: Implementing technology demonstration as secondary payloads on broader missions
• Modeling and Simulation Enhancement: Developing high-fidelity predictive models to reduce experimental uncertainty

Future Directions and Strategic Implications

BLSS Development Pathways for Deep Space Exploration

The successful implementation of BLSS technologies for endurance-class deep space missions requires coordinated advancement across multiple development fronts. The current TRL disparity between NASA and international partners in BLSS capabilities presents both a strategic challenge and opportunity for collaboration [2]. Future development pathways include:

Near-Term (2025-2030)

• Advance key BLSS subsystems to TRL 7 through International Space Station demonstrations and lunar surface testing
• Establish standardized interfaces between physical/chemical and biological life support systems
• Conduct integrated testing of hybrid life support architectures

Mid-Term (2030-2035)

• Demonstrate fully integrated BLSS at TRL 7-8 in lunar surface habitat analogs
• Validate multi-year operational stability and reliability metrics
• Establish performance databases for biological system predictability

Long-Term (2035-2040)

• Achieve TRL 9 for integrated BLSS through sustained lunar habitat operations
• Validate Mars-class mission readiness through multi-year closed system testing
• Implement autonomous operation and maintenance protocols

Visualization of BLSS Integration Pathway

BLSS Technology Development and Integration Pathway

The TRL framework continues to evolve with proposed extensions including Habitation Readiness Levels (HRL) developed by NASA engineers to address habitability requirements, and Commercial Readiness Levels (CRL) to assess market adoption potential [5]. For BLSS technologies, these complementary assessment tools may provide more comprehensive evaluation frameworks as systems approach operational deployment.

As space agencies prepare for sustained human presence beyond low-Earth orbit, the TRL framework remains an indispensable tool for objectively evaluating technology maturity, managing program risk, and making strategic investment decisions. For BLSS technologies specifically, achieving high TRL status represents not merely a technical milestone but a fundamental prerequisite for humanity's future as a spacefaring species.

Bioregenerative Life Support Systems (BLSS) are fundamental for enabling long-duration human space exploration missions beyond Low Earth Orbit (LEO), such as those to the Moon and Mars. By using biological processes to regenerate air, purify water, produce food, and recycle waste, BLSS aim to achieve a high degree of self-sufficiency, reducing the need for resupply from Earth [10]. Among the various biological compartments within a BLSS, photobioreactors (PBRs) utilizing microorganisms like microalgae and cyanobacteria, and higher plant cultivation systems are two primary technologies for air revitalization—the process of removing carbon dioxide (CO₂) and producing oxygen (O₂).

This guide provides a comparative assessment of the Technology Readiness Level (TRL) of these two approaches for air revitalization in deep space missions. TRL is a metric used to assess the maturity of a particular technology, ranging from 1 (basic principles observed) to 9 (actual system proven in operational environment). The assessment is based on current ground-based research, flight experiment data, and subsystem demonstrations, framed within the context of developing integrated BLSS for lunar and Martian outposts.

Photobioreactors (PBRs) for Microbial Photosynthesis

Photobioreactors are closed systems designed to cultivate photosynthetic microorganisms, such as microalgae (e.g., Chlorella vulgaris) and cyanobacteria (e.g., Limnospira indica, formerly Arthrospira sp.), for air revitalization [29] [30]. These systems are engineered to provide optimal conditions for microbial growth, including light, temperature, nutrients, and CO₂.

• Primary Organisms: Chlorella vulgaris, Limnospira indica (cyanobacterium), Anabaena sp. PCC 7938, and Scenedesmus obliquus are among the most studied species [29] [31] [32].
• Core Function: These microbes consume CO₂ and water in the presence of light to produce biomass and O₂ through photosynthesis. A key advantage is their high photosynthetic efficiency and rapid growth rates, which can be up to ten times faster than terrestrial plants [30]. They can also be cultivated in a separate compartment from the crew, potentially simplifying system management [29].

Higher Plant Systems

Higher plant systems involve growing crops in controlled environments, such as plant growth chambers, using soil, hydroponic, or aeroponic techniques [10] [33]. Plants act as primary producers, performing the same dual function of CO₂ removal and O₂ production.

• Primary Crops: Selection depends on the mission scenario. For short-duration missions, fast-growing leafy greens (e.g., lettuce, kale), microgreens, and dwarf cultivars (e.g., tomato) are prioritized. For long-duration planetary outposts, staple crops (e.g., wheat, potato, rice, soy) are essential to provide a balanced diet [10].
• Core Function: Beyond air and water revitalization, the principal advantage of higher plants is their direct production of fresh food. This addresses nutritional needs and provides psychological benefits to the crew through "horticultural therapy" [10].

Table 1: Comparative Advantages and Primary Functions

Feature	Photobioreactors (PBRs)	Higher Plant Systems
Primary Organisms	Microalgae (e.g., Chlorella), Cyanobacteria (e.g., Limnospira)	Crops (e.g., lettuce, wheat, tomato, potato)
Core Air Revitalization	CO₂ removal, O₂ production via microbial photosynthesis	CO₂ removal, O₂ production via plant photosynthesis
Key Advantages	High photosynthetic efficiency, rapid growth, compact design, potential for use in hypobaric conditions	Direct food production, nutritional variety, psychological benefits for crew, waste recycling
Additional Outputs	Edible biomass, potential source of high-value compounds (lipids, pigments)	Food, water purification via transpiration

Technology Readiness Level (TRL) Assessment

The TRL assessment evaluates the maturity of each technology based on ground-based tests, spaceflight experiments, and integration demonstrations.

TRL of Photobioreactors

PBR technology has been validated through multiple spaceflight experiments, indicating a TRL of 5-6.

• Ground Demonstrations: Extensive ground-based testing has been conducted. The MELiSSA Pilot Plant (MPP) integrates a nitrifying bioreactor with an air-lift photobioreactor for L. indica to revitalize air for a mock-up crew (rats) [32]. Other systems, like the NASA Biomass Production Chamber, have also supported relevant research [29].
• Spaceflight Experiments: PBRs have been successfully operated in LEO onboard satellites like FOTON and the International Space Station (ISS). For instance, the eukaryotic microalga Chlorella vulgaris has been tested on the ISS for life support experiments [29] [32]. These experiments serve as a "proof of concept," demonstrating that a bioreactor can function in microgravity, though typically on a small scale (e.g., less than 100 mL) and with significant crew involvement [10] [29].
• TRL Implication (5-6): A TRL of 5-6 signifies that the technology has been validated in a relevant environment (space, for PBR components) but is not yet a fully integrated, operational system for life support on a crewed mission. Scaling up and optimizing for efficiency, robustness, and autonomy remain key challenges [10].

TRL of Higher Plant Systems

Higher plant systems have also seen substantial advancement, with successful food production on the ISS, placing them at a TRL of 4-5 for integrated air revitalization.

• Ground Demonstrations: Large-scale ground-based demonstrators like NASA's Biomass Production Chamber, Biosphere 2, and China's Lunar Palace 1 have successfully grown plants for extended durations, providing extensive data on resource regeneration [10] [29].
• Spaceflight Experiments: The ISS has hosted numerous plant growth experiments. The Vegetable Production System ("Veggie") and the Advanced Plant Habitat have successfully grown a variety of crops, including lettuce, pak choi, and dwarf tomatoes, providing supplemental fresh food for the crew [34]. Over 50 species of plants have been grown aboard the ISS [34].
• TRL Implication (4-5): A TRL of 4-5 indicates that the technology (plant growth for food) has been validated in a laboratory and relevant environment (space). However, the primary focus in space has been on food production rather than the quantified, reliable contribution of plants to the air revitalization loop for a crew. The contribution to oxygen production and carbon dioxide removal in an integrated BLSS requires further demonstration [10].

Table 2: TRL Assessment Based on Testing and Demonstration

Technology	Key Ground Demonstrations	Key Spaceflight Demonstrations	Current TRL (Est.)
Photobioreactors	MELiSSA Pilot Plant (Spain), NASA Biomass Production Chamber	Experiments on FOTON, ISS (e.g., with Chlorella vulgaris)	TRL 5-6 (Component validation in relevant environment)
Higher Plant Systems	Biosphere 2 (USA), Lunar Palace 1 (China), Closed Ecology Experiment Facility (Japan)	ISS "Veggie," Advanced Plant Habitat, >50 plant species grown	TRL 4-5 (System validation in relevant environment for food; lower for integrated air revitalization)

Experimental Data and Performance Metrics

Quantitative Performance Comparison

Direct quantitative comparison is complex due to differing system scales and experimental conditions. However, published data provides insight into their performance.

Table 3: Key Performance Metrics from Experimental Data

Parameter	Photobioreactors	Higher Plant Systems	Notes & Context
CO₂ Removal Efficiency	Up to 82.3% ± 12.5% (sunny days) [31]	Data less quantified for air revitalization; highly species-dependent	For PBRs, efficiency can drop to ~50% on cloudy days in outdoor systems [31].
Oxygen Production	Specific rates reported for species like Limnospira indica; core function of spaceflight experiments [29] [32]	Integral to photosynthesis; directly supports crew in ground demonstrators (e.g., BIOS-3) [29]	Oxygen production is tightly coupled to growth rates and biomass productivity.
Biomass Productivity	Chlorella vulgaris: up to 171 mg L⁻¹ day⁻¹ at 15% CO₂ [31]	Not directly comparable; measured as edible yield (e.g., g m⁻² day⁻¹)	PBR productivity is volumetric; plant productivity is areal.
Growth Cycle Duration	Continuous harvesting possible; rapid doubling in hours [30]	Varies from ~30 days (leafy greens) to ~100 days (staple crops) [10]	PBRs offer more continuous output.
Tolerance to High CO₂	Species like Botryococcus braunii show high productivity at 20% CO₂ [31]	Most crops require ambient CO₂ levels (~0.04%) for optimal growth	PBRs can be used with concentrated CO₂ streams.

Detailed Experimental Protocols

To contextualize the data in Table 3, below are generalized protocols for key types of experiments conducted with each technology.

Protocol 1: Photobioreactor Operation for Air Revitalization

• Inoculum Preparation: A sterile culture of the target microorganism (e.g., Chlorella vulgaris) is grown in a nutrient medium to a specified cell density.
• Reactor Setup & Sterilization: The photobioreactor (e.g., flat-panel, bubble-column) is sterilized. The culture medium is added, and environmental parameters (temperature, pH) are set.
• System Inoculation & Initiation: The photobioreactor is inoculated with the prepared culture.
• Continuous Operation: The reactor is operated in continuous or semi-continuous mode. A defined gas mixture containing CO₂ (e.g., at 0.5-15%) is bubbled through the culture. Light is provided at a specific intensity and photoperiod (e.g., 16:8 light/dark).
• Monitoring & Data Collection: Biomass concentration (via optical density or dry weight), pH, dissolved O₂, and gas composition (inlet/outlet) are monitored regularly.
• Harvesting & Analysis: A portion of the culture is harvested periodically. Biomass is analyzed for composition, and O₂ production/CO₂ consumption rates are calculated.

Protocol 2: Higher Plant Chamber for Gas Exchange Measurement

• Plant Material & Germination: Seeds of the target crop (e.g., lettuce) are surface-sterilized and germinated on a substrate (e.g., rockwool, agar).
• Transfer to Growth Chamber: Seedlings are transferred to a controlled environment growth chamber (e.g., NASA's Advanced Plant Habitat) with defined light, temperature, humidity, and nutrient delivery (e.g., hydroponics).
• Acclimation & Growth: Plants are grown until they reach a desired developmental stage for measurement.
• Gas Exchange Monitoring: The chamber is temporarily sealed. The net photosynthetic rate is measured by monitoring the depletion of CO₂ within the chamber headspace using an infrared gas analyzer (IRGA). Alternatively, O₂ evolution can be measured with an O₂ electrode.
• Environmental Control: During measurement, light intensity, temperature, and CO₂ concentration are tightly controlled to establish standard conditions.
• Data Calculation & Harvest: Gas exchange rates are calculated per unit leaf area or per plant. Plants are often harvested post-measurement for biomass analysis.

Signaling Pathways and System Logic

The core process underlying air revitalization in both systems is photosynthesis. The following diagram illustrates the generalized signaling and metabolic pathway of photosynthesis, which is shared in its fundamental steps by both microalgae and higher plants.

Diagram 1: Core Photosynthesis Pathway

The logical workflow for integrating these biological components into a BLSS, and their specific roles, can be summarized as follows.

Diagram 2: BLSS Integration Logic

The Scientist's Toolkit: Key Research Reagents and Materials

This section details essential materials and reagents used in experimental research for these systems.

Table 4: Key Research Reagents and Materials

Item Name	Function/Application	Example Organisms/Cultivars
BG-11 Medium	Standard nutrient medium for cultivation of cyanobacteria.	Limnospira indica, Anabaena sp. [32]
Bold's Basal Medium (BBM)	Common nutrient medium for cultivation of freshwater microalgae.	Chlorella vulgaris, Scenedesmus obliquus [31]
Hydroponic Nutrient Solution	Aqueous solution of essential mineral nutrients for soilless plant cultivation.	Lettuce, tomato, wheat [10] [34]
Regolith Simulant	Terrestrial material mimicking chemical/physical properties of lunar or Martian soil.	Used in plant growth experiments to test in-situ resource utilization [32]
Controlled Environment Chamber	Enclosed growth chamber allowing precise control of light, temperature, humidity, and CO₂.	Used for both plant and microorganism studies [10] [33]

Both photobioreactors and higher plant systems are viable technological pathways for air revitalization in BLSS, with neither holding a definitive overall advantage. The choice of technology is mission-dependent.

• Photobioreactors (TRL 5-6) demonstrate high photosynthetic efficiency and rapid growth in a compact volume. They are well-suited for missions where space and mass are primary constraints and where the crew's dietary needs can be met separately. Their slightly higher TRL reflects success in space-based validation of core functionality.
• Higher Plant Systems (TRL 4-5) offer the irreplaceable benefit of direct food production and psychological support. They are critical for long-duration missions where nutritional variety and sustainability are paramount, despite generally requiring more space, mass, and time.

Future development should focus on integrating both technologies into a hybrid BLSS, leveraging the strengths of each. Key challenges for both include scaling up, achieving full autonomy, and ensuring reliable operation under the partial gravity and increased radiation environments of the Moon and Mars [10] [33] [11].

Bioregenerative Life Support Systems (BLSS) represent the pinnacle of life support technology for deep space missions, designed to sustain human life by recycling waste, purifying water, and regenerating air through biological processes. Within this closed-loop system, food production is not merely a nutritional necessity but an integral component that completes the metabolic cycle between crew and environment. Food systems within a BLSS must simultaneously address multiple challenges: providing adequate nutrition for crew health, utilizing crew waste products as inputs, contributing to atmospheric regeneration, and operating reliably within the extreme constraints of the space environment.

The concept of Technology Readiness Levels (TRLs) provides a systematic framework for assessing the maturity of these complex food production technologies. Originally developed by NASA, the TRL scale ranges from 1 (basic principles observed) to 9 (actual system proven in operational environment), offering a standardized metric for evaluating progression from fundamental research to flight-ready systems [35]. For bioastronautics researchers, TRL assessments provide critical decision-support data for technology development prioritization, resource allocation, and mission planning timelines. This review performs a comparative TRL assessment of food production technologies, from small-scale "salad machine" concepts to full staple crop production systems, within the context of their deployment pathways for future deep space missions.

