Validating Long-Term Material Closure: Key Insights from the Lunar Palace 365 Experiment for Biomedical and Pharmaceutical Stability

Abstract

This article analyzes the groundbreaking Lunar Palace 365 experiment, a 370-day closed-environment study, to extract critical methodologies and insights for long-term material stability validation. Tailored for researchers and drug development professionals, we explore the Bioregenerative Life Support System's (BLSS) 98.2% closure rate, microbial community dynamics, psychological health interactions, and advanced monitoring techniques. The findings provide a novel framework for designing robust stability testing protocols, predicting product shelf-life, and managing complex biological systems in confined environments relevant to pharmaceutical manufacturing and long-duration space missions.

The Lunar Palace 365 Mission: Foundation of Closed Ecosystem Stability Research

Lunar Palace 1 (LP1), also known as Yuegong-1, is China's first and the world's third Bioregenerative Life Support System (BLSS) test facility. Established by a team led by Professor Liu Hong at Beihang University, it represents a critical step toward enabling long-term human survival in extraterrestrial environments by creating a closed ecosystem where humans, plants, animals, and microorganisms coexist. Its primary purpose is to achieve the long-term, stable recycling of air, water, and food with a high degree of closure, supporting human life without external inputs other than energy [1] [2] [3].

The system was designed to demonstrate and verify the technologies required for permanent astrobases on the Moon or Mars. Ground-based experiments in LP1 are essential for developing the operational and control strategies needed for future space exploration. To date, it has hosted two landmark missions: a 105-day initial experiment in 2014 and the record-breaking "Lunar Palace 365" experiment that lasted 370 days from 2017 to 2018 [1] [3].

The architecture of Lunar Palace 1 is designed as an integrated, closed-loop artificial ecosystem. The facility covers 160 m² with a total volume of 500 m³ and is composed of several specialized cabins that work in concert [4] [2] [5].

• Integrated Cabin System: The layout includes four main functional areas.

  • Plant Cabin I and II: These two cabins, each measuring 10m x 6m x 3.5m, are dedicated to the highly efficient cultivation of a variety of plants. The crops grown include five cereals (wheat, corn, soybeans, peanuts, lentils), 15 vegetables (carrots, cucumbers, water spinach), and one fruit (strawberries). These plants are fundamental for oxygen regeneration, water purification, and providing the majority of the crew's caloric and nutritional intake [1] [2].
  • Comprehensive Cabin: This 42m² cabin (14m x 3m x 2.5m) serves as the living quarters for the crew. It contains four private bedrooms, a living room, a bathroom, and an insect culturing room. The insects, specifically yellow mealworms, were cultivated as a primary source of protein for the crew [2] [5].
  • Solid Waste Treatment Cabin: This cabin is dedicated to the bioconversion and composting of all solid waste produced within the system, completing the material cycle [5].

• The BLSS Loop: The core innovation of LP1 is its bioregenerative approach. Unlike the physicochemical systems used on the International Space Station (ISS), a BLSS relies on biological processes [6] [7].

  • Human-Plant-Microbe Interaction: Plants consume carbon dioxide and produce oxygen through photosynthesis for the crew. Conversely, crew respiration provides CO₂ for the plants. Plant and food waste, as well as human waste, are processed by microorganisms and through composting. The resulting nutrients are then used to fertilize the plants, closing the nutrient loop [1] [8].
  • Water Recycling: A dedicated water recycle system treats all wastewater, including condensate from humidity control, domestic wastewater, urine, and used nutrient solutions from plant cultivation. The purified water is reused for drinking, irrigation, and other needs [6].

Experimental Protocols and Performance Data from Lunar Palace 365

The "Lunar Palace 365" experiment was a 370-day mission designed to validate the long-term stability and reliability of the BLSS. Eight volunteers were divided into two groups, undertaking the mission in three shifts to study system response to crew changes with different metabolic states [1] [4].

Water Recycling and Treatment Performance

The water recycle system employed Membrane Biological Activated Carbon Reactors (MBARs) to treat different waste streams separately. The 370-day operational data demonstrated high efficiency in water reclamation [6].

Table 1: Water Treatment Performance in Lunar Palace 365 [6]

Wastewater Stream	Treatment Process	Key Performance Metrics
Condensate Wastewater	Aerobic MBAR (CW-MBAR)	CODMn reduced to 0.74 ± 0.15 mg/L, meeting drinking water standards.
Domestic Wastewater	MBAR (DW-MBAR)	85.7% ± 10.2% organic contaminant removal; stable nitrification with effluent NO₃⁻-N at 145.57-328.59 mg/L.
Urine	MBAR (Urine-MBAR)	Achieved hydrolysis of urea to NH₄⁺-N, enabling partial recovery of nitrogen for plant fertilization.
Used Nutrient Solution	MBAR	Effective purification for reuse in the hydroponic plant cultivation system.