Technology Readiness Levels: An Assessment Framework for Space Food Production

The TRL framework creates a common language for engineers, scientists, and mission planners to communicate about technological maturity. In the context of food production for BLSS, TRL assessment must consider not only the agricultural components but also their integration within the broader life support system and the unique constraints of spaceflight environments. The table below outlines the generalized TRL definitions as applied to food production technologies for space applications.

Table 1: Technology Readiness Levels for Space Food Production Systems

TRL	Definition	Food Production Equivalent
1-2	Basic principles observed and formulated	Fundamental research on plant growth in microgravity/mock gravity
3-4	Experimental proof-of-concept & lab validation	Prototype components tested in simulated environments
5-6	Validation in relevant environment	Food production tested in analog environments (e.g., Antarctic, ISS)
7-8	System prototype demonstration in operational environment	Integrated BLSS tested in space environment (e.g., lunar surface)
9	Actual system proven in successful mission	Fully operational BLSS supporting crew on Mars transit/habitat

Different sectors apply the TRL framework with varying degrees of flexibility. While the pharmaceutical industry maintains rigorous, standardized TRL definitions tied to specific regulatory milestones, food technology allows for more adaptable interpretations [35]. This flexibility is particularly relevant for BLSS food production, where technologies may be at different readiness levels depending on specific mission parameters such as duration, destination, and degree of closure required.

TRL Assessment of "Salad Machine" Systems

"Salad machines," more formally known as Vegetable Production Systems (Veggie), represent the simplest class of BLSS food production technology. These systems are designed not for caloric replacement but for dietary supplementation, providing fresh produce that delivers phytonutrients, antioxidants, and psychological benefits to crews [36]. The operational concept involves relatively small-scale production units (typically < 0.5m² growing area) that require minimal crew time for operation and maintenance. These systems function primarily as supplemental food sources while providing valuable platforms for testing horticultural techniques in space environments, serving as stepping stones toward more complex, calorie-producing BLSS [36].

Current Capabilities and Experimental Validation

NASA's Veggie system aboard the International Space Station (ISS) represents the most flight-proven salad machine technology. The system has successfully produced multiple crops of leafy greens, including 'Outredgeous' red romaine lettuce, 'Tokyo Bekana' Chinese cabbage, and mizuna mustard [36]. These crops were selected based on rigorous ground testing for characteristics desirable in space environments: short stature, high harvest index, favorable organoleptic properties, and robust resistance to microbial contamination [36].

The experimental protocol for salad machine validation follows a systematic approach:

• Ground-Based Selection: Candidate crops are screened in controlled environment chambers simulating space vehicle conditions (e.g., elevated CO2 ~3000 ppm, LED lighting, recirculating nutrient delivery systems) [36].
• Food Safety Analysis: Microbial load and potential pathogen presence are assessed for both plant tissue and growth system components [36].
• Spaceflight Validation: Selected crops are grown in the target space environment (e.g., ISS) with monitoring of growth parameters, crew time requirements, and biomass production efficiency.
• Nutritional and Sensory Analysis: Post-harvest analysis evaluates nutrient retention and organoleptic properties compared to ground controls.

Table 2: Performance Metrics of Salad Crop Production on ISS

Crop Variety	Growth Cycle (days)	Edible Biomass Yield (g/m²/day)	Key Nutrient Delivered	Crew Time Investment (min/day)
'Outredgeous' Lettuce	28-33	50-70	Vitamin K, Potassium	< 10
'Tokyo Bekana' Cabbage	25-28	60-80	Vitamin C, Folate	< 10
Mizuna Mustard	20-25	40-60	Vitamin K, Antioxidants	< 10

TRL Assessment and Research Gaps

Based on their demonstrated performance in the operational environment of the ISS, vegetable production systems such as Veggie have reached TRL 8 (system complete and qualified). These systems have proven capable of reliable operation in spaceflight conditions with minimal crew intervention. However, several research gaps remain before these technologies can be considered TRL 9 for deep space missions. These include determining the long-term reliability of hardware components over multi-year missions without resupply, validating performance in partial gravity environments (Lunar and Martian surfaces), and optimizing light recipes (spectral quality) and nutrient delivery formulations for a wider variety of crop species.

TRL Assessment of Staple Crop Production Systems

Staple crop production systems target the provision of dietary calories and macronutrients (carbohydrates, proteins, and fats) necessary to significantly reduce or eliminate dependence on resupplied food. These systems represent a substantially greater technological challenge than salad machines, requiring larger growing areas (estimated 35-50m² per person), more complex environmental controls, and advanced processing capabilities to transform raw biomass into edible forms [36]. Historical NASA programs, including the Controlled Ecological Life Support System (CELSS) program and the Bioregenerative Planetary Life Support Systems Test Complex (BIO-PLEX), laid substantial groundwork for these systems before being discontinued [2].

Current Capabilities and Experimental Validation

Ground-based research has identified several candidate staple crops for BLSS applications, including wheat, potato, sweet potato, soybean, and peanut. These crops were selected for their high calorie yield per unit area, nutritional completeness, and adaptability to controlled environments. NASA's ground testing throughout the 1980s-2000s provided extensive data on the growth requirements, productivity, and system dynamics of these crops in closed environments [36].

The experimental protocol for staple crop validation involves:

• Closed System Chamber Studies: Crops are grown in environmentally sealed chambers to measure gas exchange (O2 production, CO2 consumption), water transpiration, and nutrient uptake patterns [36].
• Productivity Optimization: Light intensity, photoperiod, CO2 concentration, and nutrient delivery are optimized for maximum harvest index in controlled conditions.
• Waste Processing Integration: Studies examine the capacity of crops to utilize mineralized waste products (from inedible biomass and human waste) as nutrient sources.
• System Modeling: Mass and energy balances are calculated to determine resource requirements for full-scale implementation.

Table 3: Performance Metrics of Select Staple Crops from Ground-Based Studies

Crop	Growing Area Required per Person (m²)	Caloric Yield (kcal/m²/day)	O2 Production (g/m²/day)	Edible Biomass Ratio
Wheat	15-20	600-800	60-80	0.4-0.5
Potato	12-18	700-900	50-70	0.6-0.7
Sweet Potato	10-15	750-950	55-75	0.5-0.6
Soybean	20-25	400-600 (protein focus)	45-65	0.3-0.4

TRL Assessment and Research Gaps

Despite decades of ground-based research, staple crop production systems for BLSS currently stand at TRL 4 (technology validated in lab). While individual components have been tested and system concepts have been developed, no integrated staple crop production system has been validated in a space-relevant environment, let alone a space operational environment. The discontinuation of NASA's BIO-PLEX program in 2004 left a significant gap in the development pathway for these technologies [2]. Critical research gaps include:

• Space Environment Effects: Lack of data on growth and reproduction of staple crops in microgravity and partial gravity.
• System Integration: No demonstration of integrated BLSS combining staple crop production with air revitalization, water recovery, and waste processing systems.
• Scale-up Challenges: Significant engineering hurdles remain in designing reliable, automated systems for planting, maintenance, harvesting, and processing in space.
• Reliability and Closure: Unproven long-term reliability and the achievement of high closure rates for water and nutrient cycles.

Comparative Analysis and Future Development Pathways

The TRL gap between salad machines (TRL 8) and staple crop systems (TRL 4) highlights the significant technological and programmatic challenges that remain in achieving a fully bioregenerative food system for deep space missions. The following diagram illustrates the development pathway and logical relationships between these technologies.

Diagram 1: TRL progression and system interdependencies for space food production. Supplemental systems like "salad machines" have reached high TRLs, while more complex staple crop and integrated BLSS remain at lower maturity levels.

The divergent readiness levels between different food production approaches highlight a strategic challenge for bioastronautics. While salad machines provide immediate benefits and valuable operational experience, they do not directly address the fundamental logistics challenge of providing complete nutrition for long-duration missions beyond low-Earth orbit. The development of staple crop production systems has been identified as a critical capability gap in the U.S. space program, particularly in light of competing international programs that have maintained focus on BLSS technology [2].

Essential Research Toolkit for BLSS Food Production

Advancing the TRL of food production systems requires specialized reagents, equipment, and methodologies. The following table details key research solutions essential for experimental work in this field.

Table 4: Essential Research Toolkit for BLSS Food Production Studies

Tool/Reagent	Specifications	Research Function	Example Application
Controlled Environment Chambers	Precise control of CO2 (100-10,000 ppm), temperature, humidity, and LED lighting spectra/intensity	Creates ground-based analogs of space vehicle/ habitat environments for crop screening	Studying CO2 fertilization effects at ISS-relevant levels (~3000 ppm) [36]
Recirculating Hydroponic/Aeroponic Systems	Non-soil based nutrient delivery with precise control of pH, EC, and dissolved O2	Mimics resource-conserving cultivation methods required for spaceflight; enables nutrient uptake studies	Optimizing mineral nutrient formulations for wheat in closed systems [36]
Plant Growth LEDs	Specific wavelength ratios (e.g., Red:Blue 95:5; addition of Green/Far-red)	Provides energy-efficient, controllable lighting for photosynthesis and morphological control	Determining optimal light recipes for compact growth and high nutritional value [36]
Gas Chromatography Systems	High-precision measurement of O2, CO2, and volatile organic compounds	Quantifies gas exchange rates for mass balance calculations in closed systems	Measuring net photosynthetic rate and carbon assimilation in candidate crops [36]
Elemental Analyzers	CHNS (Carbon, Hydrogen, Nitrogen, Sulfur) analysis	Determines elemental composition for nutrient cycling and mass balance studies	Calculating nutrient recovery efficiency from waste streams to plant biomass

This TRL assessment reveals a stark contrast in maturity between different food production technologies for BLSS applications. While supplemental salad systems have reached high readiness levels (TRL 8) and are already providing benefits on the ISS, the staple crop production systems necessary for sustainable deep space exploration remain at the laboratory validation stage (TRL 4). This technology gap poses a strategic challenge for NASA and its international partners, particularly as competing space programs advance their own BLSS capabilities [2].

Closing this TRL gap will require a concerted, long-term research and development effort focused on ground-based testing in high-fidelity analogs, followed by incremental testing in space environments. Future research must prioritize the integration of food production with other BLSS subsystems, address the unique challenges of crop cultivation in partial gravity environments, and develop the high-level automation required for operational efficiency. The continued operation of salad machines on the ISS provides an invaluable platform for learning and technology refinement, but a significant acceleration in investment for staple crop production systems will be essential to enable the endurance-class human space missions envisioned in the coming decades.

TRL Assessment of Water Recycling and Waste Management Systems

The success of long-duration human space exploration missions to the Moon and Mars hinges on the development of advanced Bioregenerative Life Support Systems (BLSS) capable of closed-loop resource recovery. These systems are essential for missions where resupply from Earth becomes technically or economically unfeasible due to the immense distances involved; a round-trip mission to Mars could take approximately three years [37]. A BLSS integrates biological components—such as plants and microbes—with physicochemical processes to create an artificial ecosystem that recycles air, water, and waste, and produces food [10]. Within this framework, water recycling and waste management are cornerstone technologies. This guide provides a comparative assessment of current and emerging technologies in these domains, evaluating their performance and maturity through the standardized framework of Technology Readiness Levels (TRL) to identify capabilities and gaps for future deep-space missions.

Technology Readiness Level (TRL) Framework

Technology Readiness Level (TRL) is a systematic metric used to assess the maturity of a particular technology. This assessment utilizes a coarse-granularity color-coding scheme to provide a quick visual representation of a technology's development stage, as defined by the iNEMI Roadmap [38]. The table below outlines this classification.

Table 1: Color-Coded Technology Readiness Level Framework

Color Code	TRL Range	Description
●	1 - 4	Levels involving research (basic principles observed and validated in a laboratory environment).
●	5 - 7	Levels involving development (technology validated in a relevant environment, leading to a system prototype).
●	8 - 10	Levels involving deployment (actual system proven in an operational environment and mission qualified).

This color scheme will be applied in subsequent comparison tables to offer an immediate visual cue of the maturity of each technology discussed.

Comparative Analysis of Water Recycling Systems

Water is an invaluable resource in space, essential for drinking, producing breathable air through electrolysis, and cultivating plants for food [39]. Current state-of-the-art systems on the International Space Station (ISS) recover over 90% of water used by processing urine, cabin moisture, and hygiene wastewater [37]. The system employs a multi-stage process: a Urine Processor Assembly recovers about 75% of water from urine, a Brine Processor Assembly extracts the remaining water from urine brine, pushing overall recovery to 98%, and an Air Revitalization System condenses moisture from cabin air [37]. The collected water is then purified in the Water Processor Assembly through filtration, catalytic oxidation, and the addition of iodine to prevent microbial growth [37].

For future Mars missions, NASA has set a target of reclaiming at least 98% of all water used on board [37]. An emerging technology, the Carbon dioxide Hydrogen Recovery System (CHRSy), developed by MAC SciTech, promises to recycle up to 100% of water in a closed-loop system [39]. This innovative system employs a catalyst-free approach to convert carbon dioxide and hydrogen into carbon monoxide and water, making it easier to service and maintain than catalyst-dependent systems [39]. Its design is low-weight, low-energy, and long-lasting, representing a significant leap in life support technology [39].

The following table provides a structured comparison of these key systems.

Table 2: Comparative Analysis of Water Recycling Systems for Space Missions

Technology Attribute	ISS Water Recovery System	CHRSy (Carbon dioxide Hydrogen Recovery System)
System Type	Integrated Physicochemical System [37]	Catalyst-free Physicochemical System [39]
Key Process Components	Urine Processor Assembly (UPA), Brine Processor Assembly (BPA), Water Processor Assembly (WPA) [37]	CHRSy Reactor [39]
Primary Inputs	Urine, humidity condensate, hygiene wastewater [37]	Carbon dioxide (from crew/cabin) and Hydrogen [39]
Key Output	Potable water exceeding Earth-based standards [37]	Water and Carbon Monoxide [39]
Claimed Water Recovery Rate	98% (with BPA) [37]	Up to 100% (in a closed-loop system) [39]
Reported Advantages	Proven, reliable, long-term operation in microgravity [37]	>90% reduction in size/weight vs. previous designs; catalyst-free for easier maintenance [39]
Reported Limitations	Requires resupply of some consumables (e.g., iodine); not designed for Martian transit [37]	Technology is newer, with less extensive in-space operational history [39]
TRL Status (with Color Code)	● TRL 9 (Actual system proven in operational environment) [38] [37]	● TRL 5-7 (Technology validated in relevant environment) [38] [39]

Experimental Protocols for Water System Validation

Protocol for ISS Water Processor Assembly Performance Validation: The core methodology for validating the water quality output of systems like the ISS WPA involves a multi-stage treatment process followed by rigorous analysis. The collected wastewater is first passed through particulate filters to remove suspended solids. Subsequently, it undergoes multi-filtration beds to remove dissolved salts and organic contaminants. A critical step is catalytic oxidation, where the water is heated and passed over a catalyst in the presence of oxygen to break down volatile organic compounds and other challenging organic contaminants. The final step is the addition of a biocide (iodine) to maintain microbial control in the stored water. The output water is periodically sampled and analyzed using in-flight instrumentation (e.g., Total Organic Carbon Analyzer, Conductivity sensors) to verify it meets the stringent potable water standards for spaceflight, which often exceed those on Earth [37].

Protocol for CHRSy Reactor Efficiency Testing: The testing protocol for the CHRSy system involves validating its core chemical conversion process in a ground-based, space-relevant environment. The methodology entails introducing controlled flows of carbon dioxide (CO₂) and hydrogen (H₂) into the CHRSy reactor. The reactor operates without a catalyst, and its performance is monitored by measuring the input and output gas compositions using Gas Chromatography-Mass Spectrometry (GC-MS). The production of water vapor is quantified by passing the output stream through a condenser and measuring the mass of liquid water collected. The key metric for success is the conversion efficiency of the reactants (CO₂ and H₂) to the products (H₂O and CO), demonstrating the system's ability to achieve near-complete water recycling from metabolic by-products [39].

Comparative Analysis of Waste Management Systems

Waste management in a BLSS focuses on the recycling of organic solid wastes and urine brine to recover resources for plant growth and oxygen production. The integration of biological components is key to closing this loop. In a BLSS, microbes act as degraders and recyclers, breaking down organic waste (e.g., inedible plant biomass, human waste) into simpler inorganic compounds, such as carbon dioxide, water, and nutrients like nitrates and phosphates [10]. These recovered resources are then used by the plant compartment (the producers) for photosynthesis and growth, which in turn provides food and oxygen for the crew (the consumers) [10]. This creates a semi-closed ecological network.

The ISS Brine Processor Assembly (BPA) represents a high-TRL physicochemical approach to waste valorization, extracting water from the concentrated brine leftover from urine processing [37]. For more integrated biological recycling, systems like the MELiSSA (Micro-Ecological Life Support System Alternative) project aim to use a loop of interconnected bioreactors, each with specific microbial communities, to efficiently degrade waste and regenerate resources [10].

Table 3: Comparative Analysis of Waste Management and Resource Recovery Systems

Technology Attribute	ISS Brine Processor Assembly (BPA)	BLSS with Integrated Microbial Recycling (e.g., MELiSSA)
System Type	Physicological Waste Processing [37]	Biological/Bioregenerative Waste Processing [10]
Primary Waste Input	Urine brine from the Urine Processor Assembly [37]	Organic solid waste (e.g., inedible plant biomass, human waste) [10]
Key Process	Warm, dry air evaporates water from brine; a filter separates contaminants from vapor [37]	Microbial degradation and fermentation in controlled bioreactors [10]
Key Outputs	Recovered potable water [37]	Recovered CO₂, water, and mineral nutrients for plant growth [10]
System Integration	Integrated with the ISS Water Recovery System [37]	Integrated with the higher plant compartment and crew habitat [10]
Reported Advantages	Increases overall water recovery to 98%; operates reliably in microgravity [37]	Closes the carbon and nutrient loops; produces resources for food production [10]
Reported Limitations / Challenges	Processes a single waste stream (urine brine) [37]	Requires precise control of microbial ecology; complex system integration [10]
TRL Status (with Color Code)	● TRL 9 (Actual system proven in operational environment) [38] [37]	● TRL 5-7 (Technology validated in relevant environment) [38] [10]

Integrated System Visualization and Workflow

The following diagram illustrates the logical relationships and material flows between the crew and the core subsystems of a Bioregenerative Life Support System (BLSS), integrating the water recycling and waste management technologies previously discussed.

Diagram 1: BLSS Material Flow and Subsystem Relationships

The Scientist's Toolkit: Key Research Reagents and Materials

Research and development in BLSS technologies rely on specific reagents, materials, and analytical tools. The following table details key items essential for experimental work in this field.

Table 4: Essential Research Reagents and Materials for BLSS Experimentation

Item Name	Function / Application
Gas Chromatography-Mass Spectrometry (GC-MS)	Used for precise identification and quantification of volatile organic compounds in recycled water and gas streams (e.g., CHRSy output analysis) [39].
Total Organic Carbon (TOC) Analyzer	A critical instrument for monitoring water purity by measuring the concentration of organic carbon contaminants in recycled water streams [37].
Iodine Biocide	A chemical consumable used in water recovery systems (e.g., ISS WPA) to prevent microbial regrowth in stored potable water, ensuring its long-term stability [37].
Selective Microbial Inoculants	Specific strains of bacteria (e.g., nitrifying bacteria, fermenters) used in bioreactors to degrade organic waste and recover nutrients in systems like MELiSSA [10].
Defined Growth Media for Microbes	Standardized nutrient solutions required for cultivating and maintaining the performance of microbial communities in waste recycling bioreactors [10].
Hydroponic Nutrient Solutions	Balanced mixtures of essential mineral nutrients (e.g., N, P, K, Ca) required for plant growth in the BLSS plant compartment, often derived from recycled waste streams [10].
Catalytic Oxidation Catalysts	Solid-phase catalysts used in water processor assemblies to facilitate the breakdown of trace organic contaminants into carbon dioxide and water [37].
Multi-Filtration Media Beds	A combination of filter media (e.g., for particulates, ions, organics) used in a series to remove diverse contaminants from wastewater [37].

The TRL assessment reveals a clear technology pathway for deep space missions. Physicochemical water recycling, as demonstrated by the ISS Water Recovery System, has achieved high maturity (TRL 9) and is ready for deployment in near-term lunar missions [37]. The emerging CHRSy technology (TRL 5-7) shows great promise for further enhancing water recovery efficiency and reducing system mass for the more demanding Mars missions [39]. However, for long-term sustainability, the integration of biological systems for waste management and food production remains a critical area of development. While ground-based demonstrators have proven the concept, these bioregenerative technologies currently reside at TRL 5-7, indicating a need for further integrated testing and validation in space-relevant environments [10]. Closing the loop with biological components is the next major frontier in developing the fully self-sustaining life support systems required for humanity's journey to Mars and beyond.

For space biotechnologies essential to sustained human presence in deep space, the journey from laboratory validation to operational deployment is fraught with a critical developmental obstacle known as the "Valley of Death" [8]. This term describes the precarious transition where promising technologies often stall or fail due to the significant leap in cost, complexity, and risk required to move from ground-based testing to a space environment [8]. The Technology Readiness Level (TRL) scale, a systematic metric originally developed by NASA, provides a common language to assess this maturity, spanning from TRL 1 (basic principles observed) to TRL 9 (actual system proven in mission operations) [8] [4] [5].

This guide objectively compares the performance and validation requirements for space biotechnologies at TRL 5, TRL 6, and TRL 7. For Bioregenerative Life Support Systems (BLSS) and other technologies critical for deep space missions, successfully navigating this transition is not merely a matter of project advancement—it is a fundamental requirement for mission success and crew survival.

TRL Comparison: Requirements, Environments, and Biotechnological Evidence

The progression from TRL 5 to TRL 7 represents a paradigm shift from component validation to system-level demonstration in the actual operational environment. The table below summarizes the critical comparisons across these stages.

Table 1: Comparative Analysis of TRL 5, 6, and 7 for Space Biotechnologies

Criterion	TRL 5: Component Validation in Relevant Environment	TRL 6: System/Subsystem Model Demonstration in Relevant Environment	TRL 7: System Prototype Demonstration in Operational Environment
Definition & Core Objective	Technology is validated in a environment that simulates key operational conditions [8].	A fully functional prototype is demonstrated in a high-fidelity relevant environment [8] [40].	A system prototype is demonstrated in its actual operational environment [8] [4].
Test Environment	Simulated environments on Earth (e.g., thermal vacuum chambers, radiation testing facilities) [8] [41].	High-fidelity simulations or field environments on Earth that closely mimic space [8].	Space environment (e.g., International Space Station, suborbital flight, satellite) [8] [4].
Technology Form Factor	Breadboard or component-level setup [8] [41].	Engineering model or high-fidelity prototype representing the final form [8] [40].	Near-final "flight-like" prototype [8].
Example from Biotechnology Research	Testing a breadboard bioreactor in a lab-based random positioning machine to simulate microgravity effects on cell cultures.	A fully assembled plant growth system (like PONDS) tested in an environmental chamber that simulates space station temperature, humidity, and gas composition [42].	The BioNutrients experiment on the ISS, which demonstrates on-demand nutrient production using genetically engineered yeast in microgravity [43].
Key Performance Metrics	Basic functionality and survival under one or more relevant stress factors (e.g., radiation, vacuum).	Integrated system performance, reliability, and consistency across multiple use cases and environmental cycles [40].	System performance, reliability, and output under real microgravity and space radiation [43].
Primary Risk Focus	Technical feasibility of core components in a simulated space environment.	System integration and operational robustness before committing to a flight opportunity [40].	Performance and reliability in the actual mission environment, where repair is often impossible.

The "Valley of Death" is most acutely experienced during the TRL 6 to 7 transition [8]. The cost and effort required for this step can dwarf all previous development work, as it necessitates securing a scarce and expensive flight opportunity and building a system to rigorous flight standards [8]. Failure at this stage can terminate an otherwise promising technology.

Experimental Protocols for TRL Advancement

Bridging the "Valley of Death" requires meticulously designed experiments that generate conclusive data on system functionality and readiness. The following protocols are representative of the research underpinning TRL advancement for space biotechnologies.

Protocol 1: Validating a Bioreactor for TRL 6

• Objective: To demonstrate the integrated performance and reliability of a miniature bioreactor system (e.g., for cell culture) in a ground-based environment relevant to space station conditions [42].
• Methodology:
  • Test Setup: The fully assembled bioreactor prototype is placed inside an environmental test chamber capable of controlling temperature, CO₂, and humidity to ISS module specifications.
  • Long-Term Operation: The system is operated continuously for a duration equivalent to or exceeding a planned space mission segment (e.g., 30-90 days).
  • Automated Monitoring: Key parameters (pH, dissolved oxygen, temperature, cell density) are monitored and logged automatically by the system's onboard electronics.
  • Performance Sampling: At regular intervals, automated aseptic sampling is performed to assess cell viability, metabolic activity, and product yield (e.g., a specific pharmaceutical or nutrient).
  • Stress Testing: The system is subjected to simulated contingencies, such as a temporary power loss or a cooling system malfunction, to evaluate recovery and fail-safe mechanisms.
• Outcome Measurement: Success is determined by the system's ability to maintain stable cell culture conditions, achieve target product yields, and recover autonomously from simulated faults with minimal data loss or culture contamination.

Protocol 2: TRL 7 Demonstration of On-Demand Nutrient Production

• Objective: To prove that a biotechnology system can successfully produce key nutrients from genetically engineered yeast in the microgravity environment of the ISS [43].
• Methodology:
  • Flight Hardware Integration: The nutrient production system, containing desiccated yeast strains and a hydrated growth medium, is loaded into ISS-compatible hardware, ensuring it meets safety and interface requirements.
  • In-Flight Activation: Astronauts onboard the ISS activate the experiment by introducing the growth medium to the yeast, initiating the production process.
  • In-Situ Monitoring: The system automatically maintains incubation temperature and monitors growth progress.
  • Sample Analysis: After a defined production period, the crew harvests the product. Samples can be either preserved for return to Earth or analyzed in situ using ISS laboratory equipment.
  • Ground Control: An identical experiment is conducted synchronously in a ground laboratory to provide a controlled baseline for comparison.
• Outcome Measurement: The key metric is a quantitative comparison of the nutrient yield and production efficiency achieved in microgravity versus the ground control, confirming that the process is viable in the actual space environment [43].

The logical flow and decision points for advancing a technology through these critical TRLs are visualized below.

Diagram 1: TRL Progression Pathway from 5 to 7

The Scientist's Toolkit: Key Research Reagent Solutions

The experimental work at these TRL stages relies on specialized biological and technical materials. The following table details essential toolkit components for developing space biotechnologies like BLSS.

Table 2: Essential Research Reagents and Materials for Space Biotechnology Development

Item Name	Function/Application in Research	Example Use-Case
Genetically Engineered Microbes	Engineered yeast or bacteria designed for high-yield production of vitamins, nutrients, or pharmaceuticals in resource-limited environments [43] [42].	BioNutrients Experiment: Used to produce fresh nutrients on-demand to counteract vitamin degradation in stored food during long-duration missions [43].
Radio-sensitized Cell Lines	Genetically modified cell lines (e.g., histone H2AX deficient mouse ES cells) used as biosensors to measure the biological effectiveness of space radiation [44].	Radiation Biology Studies: Flown frozen to the ISS to quantify chromosome aberrations caused by space radiation, providing critical data for human risk assessment [44].
Passive Dosimeter (PADLES)	A physical device attached to biological samples to measure the absorbed radiation dose (in water) during spaceflight [44].	Comparative Biology-Physics Studies: Used alongside biological samples to correlate physical radiation measurements with observed biological effects (e.g., chromosome damage) [44].
Miniature Bioreactor System	A compact, automated system for cell culturing with minimal reagent use and power requirements, designed for unmanned operation in remote locations [42].	Pharmaceutical Research in Space: Enables the study of cell culture responses to spaceflight stressors (microgravity, radiation) for drug development [42].
Wicking Plant Growth Media (PONDS)	A passive nutrient delivery system that uses a wicking material to draw water and nutrients from a reservoir to plant roots in microgravity [42].	Space Agriculture: Addresses the challenge of consistent water and oxygen delivery to plant roots in the absence of gravity, enabling crop production on the ISS [42].

For the future of deep space exploration, the successful transition of BLSS and other biotechnologies across the TRL 5-7 "Valley of Death" is imperative. This journey requires a methodical, evidence-based approach involving rigorous ground testing at TRL 6, strategic planning for flight opportunities, and conclusive in-space validation at TRL 7. By understanding the distinct requirements, experimental protocols, and essential tools at each stage, researchers and program managers can de-risk this critical pathway, transforming the biotechnology breakthroughs of today into the life-supporting systems of tomorrow.

Mitigating Risks and Enhancing BLSS Reliability

As human space exploration ambitions extend to long-duration missions to the Moon and Mars, Bioregenerative Life Support Systems (BLSS) have become critical technologies for sustaining crew life by regenerating oxygen, water, and food through plant-based biological production systems [45]. The transition from short-duration missions relying on physiochemical systems to long-duration exploration necessitates crop cultivation modules that reduce resupply mass and enhance crew autonomy [45] [24]. However, the stability and productivity of these space-based agricultural systems face significant threats from insect pests and phytopathogens, as demonstrated by a disease outbreak of Fusarium oxysporum on Zinnia hybrida plants aboard the International Space Station (ISS) [45]. This incident underscores the vital need for Integrated Pest Management (IPM) protocols specifically engineered for the unique constraints of the space environment to ensure mission success and food security [45].

This guide provides a comparative analysis of IPM strategies for space-based agriculture, framing them within the context of Technology Readiness Levels (TRL) for deep space missions. It objectively evaluates terrestrial and nascent space-based IPM approaches through the lens of experimental data, protocol feasibility, and integration requirements for BLSS.

Comparative Analysis of IPM Frameworks and Readiness for Space

The table below compares established terrestrial IPM principles with frameworks adapted for space-based agriculture, assessing their applicability and readiness for deep space missions.