The MBAR technology proved to be a gravity-independent, compact solution with high automation potential, making it suitable for space missions. Microbial community analysis within the reactors identified Meiothermus, Rhodanobacter, and Ochrobactrum as the dominant genera responsible for the stable treatment performance [6].

Ecological and Biosafety Monitoring Protocols

A critical aspect of the experiment was monitoring the microbial ecology and ensuring biosafety within the closed environment. Researchers used advanced molecular techniques to profile the bacterial and fungal communities [4] [8] [5].

• Sample Collection: Surface samples and air dust were systematically collected from the Comprehensive Cabin, Plant Cabins, and Solid Waste Treatment Cabin at multiple time points throughout the 370-day mission. Sterile swabs and HEPA filters were used for collection, with strict controls including field and lab blanks [4] [5].
• DNA Analysis: Genomic DNA was extracted from the samples. For bacterial analysis, the 16S rRNA gene was sequenced. For fungal analysis (mycobiome), the Internal Transcribed Spacer 1 (ITS1) region was sequenced using primers ITS1F and ITS2R. This allowed for a detailed census of the microbial populations [4] [5].
• Biosafety Assessment: The potential risks from pathogens, antibiotic resistance genes (ARGs), and mycotoxin-producing fungi were quantified using quantitative PCR (qPCR) with specific primers [8] [5].

The results confirmed a low abundance of potential pathogens and antibiotic resistance, attributing the favorable biosafety profile to the integration of plants. The study concluded that plants were the primary source of surface fungi, and their presence helped maintain a healthy microbial balance, reducing fluctuations caused by crew turnover [8] [5].

Comparative System Performance and Validation Data

The success of Lunar Palace 1 is evident when its performance and design are compared with other life support approaches. The Lunar Palace 365 experiment set a record as the longest and most closed BLSS experiment ever conducted [1].

Table 2: Key Material Closure Validation Data from Lunar Palace 365

System Parameter	Achievement / Metric	Significance
Mission Duration	370 days	Longest stay in a self-contained BLSS, validating long-term stability [1] [3].
System Closure	High degree of closure	Achieved recycling of oxygen, water, and food, drastically reducing the need for external resupply [1] [6].
Food Production	55% produced internally	Balanced by reserves; system provided a high-protein diet from plants and insects (mealworms) [2].
Oxygen Regeneration	Fully regenerated by plants	Plant cabins continuously produced oxygen through photosynthesis for the crew [1] [3].
Water Recovery	Recycled from all waste streams	MBAR technology successfully treated condensate, domestic water, and urine to potable standards [6].
Biosafety	Low pathogen & ARG abundance	Plant integration helped maintain a safe microbial environment, crucial for crew health on long missions [8].

The data from LP1 stands in contrast to the current ISS life support model, which relies heavily on physicochemical processes and regular resupply from Earth. The BLSS approach pioneered in LP1 is considered essential for future long-duration missions to the Moon and Mars where resupply is impractical [6] [7].

The Researcher's Toolkit: Key Reagents and Materials

The experimental validation of LP1 relied on a suite of specialized reagents and materials for molecular biology and environmental monitoring.

Table 3: Key Research Reagent Solutions Used in Lunar Palace 365 Analyses

Research Reagent / Material	Function in the Experiment
FastDNA Spin Kit	Used for the extraction of high-quality genomic DNA from surface and air dust samples for subsequent sequencing [5].
Primers (16S rRNA, ITS1)	Specific DNA sequences (e.g., ITS1F/ITS2R for fungi) used to amplify target genes for microbial community profiling via Illumina sequencing [4] [5].
KOD FX Neo Enzyme	High-fidelity DNA polymerase used for the PCR amplification steps prior to sequencing, ensuring accurate replication of genetic material [5].
Membrane Biological Activated Carbon Reactor (MBAR)	The core unit for wastewater treatment, combining microbial degradation and physical filtration to reclaim water [6].
Sterile Swab & NaCl Solution	Standardized tools for collecting surface microbiome samples from various locations inside the habitat [5].
HEPA Filter (Xiaomi Air Purifier)	Device used for continuous collection of airborne microbial particles (air dust) for microbiome and biosafety analysis [4].

Workflow and System Logic

The following diagram illustrates the core logical relationships and material flows within the Lunar Palace 1 BLSS, integrating the human, plant, and technological components.

Diagram 1: Material Flow and Subsystem Relationships in Lunar Palace 1 BLSS. This diagram shows the closed-loop circulation of oxygen, food, water, and waste between crew, plants, and processing technologies.