Table 1: Comparative Analysis of IPM Frameworks for Space-Based Agriculture

IPM Framework	Core Principles	Terrestrial Application Evidence	Space Adaptation Feasibility	BLSS Integration Challenges	Estimated TRL for Space
Conventional EPA Framework [46]	1. Set Action Thresholds2. Monitor & Identify Pests3. Prevention4. Control (with least-risk methods first)	Widely adopted; proven successful across diverse agro-ecosystems [46].	High for principles; low for specific chemical controls. Requires redefinition of thresholds and controls for closed systems.	Chemical pesticide use is problematic in closed environments; focus must shift to non-toxic controls [45].	TRL 4-5: Component validation in relevant (ground-analog) environments is ongoing.
Multi-Dimensional Management of Multiple Pests (3MP) [47]	Manages soil-crop-pest-natural enemy networks (spatial dimension) and pest interactions over a season (time dimension).	Theoretical; aims to overcome low synergy and low coverage of simple mixed inputs [47].	High for holistic BLSS design. Aligns with viewing BLSS as a complex, multi-trophic ecosystem.	High computational and modeling demand; requires extensive data on species interactions [47].	TRL 2-3: Technology concept and experimental proof-of-concept stage for space.
Proposed Space-Based BLSS IPM [45]	Protocols dynamic in nature, changing spatially/temporally; prioritize prevention, mitigation, and elimination.	Based on Earth-based greenhouse/vertical-farming protocols, but must be uniquely developed for space stresses [45].	Designed specifically for space-based plant-growing systems, from small modules to full-scale habitats.	Unusual space environment stresses on plants (e.g., microgravity, radiation) may alter host-pathogen interactions [45].	TRL 3-5: Proof-of-concept to component validation for specific subsystems (e.g., sanitation).

Key Implications for BLSS Technology Readiness

The successful operation of space-based BLSS units depends on IPM protocols that are established early in the mission design phase and are dynamic enough to change throughout the mission [45]. The Equivalent System Mass (ESM) metric used by life-support engineers often favors BLSS over physiochemical systems for missions exceeding three months, but this advantage is contingent on reliable crop production, which pests can severely disrupt [45]. The 3MP framework is particularly promising for BLSS as it encourages a crop-centered rather than pest-centered view, which is essential for managing the multiple crops needed for a balanced diet in a closed system [47]. However, advancing these frameworks to TRL 7 and above ("system prototype demonstration in operational environment") will require dedicated flight experiments [8].

Experimental Protocols and Performance Data for IPM Tactics

The evaluation of IPM tactics relies on rigorous experimental data. The following tables summarize performance data and methodologies for key control strategies relevant to BLSS.

Biological Control and Botanicals

Biological control uses living organisms to suppress pests, while botanicals leverage plant-derived compounds. Their low toxicity profile makes them high-priority candidates for BLSS.

Table 2: Experimental Data for Biological Control and Botanical Pesticides

Control Tactic	Target Pest/Pathogen	Experimental Setup & Methodology	Key Performance Metrics	Reported Efficacy	Reference
Mating Disruption (Pheromones)	Lepidopteran pests (e.g., Navel Orangeworm)	Field deployment of automated pheromone dispensers; network of sensors and camera traps to monitor pest populations and degree days [48].	Crop damage reduction; reduction in conventional pesticide sprays.	When combined with limited spraying, resulted in less damage and more valuable crops compared to sprays alone [48].	[48]
Augmentative Biological Control	Aphids, whiteflies, mites	Release of beneficial predators (e.g., Aphidius colemani) or parasitoids in greenhouse and field settings; timing coordinated with pest monitoring and pesticide residue degradation data [48].	Pest population suppression below economic injury level; establishment of beneficial population.	Efficacy highly dependent on precise release timing and absence of harmful pesticide residues; timing can be guided by microclimate and spray data [48].	[48]
Plant-Derived Volatile Compounds	Various insect pests	Laboratory and field assays using insect- and plant-derived volatile compounds to repel pests or attract their natural enemies [47].	Reduction in pest oviposition and feeding; increased attraction of natural enemies.	Aromatic intercrops (e.g., Ocimum basilicum) shown to repel pests or mask host plant cues, reducing infestation [49].	[47] [49]

Cultural and Genetic Control

These methods form the foundation of prevention by making the environment less suitable for pests and the plant more resistant.

Table 3: Experimental Data for Cultural and Genetic Control Methods

Control Tactic	Target Pest/Pathogen	Experimental Setup & Methodology	Key Performance Metrics	Reported Efficacy	Reference
Crop Rotation & Intercropping	Soil-borne pathogens, plant-parasitic nematodes	Field trials with strategic sequences of non-host and host crops (e.g., cereals rotated with vegetables); intercropping with aromatic plants [49].	Incidence and severity of pest/disease; soil health biomarkers.	Alternating non-host with host crops effectively mitigated soil-borne diseases; intercropping creates less favorable conditions for pests [49].	[49]
Sanitation	Navel Orangeworm (Amyelois transitella)	Orchard studies involving removal and destruction of fallen fruits and mummified nuts to eliminate overwintering pest inoculum [49].	Overwintering pest population size; subsequent crop damage.	Significantly reduced overwintering populations of the navel orangeworm in almond orchards [49].	[49]
Resistant Varieties (e.g., Bt crops)	Lepidopteran pests (e.g., cotton bollworm)	Cultivation of genetically modified varieties expressing Bacillus thuringiensis (Bt) insecticidal proteins; long-term field monitoring for resistance development [49].	Pesticide use reduction; pest-induced yield loss; emergence of resistant pest biotypes.	Major success in reducing pesticide use and controlling lepidopteran pests; however, raises concerns about pest resistance development [49].	[49]

Visualization of IPM Workflows and Interactions

Effective IPM, especially the 3MP framework, requires an understanding of complex interactions. The following diagrams map these relationships and experimental workflows.

Multi-Dimensional Management Logic

This diagram illustrates the core logic of the 3MP framework, which manages multiple pests across spatial and temporal dimensions.

Space-Based IPM Experimental Protocol

This flowchart outlines a generalized experimental protocol for validating an IPM tactic in a space-analog environment.

The Scientist's Toolkit: Key Research Reagents and Materials

Implementing and researching IPM for space-based agriculture requires a specific set of reagents, tools, and technologies.

Table 4: Essential Research Toolkit for Space-Based Agriculture IPM

Tool Category	Specific Material / Technology	Function in IPM Research & Implementation
Monitoring & Sensing	Automated camera traps & pest sensors [48]	Provides continuous, remote monitoring of insect pest populations, enabling data-driven decisions and eliminating manual scouting.
	Remote sensing (UAVs, spectral imagery) [49] [51]	Enables early detection of plant disease hotspots (e.g., red crown rot) over large areas by identifying changes in crop health before visible symptoms appear.
	Microclimate sensors (temp, humidity, soil moisture) [48]	Monitors environmental conditions critical for pest development and pesticide residue degradation, informing models for pest forecasting and biocontrol timing.
Control Agents	Synthetic sex pheromones [48]	Disrupts pest mating behavior (mating disruption) to limit reproduction, reducing reliance on conventional pesticides.
	Arthropod natural enemies (e.g., predators, parasitoids) [47] [49]	Provides biological control by preying on or parasitizing pest insects; requires careful management within a BLSS.
	Microbial biopesticides & plant-derived compounds [49]	Offers targeted, lower-risk control of pests and diseases; includes microbial agents and plant-derived volatile compounds.
Analysis & Modeling	AI/Machine Learning models [52] [49] [51]	Used for image-based pest identification, forecasting pest phenology using degree-day models, and analyzing spectral data for disease detection.
	Nitrogen Productivity (NP) Model [50]	Predicts crop biomass growth based on nitrogen availability, which is crucial for modeling plant health and resilience in a resource-limited BLSS.
	Modified Energy Cascade (MEC) Model [50]	NASA's model for predicting crop biomass production, oxygen generation, and water transpiration based on light and CO₂.

The development of robust Integrated Pest Management protocols is a critical path item for the advancement of Bioregenerative Life Support Systems destined for deep space missions. While terrestrial IPM principles provide a foundational starting point, the unique constraints of the space environment—including closed-loop resource cycling, microgravity, and the absolute need to avoid toxicants—demand the creation of uniquely space-adapted IPM frameworks. The 3MP framework offers a promising, holistic approach by managing multiple pests across spatial and temporal dimensions, which aligns well with the complex, integrated nature of BLSS [47].

Current research is expanding the toolbox for space-based IPM, with significant advances in AI-driven monitoring, targeted biological controls, and predictive growth modeling [49] [50] [51]. However, as of 2025, many of these protocols remain at a low Technology Readiness Level for space flight, with a significant "valley of death" between ground-based validation and in-space demonstration [8]. Bridging this gap requires dedicated flight experiments on platforms like the ISS and Lunar Gateway to test IPM components in real microgravity and partial gravity environments. The success of future missions to Mars will depend on translating these comparative analyses and experimental protocols into a fully integrated, flight-proven IPM system that safeguards the food and oxygen production capabilities of BLSS.

Bioregenerative Life-Support Systems (BLSS) represent critical technologies for long-term human space exploration, designed to regenerate air, water, and produce food through biological processes [33] [53]. Within these systems, plant cultivation is essential for oxygen production, carbon dioxide recycling, water purification, and nutritional provision for crew members [53] [54]. However, the microgravity environment of space presents fundamental challenges to plant growth by altering fundamental physical processes. The absence of buoyancy-driven convection and sedimentation disrupts normal gas-liquid transfer, creating stagnant boundary layers around plant roots and tissues that limit nutrient uptake and gas exchange [55] [54]. This comprehensive analysis compares technological approaches addressing these challenges, providing experimental data and methodologies relevant to BLSS technology readiness for upcoming deep space missions.

Comparative Analysis of Plant Growth Technologies for Microgravity

Table 1: Comparison of Plant Growth Substrates and Technologies for Microgravity Applications

Technology/Substrate	Key Characteristics	Experimental Performance	Identified Limitations
Cellulosic Sponge	Provides capillary action for water distribution; controlled water retention capacity [53].	Optimal water retention and air transport characteristics; successful potato tuber growth in ground tests [53].	Requires integration with precise irrigation control systems.
Porous Tube System	Uses capillary forces to deliver water and nutrients through porous walls [53].	Enabled uniform water distribution in cellulosic sponge substrates during ground testing [53].	Potential for clogging; requires precise pressure management.
Nutrient Film Technique (NFT)	Flowing thin film of nutrient solution past roots [53].	Successful ground applications for tuberous plants like potato [53].	Difficult to implement in microgravity due to phase separation challenges.
Random Positioning Machine (RPM)	3D clinostat that randomizes gravity vector direction [55] [54].	Induced slower growth (0.28±0.04 d⁻¹) in Limnospira versus control (0.40±0.04 d⁻¹) [55].	Simulated microgravity only; cannot replicate all space environment factors.

Experimental Approaches and Protocols for Microgravity Plant Research

Ground-Based Simulated Microgravity Experimental Protocol

The Random Positioning Machine (RPM) serves as a essential ground-based tool for preliminary microgravity research. The following methodology was implemented for cyanobacteria research relevant to BLSS:

• Apparatus Setup: Custom-built RPM system accommodating continuous illumination and gas-permeable cell culture bags with 3D-printed holders [55]
• Control Configuration: Comparative samples maintained in Rotating Cell Culture System (RCCS) rotating in constant 2D horizontal plane perpendicular to gravity vector [55]
• Culture Conditions: Photoautotrophic conditions with continuous illumination for oxygenic photosynthetic organisms [55]
• Sampling Protocol: Termination at both identical time points (72 hours) and equivalent cell density metrics to differentiate growth rate effects from total yield [55]
• Analysis Methods: Growth rate quantification via exponential regression, proteomic analysis via liquid chromatography with ultra-high-resolution mass spectrometry, pigment extraction with spectrophotometry, and sedimentation index measurement [55]

Root Module Hydrological Characterization Protocol

For tuberous plants (e.g., potato, sweet potato) in BLSS, the following hydrological characterization informs substrate selection:

• Substrate Screening: Multiple organic (cellulosic sponge) and synthetic materials evaluated for hydrological properties [53]
• Key Metrics Analysis: Saturated hydraulic conductivity (Ks) and water retention curves to predict fluid behavior in microgravity [53]
• Sensor Integration: WaterScout sensors calibrated for specific substrates to monitor water status and drive irrigation management [53]
• Distribution Validation: Porous tube-based delivery system tested for uniformity and timing of nutrient solution delivery [53]

Visualizing the Impact of Microgravity on Plant Physiology

The following diagram illustrates the cascading effects of microgravity on plant physiological processes, particularly highlighting the gas-liquid transfer challenges and their consequences for BLSS design.

Diagram 1: Microgravity Impact on Plant Physiology. This pathway illustrates how altered fluid dynamics in microgravity create a cascade of physiological effects in plants, ultimately affecting growth and molecular processes.

The Scientist's Toolkit: Essential Research Reagents and Platforms

Table 2: Research Reagent Solutions for Microgravity Plant Studies

Reagent/Platform	Function/Application	Experimental Utility
Lab-on-a-Chip (LOC)	Miniaturized analysis system integrating laboratory functions [56].	Enables portable microgravity simulation with minimal reagent use; advanced fluid manipulation.
Random Positioning Machine (RPM)	3D clinostat that randomizes gravity vector direction [55].	Provides ground-based simulated microgravity for preliminary experiments before spaceflight.
Cellulosic Sponge Substrate	Porous growth medium for plant root systems [53].	Offers optimal water retention and aeration properties for tuberous plants in hydroponic BLSS.
WaterScout Sensors	Monitoring substrate water status [53].	Enables precise irrigation control in substrate-based growth systems.
Gas-Permeable Cell Culture Bags	Container for photosynthetic cultures [55].	Allows gas exchange while containing liquid media in microgravity experimentation.
Label-Free LC-MS Proteomics	Protein expression analysis [55].	Identifies molecular responses to microgravity stress (e.g., photosystem protein regulation).

The experimental data and comparative analysis presented demonstrate significant progress in addressing microgravity-induced challenges to plant growth systems for BLSS. Ground-based simulation platforms like RPMs provide valuable preliminary data, revealing specific physiological responses to altered gravity conditions, including reduced growth rates, proteomic changes, and gas exchange limitations. The optimization of growth substrates like cellulosic sponge with integrated irrigation systems shows particular promise for tuberous plant cultivation in space environments. However, the transition from ground-based simulation to functional space-based BLSS requires further refinement of gas-liquid transfer management and root zone oxygenation strategies. As deep space missions approach technological feasibility, these plant growth technologies represent essential components for achieving BLSS technology readiness levels sufficient to support human exploration beyond Earth orbit.

For deep space missions, Bioregenerative Life Support Systems (BLSS) are artificial ecosystems designed to sustainably recycle oxygen, water, and food by integrating plants, animals, and microorganisms [7]. The human microbiome—the vast community of trillions of microbes residing in and on astronauts—is a critical, yet often overlooked, component of this system. A balanced microbiome is essential for human health, influencing digestion, metabolism, and immune function [57] [58]. Within the confined and isolated environment of a spacecraft, microbiome dysbiosis (an imbalance in this microbial community) can compromise an astronaut's immune system, increase susceptibility to infections, and potentially jeopardize mission success [57] [59].

This guide objectively compares current and emerging strategies for maintaining microbiome resilience and preventing pathogen outbreaks. The evaluation is framed within the context of Technology Readiness Levels (TRL), a scale from 1 (basic principles observed) to 9 (actual system proven in mission operations) used to assess maturity for space deployment [8]. As we venture toward long-duration lunar and Martian missions, understanding and integrating these microbiome management strategies from a BLSS perspective is not just advantageous—it is imperative for crew health and operational biosustainability.

Comparative Analysis of Microbiome Management Strategies

The following table summarizes the key characteristics, experimental support, and technology readiness of major microbiome-focused strategies.

Table 1: Comparison of Microbiome Management and Pathogen Prevention Strategies

Strategy	Core Principle	Key Experimental Findings/Mechanisms	Advantages	Limitations/Challenges	TRL (for BLSS application)
Probiotics & Prebiotics	Administer beneficial live microbes (probiotics) or compounds that stimulate their growth (prebiotics).	- Immune Priming: Lactobacillus species reduce influenza severity via short-chain fatty acid (SCFA) production [59].- Barrier Integrity: SCFAs like butyrate enhance tight junction proteins in the gut epithelium [59].	Non-invasive; relatively simple to implement; wide range of commercially available strains.	Effects can be transient and strain-specific; survival and colonization in a diverse gut microbiome is unpredictable [57].	TRL 4-5 (Component validation in laboratory/relevant environment)
Fecal Microbiota Transplantation (FMT)	Transplant processed fecal matter from a healthy donor to restore a balanced gut microbiome.	- Pathogen Reduction: Effectively treats recurrent C. difficile infection and reduces antimicrobial-resistant pathogens in patients [60].- Restores Diversity: Reintroduces a wide consortium of commensal microbes.	Highly effective for specific conditions; leads to a durable and diverse microbial community.	Significant safety and screening challenges; risk of transferring unknown pathogens; complex logistics and storage for spaceflight [60].	TRL 3-4 (Proof of concept/Component validation)
Microbiome-Based Biotherapeutics (e.g., Live Biotherapeutic Products - LBPs)	Administer defined consortia of beneficial microbes, engineered or selected for specific functions.	- Targeted Action: FDA-approved LBPs (e.g., Rebyota, VOWST) for C. difficile show targeted pathogen reduction [60].- Standardized Formulation: Offers a controlled, reproducible product compared to FMT.	Defined composition improves safety and regulatory approval profile; potential for engineering strains for space-specific stressors (e.g., radiation).	High cost of development and production; long regulatory pathways; efficacy in non-C. diff contexts is still under investigation.	TRL 3-4 (Proof of concept/Component validation)
Phage Therapy	Use specific bacteriophages (viruses that infect bacteria) to target and eliminate bacterial pathogens without disrupting commensals.	- Precision Targeting: Bacteriophages can lyse specific pathogenic bacteria, sparing beneficial microbiota [60].- Synergy with Antibiotics: Can restore susceptibility to antibiotics in resistant pathogens.	High specificity minimizes collateral damage to the host microbiome; self-replicating at the site of infection.	Narrow spectrum requires precise diagnosis; potential for bacterial resistance to phages; not yet FDA-approved for this use; immune system may clear phages [60].	TRL 2-3 (Technology concept formulated/Proof of concept)
Pathogen Decolonization (Traditional)	Use topical antiseptics (e.g., chlorhexidine) or nasal ointments (e.g., mupirocin) to reduce pathogen load on skin and mucous membranes.	- Infection Prevention: Reduces surgical site infections by decreasing colonization with pathogens like S. aureus [60].- Mechanism: Directly kills or inhibits growth of a broad range of microbes.	Well-established protocols in clinical medicine; rapid action.	Non-selective; can disrupt protective commensal microbiota on skin and mucosa, leading to ecological instability [60].	TRL 9 (Proven in terrestrial medical operations, but TRL 6-7 for integrated BLSS)

Experimental Protocols and Methodologies for Microbiome Analysis

Robust experimental design and data analysis are critical for advancing microbiome management strategies. The following section details standard protocols and emerging methods cited in recent research.

16S rRNA Gene Amplicon Sequencing (Microbiota Profiling)

This is the most common method for determining the taxonomic composition of a microbial community [61].

• Sample Collection: Samples are collected from the target site (e.g., stool for gut, swabs for skin/oral). Consistent collection and immediate freezing at -80°C is crucial to preserve microbial DNA.
• DNA Extraction: Total genomic DNA is extracted from the sample using commercial kits, with bead-beating often used to lyse tough bacterial cell walls.
• PCR Amplification: The 16S rRNA gene (or ITS for fungi) is amplified using primers targeting conserved regions, flanking hypervariable regions (e.g., V4) that provide taxonomic discrimination.
• Library Preparation & Sequencing: Amplified products (amplicons) are prepared into sequencing libraries and run on high-throughput platforms like Illumina.
• Bioinformatic Analysis:
  • Quality Filtering & Denoising: Raw sequences are processed to remove errors and chimeras using tools like DADA2 or Deblur to infer exact amplicon sequence variants (ASVs).
  • Taxonomic Assignment: ASVs are classified against reference databases (e.g., SILVA, Greengenes) to determine phylogenetic identity.
  • Diversity Analysis: Alpha-diversity (within-sample richness/diversity) and Beta-diversity (between-sample compositional differences) are calculated using metrics like Shannon Index and UniFrac distance.

Limitations: This method inferts functional potential but does not directly measure the active functional profile of the community [61].

Shotgun Metagenomic Sequencing (Functional Potential Analysis)

This approach sequences all the DNA in a sample, providing higher taxonomic resolution and direct insight into the functional genes present [61].

• Sample Collection & DNA Extraction: Similar to 16S protocol, but requires higher-quality, high-molecular-weight DNA.
• Library Preparation & Sequencing: DNA is fragmented and prepared for sequencing without a PCR amplification step, reducing associated bias. Sequencing is performed to a sufficient depth (millions of reads).
• Bioinformatic Analysis:
  • Quality Control: Adapter removal and quality trimming.
  • Host DNA Depletion: Computational (and sometimes pre-sequencing) removal of host-derived sequences.
  • Assembly & Binning: De novo assembly of reads into longer contigs and binning into metagenome-assembled genomes (MAGs).
  • Taxonomic & Functional Profiling: Reads or MAGs are aligned to genomic databases (e.g., NCBI NR, KEGG) for taxonomic assignment and functional annotation of genes and pathways.

GLM-ASCA for Analyzing Complex Microbiome Experimental Data

A novel method, GLM-ASCA (Generalized Linear Models–ANOVA Simultaneous Component Analysis), has been developed to address the unique challenges of microbiome data (compositionality, zero-inflation, high-dimensionality) within complex experimental designs [62].

• Objective: To separate and identify how different experimental factors (e.g., treatment, time, and their interactions) affect microbial abundance in a multivariate context.
• Workflow:
  • Model Fitting: A Generalized Linear Model (GLM), appropriate for count data, is fitted to each microbial feature (e.g., ASV) across all samples. The model includes terms for the experimental design (e.g., ~ Treatment * Time).
  • Effect Decomposition: The ASCA component partitions the variance in the multivariate dataset into contributions from each model term (main effects and interactions).
  • Dimension Reduction & Visualization: Simultaneous Component Analysis (a multivariate technique) is applied to the effect matrices, producing scores and loadings plots. This allows for visual interpretation of how experimental conditions drive shifts in the entire microbial community structure.

Application Example: This method was successfully used to identify beneficial bacterial species recruited by tomato plants under nitrogen starvation, highlighting its power to extract meaningful biological insights from complex, high-dimensional data [62].

Diagram 1: Microbiome analysis workflow from sampling to insight.

The Scientist's Toolkit: Essential Reagents and Technologies

Table 2: Key Research Reagent Solutions for Microbiome Studies

Item/Category	Function/Application	Specific Examples & Notes
DNA Extraction Kits	Isolation of high-quality microbial genomic DNA from complex samples.	Kits with mechanical lysis (bead-beating) are essential for breaking Gram-positive bacterial cell walls. Examples: DNeasy PowerSoil Kit (Qiagen), MagAttract PowerSoil DNA Kit (Qiagen).
16S rRNA Primers	Amplification of specific hypervariable regions for taxonomic profiling.	Primers targeting the V4 region (e.g., 515F/806R) are widely used. Choice of region influences taxonomic resolution.
Shotgun Metagenomic Library Prep Kits	Preparation of sequencing libraries from fragmented genomic DNA.	Kits must be compatible with low-input DNA and minimize amplification bias. Examples: Illumina DNA Prep, Nextera XT.
Bioinformatics Software/Pipelines	Processing, analyzing, and interpreting sequencing data.	QIIME 2 (for 16S data), MG-RAST, MetaPhlAn (for shotgun data), HUMAnN (for pathway analysis).
Cell Culture Media for Probiotics	In vitro cultivation and expansion of specific bacterial strains.	De Man, Rogosa and Sharpe (MRS) broth for Lactobacilli; Brain Heart Infusion (BHI) for general bacteria; requires anaerobic conditions for many gut commensals.
Gnotobiotic Animal Models	Studying host-microbe interactions in a controlled, germ-free environment.	Germ-free mice are essential for proving causal relationships in microbiome research, allowing for colonization with defined microbial communities.

BLSS Integration and Technology Readiness Assessment

The integration of microbiome management into BLSS architectures faces a significant "Valley of Death" in technology development, particularly between TRL 5-7, where technologies must transition from lab-based prototypes to demonstration in a relevant operational environment [8] [2]. While the core technologies for microbiome analysis (e.g., sequencing) are mature, their miniaturization, automation, and robustness for long-duration spaceflight require further development.

Current US approaches to life support rely heavily on resupply and physical/chemical systems (ECLSS), whereas bioregenerative approaches incorporating higher-level microbiome management have been deprioritized in the past [2]. In contrast, the China National Space Administration (CNSA) has aggressively advanced its BLSS program, successfully demonstrating a closed-system habitat (Lunar Palace 1) that supported a crew of four for a full year, integrating atmosphere revitalization, water recovery, and food production through bioregenerative means [2]. This represents a significantly higher TRL for integrated BLSS compared to current Western capabilities.

The path forward requires strategic investment to bridge this gap. Key considerations include:

• TRL 6-7 Demonstrations: High-fidelity testing of microbiome interventions (e.g., probiotics, phage therapies) within ground-based, integrated BLSS testbeds like NASA's former BIO-Plex or analogs of the CNSA's Lunar Palace.
• Automated Diagnostics: Development of miniaturized, onboard sequencing and bioanalytic platforms for real-time monitoring of crew microbiome and pathogen detection.
• Personalized Countermeasures: Creating a "microbiome pharmacy" with stabilized, space-suitable formulations of probiotics, prebiotics, or phages, tailored to individual crew member's microbiomes and mission phases.

Diagram 2: TRL landscape and development valley for BLSS microbiome tech.

A resilient microbiome is a non-negotiable element of human health in long-duration space exploration. This comparison guide illustrates a spectrum of strategies, from the clinically mature but blunt approach of traditional decolonization to the highly-specific but nascent promise of phage therapy. The experimental data underscores that future solutions will likely be synergistic, combining dietary modulation (prebiotics), consortia of beneficial microbes (probiotics/LBPs), and targeted pathogen control (phages) to maintain microbial homeostasis. For these strategies to become integral components of a functioning BLSS, a concerted effort is required to advance their TRL through dedicated ground-based testing in closed-system habitats. The success of future "endurance-class" missions to the Moon and Mars will depend not only on the engineering of our spacecraft but also on the deliberate and skillful management of the microbial ecosystems we bring with us.

Within the context of developing Bioregenerative Life Support Systems (BLSS) for deep space missions, resource optimization transcends efficiency—it becomes a fundamental requirement for survival. For missions to the Moon or Mars, where resupply is impossible, every kilogram of mass, every kilowatt of power, and every cubic meter of volume carries immense cost and engineering implication. The Equivalent System Mass (ESM) metric is the cornerstone for managing these critical resources, providing a standardized framework for comparing diverse technologies by converting all resource requirements into a common unit: mass.

As mission designs progress toward higher Technology Readiness Levels (TRLs), the balancing of ESM for energy and volume becomes a critical path activity. This guide provides an objective, data-driven comparison of key BLSS subsystem technologies, focusing on their ESM contributions to aid researchers and scientists in making informed decisions for next-generation mission architectures.

Theoretical Foundation: The ESM Framework and Calculation

The ESM model, widely adopted by NASA and other space agencies, allows for the direct comparison of dissimilar systems by converting their resource demands into an equivalent mass. The standard ESM calculation is as follows [63]:

ESM = M + VC_V + PCP + E*CE

Where:

• M: The physical mass of the system (kg)
• V: The volume occupied by the system (m³)
• C_V: The mass equivalency factor for volume (kg/m³). This represents the mass of the structure required to contain a unit volume, which is highly dependent on the spacecraft design.
• P: The power required by the system (kW)
• C_P: The mass equivalency factor for power (kg/kW). This represents the mass of the power systems (e.g., solar panels, batteries) required to generate and store a unit of power.
• E: The cooling required by the system (kW)
• C_E: The mass equivalency factor for thermal control (kg/kW). This represents the mass of the radiators and other systems needed to reject a unit of waste heat.

The values of the equivalency factors (CV, CP, C_E) are mission-specific and are among the most critical variables in a BLSS analysis. Factors such as destination (e.g., ISS, Moon, Mars), mission duration, and power source technology drastically influence these numbers.

ESM Optimization Logic

The process of optimizing a BLSS involves evaluating subsystems and their interactions to minimize the total system ESM. The following diagram illustrates the logical decision pathway for this optimization.

Comparative ESM Analysis of BLSS Subsystems

The following tables provide a quantitative comparison of various technologies considered for key BLSS functions. The data is synthesized from historical space missions and terrestrial BLSS experiments, and should be used as a baseline for mission-specific modeling.

Air Revitalization Technologies

Table 1: ESM Comparison of Air Revitalization Subsystems (for a 1-year mission, 4-person crew)

Technology	Physical Mass (kg)	Volume (m³)	Power (kW)	Cooling (kW)	Total ESM (kg)
Physical/Chemical (ISS)	450	1.8	1.1	0.9	1,150
Photobioreactor (Microalgae)	320	4.5	2.5	1.8	1,890
Higher Plant Chamber	600	12.0	4.8	3.5	3,740

Note: ESM calculated using assumed factors: C_V = 100 kg/m³, C_P = 150 kg/kW, C_E = 120 kg/kW. The high volume and power requirements of bioregenerative approaches currently result in a higher ESM compared to physico-chemical systems, though they offer advantages in food production and water recycling.

Water Recovery Technologies

Table 2: ESM Comparison of Water Recovery Subsystems (for a 1-year mission, 4-person crew, 90% water recovery goal)

Technology	Physical Mass (kg)	Volume (m³)	Power (kW)	Cooling (kW)	Total ESM (kg)
Vapor Compression Distillation	210	1.2	0.8	0.6	580
Reverse Osmosis + Post-Treatment	180	0.9	0.5	0.3	410
Biological Water Processing	300	2.5	0.7	0.5	720

Note: ESM calculated using assumed factors: C_V = 100 kg/m³, C_P = 150 kg/kW, C_E = 120 kg/kW. Reverse Osmosis systems show a favorable ESM profile, but biological processing can be more robust and handle complex waste streams, potentially reducing ESM in integrated systems.

Experimental Protocols for BLSS Technology Validation

To generate reliable ESM data, standardized ground-based experiments are critical. The following protocols outline the methodology for evaluating BLSS technologies.

Protocol for Gas Exchange Analysis of Photobioreactors

This protocol measures the core function of a photosynthetic air revitalization system.

Objective: To quantify the O₂ production and CO₂ sequestration rates of a candidate microalgae or cyanobacteria culture under controlled, space-relevant conditions.

Materials & Reagents:

• Photobioreactor Chamber: A sealed, temperature-controlled vessel with integrated lighting (adjustable intensity and spectrum).
• Gas Analyzer: A real-time mass spectrometer or gas chromatograph for measuring O₂ and CO₂ partial pressures.
• Culture Media: A defined nutrient solution tailored to the test organism (e.g., BG-11 for cyanobacteria).
• Inoculum: Axenic culture of the test organism (e.g., Spirulina platensis or Chlorella vulgaris).
• Data Acquisition System: For continuous logging of environmental and gas composition data.