Lunar Palace 1 has successfully transitioned from a theoretical concept to a validated, ground-based prototype for a future lunar base. The data generated from the 105-day and, more importantly, the 370-day Lunar Palace 365 experiment provides a crucial corpus of evidence for the feasibility of bioregenerative life support. The project has moved beyond simple proof-of-concept to deliver quantitative performance data on water recycling, gas closure, food production, and microbial management.

The research has clarified that a stable, closed ecosystem is achievable, with plants playing a dual role as key life support providers and moderators of the microbial environment. The technological frameworks and operational protocols developed, particularly the MBAR-based water recovery and the comprehensive biosafety monitoring strategy, provide a ready foundation for the next step: testing these systems in a true space environment. The LP1 team is now actively working toward conducting small-scale BLSS experiments on lunar or Mars probes, comparing them with ground data to refine parameters for an actual extraterrestrial base [1] [9]. The architecture of Lunar Palace 1 stands as a testament to a viable pathway for achieving long-term human presence beyond Earth.

A Guide to Experimental Design in Long-Term Closed Environment Studies

The success of long-duration space missions and future planetary bases hinges on the development of Bioregenerative Life Support Systems (BLSS), which are closed artificial ecosystems that recycle oxygen, water, and food using biological and engineering processes [10]. Among the ground-based simulations for such systems, the "Lunar Palace 365" mission stands out for its unprecedented duration and sophisticated multi-crew shift design. This guide objectively compares the mission parameters of the Lunar Palace 365 experiment with other notable closed-environment studies, providing researchers with a framework for evaluating the operational designs of long-term material closure validation experiments.

The "Lunar Palace 365" was a 370-day integrated mission conducted in the Lunar Palace 1 (LP1) facility, a ground-based BLSS with a total volume of 500 m³ [10] [11]. Its primary objective was to develop technologies to maintain and adjust system stability under long-term operational conditions and crew shifts, providing key insights for optimizing life support systems for lunar bases [10].

The table below compares the core parameters of the Lunar Palace 365 mission with other significant missions in closed-environment research.

Table: Comparative Analysis of Closed Environment Mission Parameters

Mission Parameter	Lunar Palace 365	Mars 500	HI-SEAS IV
Duration	370 days [10]	520 days [11]	365 days [11]
Crew Size	8 total (4 per shift) [10]	6 crew [11]	Not Specified
Shift Design	Two shifts across three phases [10]	Not specified	Not specified
System Type	Bioregenerative Life Support System (BLSS) [10]	Confined environment simulation [11]	Confimed environment [11]
Key Closure Achievement	98.2% material closure; 100% oxygen and water recycling [10]	Not specified	Not specified
Core Research Focus	BLSS stability, crew microbiome, resource recycling, psychological health [10] [11] [12]	Human psychology and physiology [11]	Crew dynamics and technology testing [11]

Detailed Experimental Protocols in Lunar Palace 365

The verification of the 370-day duration and multi-crew shift design was supported by extensive, cross-disciplinary experimental protocols. These methodologies were critical for collecting the data that validated the system's performance and its impact on the crew.

Table: Key Experimental Methodologies in Lunar Palace 365 Research

Research Area	Sample Type	Collection Method & Frequency	Primary Analytical Technique
System Material Closure [10]	O₂, CO₂, Water, Waste	Continuous monitoring and specific recovery measurements	Mass balance calculations and performance metrics
Air Microbiome [11]	Air dust	Collected via HEPA filters from different locations and time points	16S rRNA amplicon sequencing, shotgun metagenomics, qPCR
Salivary Microbiome & Immunity [13]	Saliva	Collected from crewmembers before, during, and after enclosure	16S rRNA gene Illumina HiSeq sequencing; cytokine level analysis
Gut Microbiome & Psychology [12]	Feces	103 sets of psychological data with corresponding fecal samples	Metagenomic, metaproteomic, and metabolomic analyses
Water Recycling [6]	Condensate, domestic wastewater, urine, nutrient solutions	Treated via separate Membrane Biological Activated Carbon Reactors (MBARs)	Chemical analysis (CODMn, N compounds); 16S rDNA sequencing

Research Workflow and Analytical Logic

The following diagram illustrates the integrated experimental workflow used to validate the mission parameters and their biological impacts in the Lunar Palace 365 study.

The Scientist's Toolkit: Key Research Reagent Solutions

The rigorous experimental protocols of the Lunar Palace 365 mission relied on a suite of specific reagents and materials essential for data collection and analysis.