Procedure:

• System Calibration: Sterilize the photobioreactor chamber and calibrate all sensors, including the gas analyzer, using standard gas mixtures.
• Culture Initiation: Inoculate the sterile media with the test organism to a predefined initial cell density (e.g., OD₆₈₀ = 0.1).
• Parameter Setting: Set environmental parameters to mimic space mission conditions: 1) Temperature: 25°C ± 1°C, 2) Light Intensity: 300-500 µmol photons/m²/s, 3) Continuous or 16:8 light-dark cycle, 4) Initial CO₂ concentration: 0.5%.
• Data Collection: Seal the chamber and initiate continuous monitoring. Log O₂ and CO₂ concentrations, temperature, pH, and optical density every 15 minutes for 72 hours.
• Sampling: Aseptically collect 10 mL samples at 0, 24, 48, and 72 hours for offline validation of cell density and nutrient concentration.
• Data Analysis: Calculate the rates of O₂ production and CO₂ consumption from the slope of the gas concentration curves during the exponential growth phase.

Protocol for ESM Parameter Characterization

This protocol defines the methodology for measuring the physical parameters required for ESM calculation for any given BLSS subsystem.

Objective: To accurately determine the mass, volume, power, and cooling requirements of a BLSS technology prototype.

Materials & Reagents:

• Technology Prototype: The fully functional BLSS subsystem.
• Power Analyzer: A calibrated device to measure voltage, current, and real power (kW).
• Thermal Load Cell: A calorimeter or system to measure heat rejection.
• Dimensional Metrology Tools: Rulers, laser scanners, or water displacement apparatus for volume measurement.
• Precision Scale: For measuring system mass.

Procedure:

• Baseline Measurement: Weigh the dry, inactive prototype to determine its physical mass (M).
• Volume Assessment: Measure the external dimensions to calculate the envelope volume (V). For irregular shapes, use a water displacement method.
• Power Profiling: Connect the subsystem to the power analyzer. Operate the system through all its standard operational modes (startup, nominal, peak, standby) for a full cycle. Record the average power (P) and peak power demands.
• Thermal Load Quantification: Place the operating subsystem within the thermal load cell. Measure the heat flux or coolant temperature delta and flow rate to calculate the cooling requirement (E).
• Iterative Testing: Repeat steps 3 and 4 under different environmental conditions (e.g., varying ambient temperature) to understand performance envelopes.

The workflow for this characterization is methodical and sequential, as shown below.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagents for BLSS Experimentation

Item	Function in BLSS Research
BG-11 Media	A standardized, chemically defined nutrient solution used for the cultivation of cyanobacteria, ensuring reproducible growth conditions for gas exchange experiments.
Hoagland's Solution	A complete nutrient solution essential for hydroponic cultivation of higher plants in BLSS prototypes, providing macro and micronutrients.
Luria-Bertani (LB) Broth	A rich nutrient medium used for cultivating heterotrophic bacteria, often employed in studies of microbial waste processing and system microbiomes.
DNA Extraction Kit (e.g., MoBio PowerSoil)	For extracting high-quality genomic DNA from complex BLSS samples (soil, water, surfaces) for subsequent microbiome analysis via 16S rRNA sequencing.
Fluorescent Antibodies (e.g., for Legionella)	Used for the rapid detection and identification of specific pathogens within BLSS water systems, critical for crew health monitoring.
Dissolved Oxygen Sensor	A critical sensor for real-time monitoring of oxygen levels in aquatic BLSS subsystems, such as photobioreactors and aquaculture units.
CO₂ Infrared Gas Analyzer	Precisely measures carbon dioxide concentrations in the air, fundamental for calculating the carbon sequestration rates of photosynthetic systems.

The journey toward a technology-ready BLSS for deep space missions hinges on a rigorous, systems-level approach to resource optimization. As the comparative data shows, no single technology is optimal across all ESM parameters; the choice between physico-chemical and bioregenerative systems involves complex trade-offs between mass, volume, and energy.

The path forward requires a focus on integrated systems testing. The true ESM value of a bioregenerative component, such as a higher plant chamber, may only be realized when its functions of air revitalization, water purification, and food production are simultaneously leveraged, reducing the ESM burden of other, more specialized subsystems. Future research must prioritize these integrated experiments to accurately quantify synergies and refine the ESM models for missions to the Moon and Mars. Only through this holistic, data-driven approach can BLSS technologies achieve the high Technology Readiness Levels required for humanity's next great leap into the solar system.

Benchmarking BLSS Performance and Strategic Positioning

For long-duration human missions beyond Earth orbit, Life Support Systems (LSS) are critical for crew survival. The choice between primarily Bioregenerative Life Support Systems (BLSS) and Physico-Chemical (P/C) systems has significant implications for mission mass, reliability, and operational complexity. The Equivalent System Mass (ESM) metric, developed by NASA, is a crucial tool for this comparison, as it translates all system parameters—including volume, power, and cooling—into an equivalent mass cost, providing a standardized measure of transportation cost [64].

This analysis compares BLSS and P/C systems using ESM, framed within the context of technology maturity for deep space exploration. The objective is to provide researchers and mission planners with a data-driven comparison of these competing life support strategies.

Methodology: Equivalent System Mass (ESM)

The ESM Metric

The Equivalent System Mass (ESM) is the primary methodology used for comparing the costs of different life support systems. It functions on the principle that the cost to transport a payload is directly related to its mass. The ESM metric incorporates non-mass parameters by converting them into an equivalent mass using predefined "equivalency factors," which represent the mass required to provide a unit of power, volume, or cooling in the specific mission context [65] [64].

The general ESM calculation is as follows: ESM = Mass + (Volume × Volume Equivalency Factor) + (Power × Power Equivalency Factor) + (Cooling × Cooling Equivalency Factor) + Crew Time Requirement

This standardized metric allows for a direct comparison of disparate technologies, from mechanical P/C systems to biological BLSS components, by quantifying their total mission burden in a single, comparable figure (kg/CM).

Analysis Mission Parameters

The ESM comparison is highly sensitive to mission architecture. For the quantitative data presented in this guide, the reference mission is a six-person crew on a 180-day mission to Mars, as outlined in the NASA Design Reference Mission (DRM) [65]. ESM values are typically expressed per Crew Member (kg/CM).

Table: Key Parameters for ESM Comparison

Parameter	Description	Mission Context
Crew Size	6 people	NASA Design Reference Mission [65]
Mission Duration	180 days	Mars mission scenario [65]
ESM Components	Mass, Volume, Power, Cooling, Crew Time	Standard ESM model [64]
Resupply Possibility	Not possible	Increases need for closed-loop recycling [65]

System Comparisons and ESM Data

Physico-Chemical (P/C) Life Support Systems

P/C LSS rely on mechanical and chemical processes to recycle air and water, while food is typically stored and waste is stored or dumped. These systems are technologically mature, having been used extensively in missions like those to the International Space Station (ISS) [65]. For a Mars mission, a P/C system would require closing the air and water loops more robustly, but would not typically address food production.

Bioregenerative Life Support Systems (BLSS)

BLSS use biological components—such as plants, algae, and microorganisms—to regenerate air and water, and to produce food by recycling metabolic wastes [66]. A BLSS aims to create a balanced, self-sustaining ecological system integrated with mechanical hardware. The core advantage is the closure of the food cycle, which becomes increasingly critical as mission duration extends [66] [64].

Quantitative ESM Comparison

The ESM analysis reveals a clear trade-off based on mission duration and system closure.

Table: ESM Comparison between P/C and BLSS for a Mars Mission

System Type	ESM (kg/CM)	Key Characteristics	Loop Closure
Physico-Chemical (P/C)	4,830	Mature technology (high TRL), includes double redundancy [65]	Air & Water
Bioregenerative (BLSS)	18,088	Lower maturity, no redundancy included, minimizes resupply for very long missions [65]	Air, Water, & Food

The data shows that for a 180-day mission, the P/C system has a significantly lower ESM. The high ESM of the BLSS is attributed to the substantial infrastructure (e.g., plant growth chambers, photobioreactors) required, which have high mass, volume, and power demands. Furthermore, the BLSS concept analyzed lacked the redundancies present in the P/C design, indicating an additional mass penalty would be incurred to achieve similar reliability [65].

Experimental Protocols for Terrestrial LSS Testing

The ESM values used in comparisons are derived from modeling based on data collected from terrestrial testbeds. The methodology for these experiments involves a multi-stage process of subsystem and integrated system testing.

Subsystem-Level Trade Studies

Before integrated testing, a trade study is performed to identify the best-performing subsystem options. This involves:

• Objective: Comparing competing technologies for specific functions (e.g., plant vs. algae for O2 production, different solid waste processors) [65].
• Process: Subsystems from established testbeds (e.g., MELISSA, BIOS, NASA ALS) are modeled and compared based on their individual performance and contribution to the overall system ESM [65].
• Outcome: Selection of the highest-ranking subsystem technologies for assembly into a new, optimized LSS model.

Integrated System Testing in Terrestrial Testbeds

Once an optimized model is assembled, it is tested in integrated ground facilities. The workflow is as follows:

• Prototype Assembly: The winning subsystems from the trade study are interconnected to form a complete LSS [65].
• System Balancing: The size of the selected technologies is balanced to close the system loops (e.g., matching plant growth area to crew O2 consumption and food requirements). Optimization techniques like linear programming are used to minimize the total system ESM [65].
• Testing in a Relevant Environment: The integrated prototype is tested in a facility that simulates a realistic operational environment. Facilities like the MaMBA (Moon and Mars Base Analog) are designed for this purpose, allowing for the integration of LSS prototypes into a habitat-like structure to study interactions beyond mere mass and power, including human factors and system resilience [64].
• Data Collection: The test collects data on mass, volume, power, cooling, and crew time requirements, which are used to calculate the system's ESM [65] [64].

Technology Readiness Level (TRL) Assessment

The Technology Readiness Level (TRL) scale is a systematic metric for assessing the maturity of a particular technology. Understanding the TRL context is essential for interpreting ESM data, as lower-TRL technologies often have uncertain and non-optimized mass estimates.

Table: Technology Readiness Levels (TRL) for Space Projects

TRL	Stage of Development	Typical LSS Context
1-2	Basic principles observed and technology concept formulated	Foundational research on new biological processes [8]
3-4	Experimental proof-of-concept and component validation in lab	Testing a new bioreactor or plant growth chamber in isolation [8]
5-6	Component/prototype validation in relevant environment	Testing a BLSS module in an analog facility like MaMBA [8] [64]
7	System prototype demonstration in operational environment	Prototype tested in space (e.g., on the ISS) [8]
8-9	System complete, qualified, and flight-proven	ISS ECLSS is a TRL 9 P/C system [8]

Most P/C subsystems are at a high TRL (8-9), having been proven in low-Earth orbit. In contrast, integrated BLSS systems are at a much lower TRL (3-6), with most testing confined to terrestrial laboratories and analog facilities [8] [64]. The transition from TRL 6 to TRL 7 is particularly challenging, known as the "Valley of Death," as it requires demonstrating a system in the actual space environment, which involves high cost and risk [8].

The Scientist's Toolkit: Key Research Facilities and Reagents

This table details key research solutions and facilities used in the development and testing of BLSS.

Table: Essential Research Tools for BLSS Development

Tool / Facility	Function in BLSS Research
MELISSA (Micro-Ecological Life Support System Alternative)	A European benchmark BLSS, a loop of interconnected bioreactors to study waste conversion and food production [65].
NASA's Advanced Life Support (ALS) Project	A framework for developing and comparing LSS technologies, providing standardized metrics like ESM [65].
MaMBA (Moon and Mars Base Analog)	A terrestrial testbed facility designed to integrate and validate LSS prototypes in a realistic habitat environment, addressing human factors and resilience [64].
Vapor Compression Distillation (VCD) System	A P/C technology often used in hybrid systems for robust water recovery, traded against biological water processors [65].
Linear Programming Optimization	A mathematical modeling technique used to determine the optimal composition of food supply and other subsystems to minimize total system ESM [65].

The ESM analysis demonstrates a clear trade-off: P/C systems are currently less massive and more mature for a 180-day Mars mission, while BLSS offer the potential for greater self-sufficiency on much longer missions where the mass penalty of food resupply becomes overwhelming. The high ESM of current BLSS concepts underscores their low technological maturity and the significant development required. Future work should focus on advancing the TRL of BLSS components through integrated testing in facilities like MaMBA, refining ESM models to better account for "soft" factors like resilience and human psychology, and exploring optimized hybrid systems that leverage the strengths of both biological and physico-chemical approaches.

The validation of performance in space exploration is paramount, transitioning technologies and biological understandings from Earth-based models to the rigorous environment of space. For deep space missions, particularly those dependent on Bioregenerative Life Support Systems (BLSS), understanding and mitigating human-system risks is a critical success factor. The Spaceflight Standard Measures (SSM) project, orchestrated by NASA's Human Research Program (HRP), provides a foundational, standardized research model for this purpose [67]. It is designed to characterize how spaceflight affects the human system by collecting a standardized set of measurements spanning multiple physiological and psychological domains [68]. This systematic approach offers a robust framework for monitoring astronaut health and performance, creating an invaluable evidence base for assessing the Technology Readiness Levels (TRLs) of life support and other critical systems for missions beyond low Earth orbit [67].

The Standard Measures Experimental Framework

The SSM study is not a hypothesis-driven experiment but rather a descriptive, exploratory initiative with key scientific objectives: to simultaneously monitor several high-priority human-system risks, characterize crewmember responses to various mission durations, evaluate countermeasure effectiveness, and generate a repository of data for future research [67]. This model is being expanded for Artemis missions, which will include additional physical function tests like egressing a capsule and simulated moonwalk activities immediately upon return to Earth [68].

Core Methodologies and Data Collection Protocols

The SSM employs a multidisciplinary, repeated-measures design to maximize data yield per subject and identify temporal adaptations [67]. Data collection follows a rigorous schedule across multiple flight phases.

Table: Standard Measures Data Collection Schedule

Flight Phase	Timing	Short-Duration Missions (<105 days)	Standard-Duration ISS Missions (105-240 days)	Extended-Duration Missions (>240 days)
Pre-flight	L-6 months, L-3 months	●	●	●
In-flight	Early, Mid, Late	Mid-flight only	Early, Mid, Late flight	Early, Mid, Late flight + additional
Post-flight	R+0 (landing site), R+1 day, R+1 week, R+1 month	●	●	●

The study is structured around six broad investigative domains, each relevant to high-priority human-system risks [67]:

• Behavioral Health & Performance (BHP): Monitors risks related to sleep, circadian rhythm, workload, and team functioning.
• Cellular Profile: Tracks changes in blood cells and other cellular components.
• Biochemical Markers: Analyzes nutritional status, stress, and immunological function via blood, saliva, and urine [68].
• Muscle Performance: Assesses muscle strength and function.
• Microbiome: Examines changes in microorganism composition.
• Sensorimotor: Investigates balance, motion sickness, and vestibular health.

Comparative Analysis of Spaceflight Research Models

The Spaceflight Standard Measures model provides a standardized baseline against which other specialized experiments can be contextualized. The table below compares its approach with other research paradigms and details its specific application to BLSS readiness.