Table: Essential Research Reagents and Materials in Lunar Palace 365

Reagent/Material	Primary Function in Research
HEPA Filters [11]	Collection of air dust samples for subsequent microbiome and antibiotic resistance gene (ARG) analysis.
DNA Extraction Kits [11]	Isolation of high-quality genomic DNA from diverse sample types (air, saliva, feces, water) for sequencing.
16S rRNA Gene Primers [13]	Amplification of specific bacterial gene regions for taxonomic identification and diversity analysis of microbiomes.
Illumina HiSeq Sequencing Kits [13]	High-throughput sequencing of prepared DNA libraries for metagenomic, metaproteomic, and amplicon analysis.
Membrane Biological Activated Carbon Reactors (MBARs) [6]	Core bioreactor technology for treating various wastewaters (condensate, domestic, urine) and recovering water and nutrients.
Psychological Assessment Tools [12]	Standardized questionnaires, including the Symptom Checklist-90 (SCL-90) and Profile of Mood States (POMS), to quantify crew mental health.

Key Insights for Future Mission Design

The 370-day duration and structured multi-crew shift design of the Lunar Palace 365 mission provided unparalleled data on the long-term operation of a BLSS. Key findings demonstrate that a well-designed mission can achieve a high degree of material closure (98.2%) with robust system stability [10]. Furthermore, the shift change design proved critical for understanding the impact of crew rotation on the system's microbial ecology, revealing that human presence is the primary driver of airborne microbial succession [11]. Importantly, the mission yielded evidence that long-term habitation in a strictly controlled enclosed environment does not lead to persistent, detrimental changes in human salivary microbiota and oral immunity [13], a vital finding for crew health planning. Finally, the project successfully identified specific gut microbiota, or "psychobiotics," correlated with positive mood regulation, offering potential avenues for mitigating psychological risks on long-duration missions [12].

The "Lunar Palace 365" mission represents a landmark achievement in bioregenerative life support system (BLSS) research, demonstrating unprecedented material closure for long-term human space habitation. Conducted in the ground-based "Lunar Palace 1" facility at Beihang University, this 370-day integrated experiment achieved 98.2% closure of materials crucial for human survival, marking a critical milestone for future lunar bases and deep space exploration [10]. This high-closure system successfully maintained eight crew members across multiple shift changes while regenerating essential life support resources through biological processes [10].

As space agencies worldwide plan for sustained lunar presence, achieving near-complete material recycling has become strategically essential. The Lunar Palace 365 experiment provides the most comprehensive validation to date of BLSS viability, significantly advancing beyond previous physical-chemical life support systems that depend heavily on Earth resupply [14]. This analysis examines the specific oxygen, water, and food recycling metrics that contributed to this record-breaking closure rate, comparing system components and detailing the experimental protocols that enabled this achievement.

The Lunar Palace 365 mission established new benchmarks for closed-loop life support across multiple resource categories. The system's overall performance demonstrates significant progress toward sustainable human habitation in extraterrestrial environments.

Table 1: Overall Resource Closure Metrics in Lunar Palace 365

Resource Category	Closure Rate	Key Achievement	Comparison to Previous Systems
Overall System Materials	98.2%	Recycling of materials crucial for human survival	Higher than 97% in earlier 105-day mission [10]
Oxygen	100%	Complete biological regeneration through plants	Superior to physicochemical systems on ISS requiring resupply [14]
Water	100%	Complete recycling for human use including drinking	Advanced biological treatment vs. ISS physical-chemical systems [6]
Plant-Based Food	100%	Fully met crew nutritional requirements	Beyond snack-level production in current space systems [10]
Urine	99.7%	High recovery of water and nitrogen	More comprehensive than urine processing on ISS [10]
Solid Waste	67%	Bioconversion to soil-like substrate	Integrated approach not found in current space habitats [10]

Table 2: Atmospheric Gas Management Performance

Parameter	Performance	Regulation Method
CO₂ Concentration	Maintained between 246-4131 ppm	Soybean photoperiod adjustment [10]
O₂ Concentration	Stable within breathable range	Balanced plant cultivation [10]
System Robustness	Quick recovery from disturbances	Solid waste reactor activity control [10]

Oxygen Recycling: Achieving 100% Closure

Experimental Protocols and System Design

The Lunar Palace 1 facility maintained atmospheric balance through precisely managed plant cultivation systems. The facility incorporated two plant cabins containing 35 carefully selected plant species optimized for oxygen production and carbon dioxide consumption [10]. The photosynthetic oxygen production was quantitatively matched to crew respiratory requirements through real-time monitoring and adjustment of cultivation parameters.

Gas balance maintenance employed two strategic regulation methods: soybean photoperiod manipulation and solid waste reactor activity control [10]. The system demonstrated strong robustness, quickly minimizing effects of disturbances such as crew shift changes and equipment operations. The selected plants exhibited high production efficiencies while growing effectively within the system constraints.

Comparative Performance Analysis

The 100% oxygen regeneration achievement surpasses the capabilities of current International Space Station (ISS) systems, which rely primarily on electrolysis of water and imported oxygen rather than biological regeneration [14]. The Lunar Palace approach eliminates dependency on Earth resupply for atmospheric constituents, a critical requirement for long-duration lunar missions where resupply opportunities are limited and costly.