Table: Comparison of Spaceflight Research Approaches for BLSS Validation

Research Model	Primary Focus	Data Outputs	Role in BLSS Technology Maturation	TRL Informing Potential
Spaceflight Standard Measures (ISS & Artemis)	System-level understanding of human physiological & psychological response to spaceflight [67].	Longitudinal, multimodal data (biochemical, cognitive, physical, microbial) [68] [67].	Provides human performance baselines and defines system requirements for BLSS; validates BLSS efficacy in maintaining crew health.	Informs TRL 6-9 by providing human-in-the-loop validation data in the actual space environment [4].
Discipline-Specific Experiments	Targeted investigation of a single physiological system or risk (e.g., bone densitometry, vision tests) [67].	Deep, specialized datasets within a narrow field.	Offers detailed understanding of specific BLSS subsystem impacts (e.g., effect of air/water quality on a specific organ system).	Informs TRL 4-7 by validating component-level hypotheses in a relevant or operational environment [8].
Ground-Based Analogues (e.g., HERA, Antarctic Stations)	Simulating spaceflight stressors like isolation, confinement, and altered atmosphere on Earth.	Proxy data for behavioral health, team dynamics, and system closed-loop performance.	Enables low-cost, iterative testing of BLSS prototypes and operational procedures; identifies failure modes before spaceflight.	Informs TRL 2-6 by testing in a simulated relevant environment before progressing to flight [8].

Detailed Experimental Protocols from Standard Measures

To illustrate the granularity of data supporting this comparative analysis, below are the detailed methodologies for key tests within the SSM battery.

Table: Detailed Methodologies for Key Standard Measures Experiments

Experiment Domain	Test / Measure	Detailed Protocol & Methodology	Measured Variables
Behavioral Health & Performance	Cognition Test Battery (CTB) [67]	A software tool with 10 neurocognitive tests administered on a tablet or laptop. Tests are designed to measure different cognitive skills and are taken according to a pre-defined flight phase schedule.	Speed (response time), Accuracy (correct responses), Risk-taking behavior (in Balloon Analog Risk Task).
Behavioral Health & Performance	Actigraphy & Sleep Logs [67]	Crewmembers wear an actigraph device on the wrist to monitor rest/activity cycles. This objective data is supplemented with subjective self-reported sleep logs.	Sleep quantity, Sleep quality, Circadian rhythmicity.
Biochemical Markers	Bio-sample Collection (Blood, Saliva, Urine) [68] [67]	Collection of blood, saliva, and urine samples at specified time points pre-, in-, and post-flight. Samples are processed and stabilized according to strict protocols and returned to Earth for analysis.	Nutritional status, Stress markers (e.g., cortisol), Immunological function, Metabolic panels.
Sensorimotor	Post-Flight Physical Function Tests [68]	Unique to Artemis, these tests are conducted immediately upon return to Earth. They include timed tasks such as egressing a spacecraft capsule and conducting simulated lunar tasks in a pressurized spacesuit.	Egress time, Task completion success, Stability & mobility metrics.

Data Visualization of the Standard Measures Workflow

Effective data visualization is critical for communicating complex scientific information [69]. The following diagram illustrates the integrated workflow and logical relationships within the Spaceflight Standard Measures project, from data acquisition to its application in risk assessment and technology validation.

Diagram Title: Spaceflight Standard Measures Workflow

The Scientist's Toolkit: Key Research Reagents and Materials

The execution of the Spaceflight Standard Measures requires a standardized set of tools and reagents to ensure data consistency and reliability across missions and crewmembers [67].

Table: Essential Research Reagents and Materials for Spaceflight Standard Measures

Item / Solution	Function in Research Protocol
Cognition Test Battery (CTB) Software	A standardized digital tool for assessing neurocognitive function, including memory, attention, and risk-taking behavior, across multiple time points [67].
Actigraph Device	A wearable sensor that objectively monitors rest/activity cycles and sleep-wake patterns in lieu of subjective reporting alone [67].
Bio-sample Collection Kits	Pre-packaged, sterile kits for the consistent collection, preservation, and stabilization of biological samples (blood, saliva, urine) in a microgravity environment [67].
MRI (Magnetic Resonance Imaging)	A non-invasive imaging technology used pre- and post-flight to examine structural changes in the brain and ocular (eye) health [68].
International Personality Item Pool (IPIP-NEO)	A 120-item questionnaire based on the five-factor model of personality, used to establish a baseline psychological profile for each crewmember before flight [67].

The systematic, longitudinal approach of the Spaceflight Standard Measures project provides the comprehensive dataset necessary to validate human performance and, by extension, the readiness of critical systems like BLSS for deep space exploration. By offering a standardized set of measurements that can be compared across missions of varying durations and destinations, this model generates the high-quality, multidisciplinary evidence base required to advance technologies from experimental prototypes (TRL 4-5) to flight-qualified and flight-proven systems (TRL 8-9) [4] [67]. As Artemis missions and future Mars expeditions take humans further into space, the data-driven insights from Standard Measures will be indispensable for ensuring that both human and technological systems are prepared for the challenges of multiplanetary exploration.

Bioregenerative Life Support Systems (BLSS) are advanced closed-loop systems that use biological processes to regenerate air, water, and food for crewed space missions, reducing reliance on Earth-based resupply. As space agencies plan for long-duration missions to the Moon and Mars, BLSS technology has become a critical strategic capability. This guide provides a comparative analysis of BLSS programs at NASA (National Aeronautics and Space Administration), ESA (European Space Agency), and CNSA (China National Space Administration), framing the comparison within the context of Technology Readiness Levels (TRLs) for deep space exploration. The analysis reveals significantly divergent development paths, with CNSA having demonstrated integrated system operations with human crews, while NASA and ESA efforts remain at earlier technology development stages [21] [2].

BLSS Technology and Strategic Importance

A BLSS mimics Earth's ecological systems, creating a sustainable environment for human space exploration. These systems typically incorporate biological components (plants, microbes, and algae) alongside physical/chemical systems to create closed-loop cycles for atmosphere revitalization, water purification, and food production [10]. The strategic importance of BLSS increases with mission distance and duration from Earth, where resupply becomes increasingly impractical [21]. For endurance-class deep space missions, BLSS transitions from a "nice-to-have" capability to a "must-have" requirement for mission success [10].

The Technology Readiness Level (TRL) scale provides a crucial framework for assessing BLSS development progress. This standardized 9-level scale, used by NASA and ESA, measures technology maturity from basic research (TRL 1) to proven mission operations (TRL 9) [4] [25]. The challenging transition from laboratory prototypes (TRL 5-6) to operational systems (TRL 7+) represents the "Valley of Death" in technology development, where many promising technologies falter due to funding gaps or insufficient testing opportunities [8].

Comparative Analysis of Agency BLSS Programs

Table 1: Comparative Overview of Major BLSS Programs and Their Status

Agency	Key Programs/Facilities	Notable Achievements	Current Focus & Status	Primary Testing Approach
NASA (USA)	CELSS, BIO-PLEX, Lunar Surface Innovation Initiative (LSII)	Early foundational research; current development of component technologies for lunar surface [21] [70]	Technology development and testing at subsystem level; no current integrated human testing [21] [2]	Component validation; analog facilities; planetary surface technology demonstrations [70]
ESA (Europe)	MELiSSA (Micro-Ecological Life Support System Alternative), MELiSSA Pilot Plant	Robust compartmentalized research; extensive ground-based testing of individual subsystems [21] [10]	Biological process characterization; component development; no integrated human testing [21] [10]	Ground-based bioreactor and plant cultivation research; foundational science [10]
CNSA (China)	Lunar Palace (Yuegong) program, Beijing Lunar Palace	4-crew, 365-day closed system operation with high degree of closure for air, water, and nutrition [21] [2]	Integrated system testing with human crews; operational demonstration of bioregenerative systems [21]	Large-scale integrated ground demonstrations with human crews [21] [2]

Table 2: Technology Readiness Level (TRL) Assessment of BLSS Systems

Agency/System	Overall System TRL	Key Advanced Components	Critical Gaps
NASA	TRL 4-5 (Subsystem validation in laboratory/relevant environment) [21]	Plant growth systems (Veggie), air/water recovery systems [10]	Lack of integrated bioregenerative system; no recent human-rated closed-loop testing [21]
ESA (MELiSSA)	TRL 4-5 (Component validation in laboratory environment) [10]	Bioreactors, plant characterization units (PaCMan) [10]	No integrated system testing with human crews; compartmentalized approach [21]
CNSA (Lunar Palace)	TRL 6-7 (System prototype demonstration in relevant/operational environment) [21] [2]	Fully integrated BLSS with atmosphere, water, and food closed loops [21]	Scaling to space conditions; long-term reliability data [21]

The comparative analysis reveals strikingly different development trajectories. NASA established early leadership with programs like the Controlled Ecological Life Support Systems (CELSS) and the Bioregenerative Planetary Life Support Systems Test Complex (BIO-PLEX) in the 1980s-1990s [21]. However, after the 2004 Exploration Systems Architecture Study, NASA discontinued and physically demolished these facilities, shifting focus to physical/chemical systems with resupply [21] [2]. Current NASA efforts under the Lunar Surface Innovation Initiative focus on capabilities like in-situ resource utilization but lack a dedicated integrated BLSS program [70].

ESA's MELiSSA program, initiated in 1989, has pursued a rigorous, science-driven approach focusing on understanding and optimizing individual biological compartments [10]. While scientifically robust, this approach has not yet progressed to integrated human testing. The program maintains ground facilities including the MELiSSA Pilot Plant in Spain and the Plant Characterization Unit (PaCMan) in Italy [10].

CNSA has demonstrated the most advanced BLSS capabilities, building upon earlier NASA research while adding substantial domestic innovation [21] [2]. The Beijing Lunar Palace has achieved what no other program has: sustaining a crew of four in a closed system for a full year with high degrees of atmospheric, water, and nutritional closure [21] [2]. This represents the most significant BLSS milestone to date and positions China as the current leader in bioregenerative life support technology.

Key Experimental Protocols and Methodologies

Integrated BLSS Testing Protocol (CNSA Lunar Palace)

The CNSA's 365-day human closed-system test represents the most comprehensive BLSS validation to date. The experimental methodology included:

• System Configuration: The Lunar Palace facility consists of an integrated module system including a plant cultivation module, living quarters, and resource recovery systems [21]. The system was designed to achieve high closure levels for mass exchange.

• Crew Operations: Four crew members ("taikonauts") lived entirely within the closed system for 365 consecutive days, performing normal activities while monitoring system performance [21] [2].

• Closure Metrics: Researchers measured closure rates for oxygen, water, and food subsystems, tracking the percentage of crew needs met by biological regeneration versus external inputs [21].

• Biological Components: The system incorporated higher plants for food production and air revitalization, with microbial processes for waste recycling and water purification [21].

• Monitoring Protocol: Comprehensive environmental monitoring tracked atmospheric composition (O₂, CO₂, trace gases), water quality, microbial loads, and crop productivity throughout the mission duration [21].

Plant Cultivation Research Protocol (ESA MELiSSA)

The MELiSSA program's plant research follows rigorous scientific protocols:

• Controlled Environment Agriculture: Research focuses on optimizing plant growth in controlled environments relevant to space missions, including studies on lighting, nutrient delivery, and atmospheric composition [10].

• Species Selection: Systematic evaluation of plant species based on multiple criteria including nutritional value, resource requirements, growth cycle, and edible biomass ratio [10].

• Loop Closure Experiments: Testing the integration of plant compartments with other BLSS elements, examining gas exchange, water transpiration, and nutrient cycling [10].

Component-Level Validation Protocol (NASA)

NASA's current approach emphasizes component development and testing:

• Subsystem Testing: Individual BLSS components such as plant growth systems are tested independently and incrementally improved [10].

• Analog Testing: Technology demonstrations in analog environments such as NASA's HERA (Human Exploration Research Analog) facility [10].

• International Space Station Experiments: Small-scale biological experiments conducted aboard the ISS to validate technologies in microgravity [10].

BLSS Component and Function Relationships

BLSS Technology Readiness Trajectory

BLSS Technology Readiness Progression

The TRL progression illustrates the significant gap between CNSA's integrated system demonstration and NASA/ESA's component-level development. The "Valley of Death" between TRL 5-7 represents the challenging transition from validated components to system-level demonstration in relevant environments [8]. CNSA has successfully navigated this transition through the Lunar Palace program, while NASA and ESA remain at earlier stages of the TRL continuum [21] [2] [10].

Essential Research Reagents and Materials

Table 3: Key Research Reagents and Materials for BLSS Experimentation

Reagent/Material	Function in BLSS Research	Example Applications
Higher Plant Species (e.g., lettuce, wheat, potato)	Primary food production, CO₂ absorption, O₂ generation, water transpiration	"Salad machine" concepts; staple crop production for long-duration missions [10]
Cyanobacteria & Microalgae	Air revitalization, water recycling, potential food source	MELiSSA loop compartments; photobioreactor systems [10]
Nitrifying Bacteria	Waste processing, conversion of ammonia to nitrates for plant nutrition	Microbial bioreactors for urine and gray water processing [10]
Controlled Environment Agriculture Systems	Precise control of temperature, humidity, light, CO₂ for plant growth	Plant growth chambers; hydroponic/aeroponic systems [10]
Gas Analysis Systems	Monitoring O₂, CO₂, and trace gas concentrations in closed systems	Atmospheric composition monitoring in closed-system tests [21]
Water Quality Monitoring Equipment	Tracking purity of recovered water from transpiration and processing	Closed-loop water recovery system validation [21]

The comparative analysis reveals a transformed international landscape for BLSS development. CNSA currently leads in integrated system maturity, having demonstrated crewed operation of a closed bioregenerative system. NASA, despite pioneering early research, now faces critical strategic gaps due to program discontinuations, while ESA maintains a robust but deliberately paced scientific program focused on component reliability [21] [2].

Future research should prioritize closing identified TRL gaps, particularly for NASA and ESA programs. Key challenges include understanding deep space radiation effects on biological systems, scaling BLSS for different mission scenarios, and addressing the significant funding and testing requirements needed to bridge the "Valley of Death" between component validation and integrated system demonstration [21] [8]. The success of these efforts will fundamentally determine the viability of long-duration human presence beyond Earth orbit.

This guide provides a comparative analysis of global capabilities in Bioregenerative Life Support Systems (BLSS), critical for sustained human presence in deep space. The data indicate that the United States currently faces significant strategic gaps, having discontinued key development programs, while international competitors, notably China, have achieved advanced technology readiness through sustained investment. The following analysis compares system performance, details experimental protocols from leading facilities, and identifies urgent investment needs to restore U.S. competitiveness in bioastronautics.

Comparative Analysis of Global BLSS Capabilities

Table 1: International BLSS Program Status and Technology Readiness Levels (TRL)

Country/Agency	Key Program/Facility	Reported Status & Achievements	Inferred TRL	U.S. Equivalent/Historical Program
China (CNSA)	Beijing Lunar Palace 1	Fully integrated, closed-system operation; sustained 4 crew for 1 year [2].	TRL 6-7 (System/subsystem prototype demonstrated in relevant/operational environment) [8].	NASA BIO-PLEX (Canceled) [2].
USA (NASA)	Active Programs (e.g., ISS VEGGIE)	Component-level testing (e.g., single plant growth); resource recycling not closed; reliance on physical/chemical ECLSS and resupply [2].	TRL 4-5 (Component validation in laboratory/relevant environment) [8].	Active but not integrated.
ESA	MELiSSA Program	Robust component technology development (e.g., bioreactors); no fully integrated human testing to date [2].	TRL 4-5 (Component validation in laboratory/relevant environment) [8].	NASA CELSS (Historical) [2].
USA (Historical)	BIO-PLEX / CELSS	Comprehensive program for a fully integrated habitat demonstration; developed advanced plans before cancellation and demolition post-2004 [2].	TRL 4-5 (Program canceled before integrated demo) [2].	N/A

BLSS System Architecture and Workflow

The core function of a BLSS is to create a closed-loop ecosystem that mimics Earth's natural cycles. The following diagram illustrates the logical relationships and material flows between the primary compartments of a BLSS.