Water Recycling: Comprehensive Treatment and Reuse

Advanced Water Processing Methodologies

The water recycle system in Lunar Palace 365 implemented sophisticated separation and treatment processes for different wastewater streams. The system employed Membrane Biological Activated Carbon Reactors (MBARs) specifically configured for four distinct wastewater types: condensate wastewater, domestic wastewater, urine, and used nutrient solutions [6].

Table 3: Water Treatment System Performance

Waste Stream	Treatment Technology	Treatment Performance	End Use
Condensate Wastewater	Aerobic MBAR (CW-MBAR)	CODMn reduced to 0.74 ± 0.15 mg/L	Potable water meeting drinking standards [6]
Domestic Wastewater	MBAR (DW-MBAR)	85.7% ± 10.2% organic contaminant removal	Irrigation and system uses [6]
Urine	Urine-MBAR	High-efficiency urea hydrolysis with nitrogen recovery	Nutrient solution for plant growth [6]
Used Nutrient Solutions	MBAR technology	Purification of hydroponic solutions	Reuse in plant cultivation systems [6]

The condensate wastewater treatment achieved particularly notable results, with purified water meeting stringent drinking water standards [6]. The urine processing system successfully converted urea-nitrogen to ammonium-nitrogen, enabling partial recovery of nitrogen for agricultural reuse within the closed ecosystem.

Microbial Community Management

The water system maintained functionality through careful management of microbial communities. 16S rDNA sequencing revealed that Meiothermus, Rhodanobacter, and Ochrobactrum emerged as dominant microorganisms in the various MBARs [6]. These microbial populations demonstrated stable performance throughout the 370-day operation, contributing to both organic contaminant removal and nitrification processes.

Food Production and Waste Recycling

Bioregenerative Food Production

The food production component achieved complete satisfaction of crew plant-based food requirements through efficient cultivation of the 35 plant species within the system's two plant cabins [10]. The BLSS plant production efficiency fully met the crew's nutritional needs throughout all mission phases, demonstrating the viability of continuous agricultural production within closed environments.

The system also incorporated animal protein production through insects, complementing the plant-based diet and providing nutritional diversity essential for long-term crew health [10]. This integrated approach to food production represents a significant advancement over current space food systems, which rely primarily on prepackaged foods with limited fresh supplementation.

Waste Transformation and Nutrient Recovery

Solid waste management achieved 67% recovery through bioconversion to soil-like substrate [10]. This waste transformation process enabled nutrient recycling back into the agricultural system, completing essential elemental cycles. Meanwhile, urine processing reached 99.7% recovery, efficiently reclaiming both water and nitrogen components [10].

The system demonstrated excellent stability in managing waste streams while maintaining environmental safety. Bacterial population dynamics monitoring revealed a low abundance of potential pathogens, minimal antibiotic resistance, and negligible impact of isolated bacterial strains on equipment materials [8].

Research Reagents and Essential Materials

Table 4: Key Research Reagents and Experimental Materials

Reagent/Material	Function in Lunar Palace 365	Application Context
35 Plant Species	Oxygen production, food provision, CO₂ consumption	Bioregenerative life support [10]
Membrane Biological Activated Carbon Reactors (MBARs)	Wastewater treatment and purification	Water recycling system [6]
Soybean Cultivars	Photoperiod-based gas regulation	Atmospheric management [10]
Insect Species	Animal protein production	Nutritional supplementation [10]
16S rDNA Sequencing	Microbial community analysis	System safety and performance monitoring [6]
High-Efficiency Particulate Absorbing (HEPA) Filters	Air dust collection for microbiome studies	Environmental monitoring [11]

System Workflow and Integration

The overall functionality of the Lunar Palace 365 BLSS depends on the precise integration of its biological and technological components. The system represents a complex network of interdependent processes that maintain human life through continuous resource regeneration.

The Lunar Palace 365 experiment represents a transformative achievement in bioregenerative life support, validating the technical feasibility of near-complete material closure for long-duration space missions. The documented metrics—100% oxygen and water recycling, 100% plant-based food production, and 98.2% overall system closure—establish new benchmarks for life support system performance [10].

These results have profound implications for future lunar habitation and deep space exploration. The demonstrated technology reduces dependency on Earth resupply, enabling more sustainable and economically viable long-duration missions. As space agencies worldwide pursue lunar exploration programs, the integration of such bioregenerative systems will be essential for establishing permanent human presence beyond Earth orbit [14].

The success of Lunar Palace 365 positions BLSS technology as a cornerstone for the next era of human space exploration, potentially supporting not only lunar bases but also future missions to Mars and beyond. The experimental protocols, system architectures, and performance metrics established in this groundbreaking mission provide a validated foundation for the life support systems that will sustain humanity as we become a multi-planetary species.