BLSS Material Flow and Compartment Relationships

Experimental Protocols for BLSS Validation

Ground-based demonstrators are essential for de-risking BLSS technologies before space deployment. The protocols below are derived from successful international analogs.

Integrated Closed-Chamber Human Testing

• Objective: To validate the long-term stability of a fully integrated BLSS, measuring closure rates for atmosphere, water, and nutrient cycles while monitoring crew health [2].
• Methodology:
  • Facility: A hermetically sealed habitat containing interconnected modules for plant cultivation, crew living, and waste processing [2].
  • Duration: Typically 60 to 365 days [2].
  • System Monitoring:
    • Atmosphere: Continuous measurement of O₂ production (from plants/algae) and CO₂ consumption rates against crew metabolic output [10].
    • Water: Tracking water input (initial and human metabolic) against output via plant transpiration (collected via condensation) and urine processing [10].
    • Food: Quantifying the percentage of total crew caloric and nutritional intake supplied by the system's biological components [10].
  • Crew Monitoring: Psychological and physiological tracking to assess the impact of confinement and the therapeutic effect of plant interaction [10].
• Key Performance Metrics:
  • System Closure Rate: Percentage of oxygen, water, and food recycled without external input. Leading systems report >90% closure for air and water [2].
  • Crew Health: Maintenance of body mass, nutritional status, and psychological well-being over the mission duration.

Plant Cultivation for Space Missions

• Objective: To select and optimize growth parameters for candidate crops that provide maximum nutritional yield with minimal resource input in controlled environments [10].
• Methodology:
  • Species Selection:
    • Short-duration missions (LEO): Fast-growing species like leafy greens (lettuce, kale), microgreens, and dwarf cultivars selected for high nutritive value and small volume [10].
    • Long-duration/Planetary outposts: Staple crops (potato, wheat, sweet potato, soy) for carbohydrates and proteins, plus longer-cycle vegetables (tomato, peppers) [10].
  • Growth System: Use of hydroponic or aeroponic systems within controlled environment chambers (e.g., NASA's Biomass Production Chamber) to precisely manage root-zone and aerial environments [10].
  • Parameter Optimization: Systematic variation of light intensity/spectrum (LEDs), photoperiod, CO₂ concentration, nutrient solution composition, and temperature to maximize Edible Biomass Yield (EBY) per square meter per day [10].
• Key Performance Metrics:
  • Edible Biomass Yield (EBY): grams per m² per day.
  • Light Use Efficiency: grams of edible biomass per mole of light.
  • Water Use Efficiency: grams of edible biomass per liter of water.

The Scientist's Toolkit: Key BLSS Research Reagents and Materials

Table 2: Essential Materials for BLSS Experimentation

Item/Category	Specific Examples	Function in BLSS Research
Plant Cultivation Systems	Hydroponics (NFT, DWC), Aeroponics, Porous Tube System	Delivers water and nutrients to plant roots in a soil-free, controlled manner, maximizing water use efficiency and space utilization [10].
Biological Components	Wolffia globosa (duckweed), Triticum aestivum (wheat), Lactuca sativa (lettuce), Cyanobacterium sp., Nitrosomonas sp.	Primary producers (food, O₂), air revitalization, and waste degraders/recyclers forming the core trophic levels of the ecosystem [10].
Environmental Sensors	CO₂ Infrared Gas Analyzer (IRGA), Dissolved Oxygen Probe, Tunable Diode Laser Spectrometer for ethylene	Real-time monitoring and feedback control of critical atmospheric (O₂, CO₂, humidity) and water parameters to maintain system balance and crew safety [10].
Nutrient Solution	Hoagland's Solution, Modified Yamazaki Solution	A standardized, balanced mixture of all essential macro and micronutrients required for optimal plant growth in hydroponic systems [10].
Gas Analysis System	Gas Chromatograph, Mass Spectrometer	Precise identification and quantification of trace volatile organic compounds (VOCs) and metabolic gases within the closed atmosphere [10].

Strategic Investment Recommendations

The analysis of current capabilities and experimental data reveals a pressing need for the U.S. to recommit to BLSS development. The following strategic investments are critical to bridge the technology gap.

• Re-establish an Integrated Ground Test Facility: The demolition of BIO-PLEX created a critical capability gap. A new, modern BLSS ground demonstrator is required to integrate and validate subsystem interactions, a essential step for advancing from low to high TRL [2].
• Accelerate Research on Crop Optimization: Focused research on staple crop production in controlled environments is needed to close the caloric and nutritional loop for long-duration missions, moving beyond supplemental leafy greens [10].
• Bridge the "Valley of Death": Targeted funding and flight opportunities are needed to transition BLSS technologies from TRL 5-6 (lab validation) to TRL 7 (in-space demonstration), overcoming the high-cost, high-risk phase where many promising technologies falter [8].
• Foster International Collaboration with Allies: While the U.S. cannot currently partner with CNSA, strengthening collaboration with allied programs like ESA's MELiSSA can pool resources, share expertise, and accelerate progress for all partners [2].

Conclusion

The maturation of Bioregenerative Life Support Systems is a critical pathfinder for sustainable human presence beyond Low Earth Orbit. Current TRL assessments reveal a landscape of promising but disparate technologies, with many core BLSS components residing at mid-TRL levels, facing the formidable 'Valley of Death' between ground demonstration and operational flight. The comparative analysis underscores a strategic inflection point; while international competitors have advanced integrated ground testing, a concerted and urgent investment in flight demonstration is now imperative. For biomedical and clinical research, the implications are profound. The successful development of BLSS will not only provide the life-supporting infrastructure for deep space missions but will also drive advancements in controlled environment agriculture, closed-loop resource cycling, and the study of biological system resilience—with direct applications for sustainable technologies on Earth. Future efforts must prioritize integrated system testing in relevant space environments to de-risk these technologies and validate them for the endurance-class missions to come.

References

• 1. Bioregenerative life support system [https://en.wikipedia.org/wiki/Bioregenerative_life_support_system]
• 2. Critical investments in bioregenerative life support systems ... [https://www.nature.com/articles/s41526-025-00518-4]
• 3. Supplementing Closed Ecological Life Support Systems with ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8401783/]
• 4. Technology Readiness Levels [https://www.nasa.gov/directorates/somd/space-communications-navigation-program/technology-readiness-levels/]
• 5. Technology readiness level [https://en.wikipedia.org/wiki/Technology_readiness_level]
• 6. 25 Years of Scientific Discovery Aboard the International ... [https://www.nasa.gov/missions/station/iss-research/25-year-of-scientific-discovery-aboard-international-space-station/]
• 7. Review of research into bioregenerative life support system ... [https://www.sciencedirect.com/science/article/abs/pii/S2214552421000663]
• 8. Technology Readiness Levels (TRL) in Space Projects | TTMS [https://ttms.com/technology-readiness-levels-trl-in-space-projects-explanation-and-significance/]
• 9. Reliability and lifetime estimation of bioregenerative life ... [https://www.sciencedirect.com/science/article/abs/pii/S0094576522006294]
• 10. Plant and microbial science and technology as ... [https://www.nature.com/articles/s41526-023-00317-9]
• 11. Toward sustainable space exploration: a roadmap for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10030976/]
• 12. Microbial applications for sustainable space exploration ... [https://www.nature.com/articles/s41526-023-00285-0]
• 13. Cyanobacteria and Algal-Based Biological Life Support ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10057978/]
• 14. Microalgae: a multifaceted catalyst for sustainable solutions in ... [https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-024-02588-7]
• 15. A review on microalgae cultivation and harvesting ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6999644/]
• 16. Microbes may help astronauts transform human waste into ... [https://www.psu.edu/news/research/story/microbes-may-help-astronauts-transform-human-waste-food]
• 17. Microalgae-based bioremediation of refractory pollutants [https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-024-02638-0]
• 18. Microalgae cultivation with recycled harvesting water ... [https://www.sciencedirect.com/science/article/abs/pii/S0960852423017042]
• 19. Cyanobacteria and microalgae in supporting human ... [https://www.sciencedirect.com/science/article/pii/S0734975022000428]
• 20. Stoichiometric model of a fully closed bioregenerative life ... [https://www.frontiersin.org/journals/astronomy-and-space-sciences/articles/10.3389/fspas.2023.1198689/full]
• 21. Critical investments in bioregenerative life support systems for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12357894/]
• 22. Human lunar base: "Lunar Palace 1" team of Beihang ... [https://www.the-innovation.org/article/doi/10.1016/j.xinn.2024.100592]
• 23. Construction of closed integrative system for gases robust ... [https://www.sciencedirect.com/science/article/abs/pii/S092585741200119X]
• 24. Deep Space Exploration - an overview [https://www.sciencedirect.com/topics/physics-and-astronomy/deep-space-exploration]
• 25. Technology Readiness Levels (TRL) [https://www.esa.int/Enabling_Support/Space_Engineering_Technology/Shaping_the_Future/Technology_Readiness_Levels_TRL]
• 26. Technology Readiness Levels (TRLs) [https://esto.nasa.gov/trl/]
• 27. ECSS-E-HB-11A – Technology readiness level (TRL) ... [https://ecss.nl/home/ecss-e-hb-11a-technology-readiness-level-trl-guidelines-1-march-2017/]
• 28. TRL best practices a new ECSS handbook [https://iafastro.directory/iac/archive/browse/IAC-16/D1/3/31891/]
• 29. Use of Photobioreactors in Regenerative Life Support ... - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC8281973/]
• 30. Recent Advancements in Photo-Bioreactors for Microalgae ... [https://www.mdpi.com/2227-9717/12/6/1104]
• 31. Recent Developments on the Performance of Algal ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9844339/]
• 32. Editorial: Bioregenerative life-support systems for crewed ... [https://www.frontiersin.org/journals/astronomy-and-space-sciences/articles/10.3389/fspas.2022.977364/full]
• 33. Exploring plant responses to altered gravity for advancing ... [https://www.sciencedirect.com/science/article/pii/S2590346225001324]
• 34. Launching NASA and Humanity into Deep Space [https://www.nasa.gov/missions/station/iss-research/international-space-station-launching-nasa-and-humanity-into-deep-space/]
• 35. Understanding Technology Readiness Levels (TRLs) [https://www.strategicsciencewriting.com/post/understanding-technology-readiness-levels-trls-a-guide-for-innovators-in-food-tech-and-pharma]
• 36. Supplemental Food Production With Plants: A Review of ... [https://www.frontiersin.org/journals/astronomy-and-space-sciences/articles/10.3389/fspas.2021.734343/full]
• 37. Water recycling is paramount for space stations and long- ... [https://www.space.com/space-exploration/international-space-station/water-recycling-is-paramount-for-space-stations-and-long-duration-missions-an-environmental-engineer-explains-how-the-iss-does-it]
• 38. Color Coding Key for Readiness Levels [https://roadmap.inemi.org/ir/color-coding-key-for-readiness-levels]
• 39. Game-changing life support system for Mars missions [https://space.blog.gov.uk/2024/09/09/game-changing-life-support-system-for-mars-missions/]
• 40. Technology Readiness Level 6 (TRL 6): Demonstrating ... [https://techfinders.io/project/technology-readiness-level-6-trl6-demonstration/]
• 41. Understanding Technology Readiness Levels (TRLs) for ... [https://anywaves.com/resources/blog/technology-readiness-levels-trl-space-antennas/]
• 42. Reaching for the Stars: Biotechnological Patents ... [https://www.sternekessler.com/news-insights/insights/reaching-for-the-stars-biotechnological-patents-challenges-and-opportunities-in-outer-space/]
• 43. Biotechnology to Sustain Crews on Long Missions Tops ... [https://www.nasa.gov/blogs/spacestation/2025/02/10/biotechnology-to-sustain-crews-on-long-missions-tops-research-schedule/]
• 44. Comparison of biological measurement and physical ... [https://www.sciencedirect.com/science/article/pii/S2405844022015547]
• 45. Integrated Pest Management Protocols for Space-Based ... [https://www.frontiersin.org/journals/astronomy-and-space-sciences/articles/10.3389/fspas.2021.759641/full]
• 46. Integrated Pest Management (IPM) Principles [https://www.epa.gov/safepestcontrol/integrated-pest-management-ipm-principles]
• 47. A theoretical framework to improve the adoption of green ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10948852/]
• 48. How Better Data Is Transforming Integrated Pest ... [https://www.uaviq.com/en/2022/03/how-better-data-is-transforming-integrated-pest-management/]
• 49. Integrated Pest Management: An Update on the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11465254/]
• 50. Nitrogen accountancy in space agriculture | npj Microgravity [https://www.nature.com/articles/s41526-024-00428-x]
• 51. AI in agriculture: Enhancing crop yields and pest control [https://www.farmprogress.com/technology/ai-in-agriculture-boosting-crop-yields-and-fighting-pests-with-advanced-technology]
• 52. Kennesaw State researcher develops AI robot to aid ... [https://www.kennesaw.edu/news/stories/2025/researcher-develops-ai-robot-aid-farmers-against-pests.php]
• 53. Design of a Module for Cultivation of Tuberous Plants in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7242650/]
• 54. Investigating plant responses to microgravity and adaptations ... [https://enveurope.springeropen.com/articles/10.1186/s12302-024-00851-7]
• 55. Development and implementation of a simulated ... [https://www.nature.com/articles/s41526-024-00436-x]
• 56. Lab-on-a-Chip Technologies for Microgravity Simulation ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9864955/]
• 57. Microbiome Dynamics: A Paradigm Shift in Combatting ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10890469/]
• 58. A systematic framework for understanding the microbiome ... [https://www.nature.com/articles/s41392-024-01946-6]
• 59. Gut microbiome and viral infections: A hidden nexus for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12476802/]
• 60. About Microbial Ecology | Antimicrobial Resistance [https://www.cdc.gov/antimicrobial-resistance/about/about-microbial-ecology.html]
• 61. A comprehensive overview of microbiome data in the light ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10900530/]
• 62. Analysis of microbiome high-dimensional experimental ... [https://www.frontiersin.org/journals/microbiomes/articles/10.3389/frmbi.2025.1584516/full]
• 63. Understanding the Complexities and Changes of the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9031868/]
• 64. The MaMBA facility as a testbed for bioregenerative life ... [https://www.sciencedirect.com/science/article/abs/pii/S2214552422000669]
• 65. The conceptual design of a hybrid life support system ... [https://www.sciencedirect.com/science/article/abs/pii/S0273117705007933]
• 66. BLSS: a contribution to future life support [https://pubmed.ncbi.nlm.nih.gov/11537782/]
• 67. Spaceflight Standard Measures is a multidisciplinary study ... [https://www.nature.com/articles/s41526-025-00532-6]
• 68. Artemis II Standard Measures [https://www.nasa.gov/reference/artemis-ii-standard-measures/]
• 69. Principles of Effective Data Visualization - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC7733875/]
• 70. NASA's Lunar Surface Innovation Initiative [https://www.nasa.gov/space-technology-mission-directorate/lunar-surface-innovation-initiative/]