The Critical Role of Plant Cabins in Atmospheric Gas Balance

In the pursuit of long-duration human space exploration, the establishment of a controlled ecological life support system is paramount. The Bioregenerative Life Support System (BLSS) represents the most advanced technology for enabling permanent human presence on the Moon and beyond, creating an Earth-like microenvironment where oxygen, water, and food are continuously recycled using biological and engineering processes [10]. Within this system, plant cabins serve as the primary biological engine responsible for atmospheric gas balance through photosynthetic carbon assimilation and oxygen generation.

The Lunar Palace 365 experiment, conducted in the Lunar Palace 1 facility at Beihang University, marked a groundbreaking 370-day mission to validate long-term material closure for survival in lunar base environments [15] [10]. This comprehensive study provided critical insights into how plant cabins maintain gas exchange homeostasis through sophisticated biological processes, engineering controls, and system management strategies that ensure crew safety and system sustainability over extended periods.

Lunar Palace 365 Experimental Framework

Facility Design and Mission Parameters

The Lunar Palace 1 facility was designed as an artificial closed ecosystem with a total area of 160m² and volume of 500m³, comprising two distinct plant cabins and one comprehensive cabin for crew living quarters and waste processing [10] [16]. The system hosted 35 different crop species including wheat, soybean, potato, carrot, and various leafy vegetables, creating a diverse agricultural ecosystem capable of supporting crew nutritional needs while driving atmospheric regeneration [15].

The 370-day experiment employed a structured crew rotation protocol with eight volunteers divided into two groups across three phases: an initial 60-day phase, a record-breaking 200-day middle phase, and a final 110-day phase [10] [16]. This design allowed researchers to evaluate system stability during metabolic transitions and crew shifts, representing realistic operational scenarios for future lunar habitats.

Table: Lunar Palace 365 Experimental Parameters

Parameter	Specification
Mission Duration	370 days
Total Facility Volume	500 m³
Plant Cultivation Area	Two dedicated plant cabins
Number of Crop Species	35 species
Crew Configuration	8 volunteers across 3 phases
Primary Gas Exchange Mechanism	Plant photosynthesis and respiration

Methodologies for Gas Balance Monitoring

The research team implemented comprehensive atmospheric monitoring throughout the mission duration. Oxygen and carbon dioxide concentrations were continuously tracked using integrated sensor arrays, with data recorded at regular intervals to capture diurnal fluctuations and long-term trends [10]. The experimental protocol included deliberate system perturbations such as power outages and equipment failures to evaluate the resilience of gas balancing mechanisms under stress conditions [16].

Plant photosynthetic performance was assessed through regular measurement of growth rates, biomass accumulation, and gas exchange characteristics of key crop species [10]. Additionally, the team employed biological modulation techniques including adjustment of soybean photoperiod and solid waste reactor activity to actively manage gas concentrations when passive biological processes proved insufficient [10].

Quantitative Analysis of Gas Balance Performance

Atmospheric Stability Metrics

Throughout the 370-day mission, the plant cabins demonstrated remarkable atmospheric control despite varying metabolic loads from crew rotations. The system maintained CO₂ concentrations between 246 and 4,131 ppm, with an average of 1,845 ppm, while oxygen levels remained within life-supporting parameters [10]. This stability was particularly noteworthy during crew shift changes, which represented significant perturbations to the system's metabolic equilibrium.

The research confirmed 100% oxygen regeneration through photosynthetic activity, with plant cabins serving as the exclusive source of oxygen replenishment for crew respiration [10]. Simultaneously, the system achieved complete carbon dioxide processing, converting respired CO₂ back to oxygen through regulated photosynthetic pathways, thus closing the atmospheric gas loop.

Table: Gas Balance Performance During Lunar Palace 365 Mission

Performance Indicator	Result	Significance
O₂ Recycling Rate	100%	Complete regeneration of breathable oxygen
CO₂ Concentration Range	246-4,131 ppm	Maintained within physiologically tolerable limits
System Closure Degree	98.2%	Near-complete material recycling
Food Self-sufficiency	High (plant-based)	Majority of nutritional needs met internally
Water Recycling Rate	100%	Complete water recovery and purification

Comparative Performance Against Alternative Systems

When evaluated against other life support technologies, the BLSS approach with dedicated plant cabins demonstrates distinct advantages in gas balance management over physical-chemical systems. Unlike the International Space Station's life support system which relies on mechanical oxygen generation and chemical CO₂ scrubbing, the Lunar Palace 1 achieved biologically-mediated gas exchange that more closely mimics Earth's natural cycles [6].

The system outperformed earlier BLSS prototypes including Russia's BIOS-3 and Japan's CEEF in terms of closure degree and operational duration [10]. The 98.2% material closure represented a significant improvement over the 97% achieved in the previous 105-day Lunar Palace experiment [10], highlighting advancements in system integration and control methodologies.

Biological Mechanisms of Atmospheric Regulation

Photosynthetic Gas Exchange Dynamics

The plant cabins functioned as photoautotrophic engines within the closed system, converting light energy into chemical energy while simultaneously consuming carbon dioxide and producing oxygen. Researchers optimized this process through species selection and cultivation management, choosing plants with complementary photosynthetic characteristics and growth patterns to ensure consistent gas exchange capacity [15].

The system employed staged cultivation protocols with overlapping growth cycles, maintaining a constant biomass of photosynthetically active plants despite periodic harvesting for crew consumption. This approach ensured continuous gas processing without the fluctuations that would occur from simultaneous planting and harvesting of all specimens [10].

Integrated Microbial Contributions

Beyond higher plants, the BLSS incorporated specialized microbial communities that contributed to gas balance through auxiliary pathways. Solid waste treatment modules employed microorganisms to convert inedible plant biomass and crew waste into soil-like substrate while releasing CO₂, thereby closing carbon loops that would otherwise represent system losses [15].

The water recycling system hosted specific bacterial genera including Meiothermus, Rhodanobacter, and Ochrobactrum that processed organic contaminants while maintaining appropriate gas equilibria in aquatic subsystems [6]. These microbial processes prevented anaerobic conditions that could lead to methane or hydrogen sulfide production, thereby avoiding atmospheric contamination.

Diagram: Atmospheric Gas Balance in the Lunar Palace 1 BLSS. The diagram illustrates the cyclic exchange of oxygen and carbon dioxide between plant cabins, crew, and microbial systems, driven by photosynthetic conversion of light energy.

Operational Protocols for Gas Balance Management

Active Regulation Strategies

The Lunar Palace 365 experiment demonstrated that biological self-organization alone is insufficient for maintaining precise gas balance in a closed system, necessitating active human intervention and control mechanisms [10]. Researchers implemented photoperiod manipulation of key crops such as soybean to modulate photosynthetic activity in response to changing CO₂ concentrations, effectively using plant growth parameters as control variables.

The mission also utilized regulated microbial activity in solid waste processing, adjusting decomposition rates to manage CO₂ release in coordination with plant consumption capacity [10]. This integrated approach represented a significant advancement over previous BLSS attempts, including the Biosphere 2 project which experienced critical imbalances in atmospheric composition [10].

Disturbance Response and System Resilience

The experimental design intentionally incorporated stress testing scenarios including power failures and equipment malfunctions to evaluate system robustness [16]. During these events, the plant cabins demonstrated remarkable resilience, with gas concentrations returning to equilibrium levels shortly after normal conditions were restored, validating the inherent stability of the biological components.

The research team documented specific recovery protocols for different disturbance types, creating a decision framework for managing atmospheric emergencies in operational lunar habitats. These protocols emphasized the rapid stabilization of plant function as the highest priority during gas balance disruptions, recognizing the photosynthetic system as the most critical component for long-term atmospheric control.

Research Reagent Solutions for BLSS Implementation

Table: Essential Research Reagents and Materials for BLSS Gas Balance Studies

Reagent/Material	Function in Gas Balance Research	Application in Lunar Palace 365
35 Crop Species	Photosynthetic gas exchange	Primary producers for O₂ generation and CO₂ consumption [15]
Membrane Biological Activated Carbon Reactors	Water purification and microbial support	Maintained water quality for plant growth and hosted gas-relevant microbes [6]
Yellow Mealworms (Tenebrio molitor)	Protein source and waste processing	Contributed to carbon cycling through consumption of plant waste [15]
Specific Microbial Consortia	Waste decomposition and gas regulation	Processed solid waste to release CO₂ for plant consumption [15]
Environmental Sensors	Continuous atmospheric monitoring	Tracked O₂ and CO₂ concentrations to inform management decisions [10]
DNA Sequencing Reagents	Microbial community analysis	Monitored functional microbiota in plant and waste systems [11]

The Lunar Palace 365 experiment represents a transformative advancement in bioregenerative life support technology, conclusively demonstrating the critical role of plant cabins in maintaining atmospheric gas balance for long-duration space missions. Through its 370-day operation, the system validated biological gas exchange as a feasible, robust, and sustainable approach to oxygen regeneration and carbon dioxide management in closed environments.

The research established that integrated biological systems combining higher plants, microorganisms, and appropriate engineering controls can achieve the stability required for human life support in extraterrestrial habitats. The experimental results provide both theoretical frameworks and practical protocols for implementing similar systems in future lunar bases, moving humanity closer to sustainable presence beyond Earth.

As spacefaring nations target extended lunar missions and eventual Martian exploration, the technologies validated in Lunar Palace 365 will form the foundation of life support architecture, with plant cabins serving as the central component for atmospheric management. The mission's success marks a pivotal milestone in the transition from physical-chemical to biological life support systems, highlighting the indispensable role of plant-based ecosystems in our future in space.

Psychological and Physiological Monitoring of Crew Members

The "Lunar Palace 365" mission, a 370-day ground-based isolation experiment conducted within the Lunar Palace 1 (LP1) facility, represents a significant advancement in understanding human adaptation to confined environments essential for long-duration space missions [10]. This Bioregenerative Life Support System (BLSS) achieved a remarkable 98.2% closure degree, recycling nearly all crucial materials for human survival, thereby creating an ideal setting for studying the intricate interplay between physiological and psychological factors in a controlled, isolated environment [10] [12]. The mission's design—featuring two crew groups undergoing rotational shifts across three phases (60, 200, and 110 days)—provided unique insights into human resilience and adaptability under conditions simulating future lunar or Martian habitats [10]. This review comprehensively compares the monitoring methodologies and findings from this pioneering experiment, offering evidence-based protocols for assessing crew health in extreme environments.

Psychological Monitoring Protocols and Findings

Standardized Psychological Assessment Tools

Researchers implemented a multi-faceted psychological monitoring regime using validated instruments to quantify crew mental health dynamics throughout the mission. The primary assessment tools included:

• Symptom Checklist-90 (SCL-90): This comprehensive instrument measured ten psychological symptom dimensions including somatization, obsessive-compulsive traits, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, and psychoticism, along with a global severity index (SUM-SCL) [12].
• Profile of Mood States (POMS): This assessment captured transient, distinct mood states across seven factors, with results synthesized into a Total Mood Disturbance (TMD) score [12].

Psychological data collection was systematically timed, with 103 sets of psychological measurements and corresponding fecal samples obtained to correlate mental health parameters with physiological biomarkers [12]. The rigorous scheduling ensured that assessments captured the psychological evolution across different mission phases and crew compositions.

Key Psychological Findings

Analysis revealed that despite the prolonged confinement, crew members maintained psychological health throughout the mission, though with dynamic, unexpected fluctuations that highlighted individual and gender-specific response patterns [12]. The most significant findings included:

• Individual Variability: Principal component analysis demonstrated significant differences (P < 0.001) in psychological responses between individuals and genders, underscoring the need for personalized mental health support strategies in space missions [12].
• Temporal Dynamics: Horizon graph visualizations of normalized psychological factor scores revealed that mood states followed complex, non-linear patterns over time, rather than showing simple progressive deterioration [12].
• Resilience Indicators: The overall maintenance of mental health across the 370-day isolation demonstrated human capacity to adapt to extreme confinement when proper life support systems and structured routines are maintained [12].

Table 1: Psychological Assessment Tools Used in Lunar Palace 365 Mission

Assessment Tool	Measured Parameters	Frequency of Administration	Key Findings
Symptom Checklist-90 (SCL-90)	10 symptom dimensions including depression, anxiety, hostility	Regular intervals throughout 370-day mission	Significant individual variability in symptom patterns
Profile of Mood States (POMS)	7 mood factors, Total Mood Disturbance (TMD) score	Coordinated with biological sampling	Dynamic, non-linear mood fluctuations over time
Automated Behavioral Monitoring	Facial expressions, interactions, communications	Continuous through video and audio recording	Correlation between behavioral markers and psychological states

Physiological Monitoring Approaches

Gut Microbiota Analysis

The Lunar Palace 365 investigation pioneered comprehensive gut microbiome monitoring as a window into crew physiological health. The experimental protocol included:

• Sample Collection: 103 fecal samples were collected from crew members at predetermined intervals, with the first sample taken 28 days after cabin entry to avoid dietary transition confounding factors [12].
• Multi-Omics Analysis: Researchers employed metagenomic sequencing (103 samples), metaproteomic analysis (90 samples), and metabolomic profiling (56 samples) to characterize the gut ecosystem at multiple functional levels [12].
• Sequencing and Annotation: Metagenomic sequencing generated 885,160.47 Mb of clean data, with taxonomic annotation achieving species-level identification for 43.67% of sequences [12].

The analysis revealed that the dominant gut phyla were Bacteroidetes, Firmicutes, and Proteobacteria, collectively accounting for over 75% of the microbial community [12]. At the genus level, significant inter-individual variation existed, with Prevotella dominating in some crew members and Bacteroides in others, suggesting personalized microbiome configurations [12].

Environmental Microbiome Monitoring

Understanding the interface between crew members and their environment required comprehensive characterization of the LP1 microbial ecosystem:

• Airborne Microbiome: Researchers collected 34 air dust samples using HEPA filters from different locations and mission phases, followed by amplicon and shotgun sequencing to track microbial community succession [11].
• Surface Mycobiome: Fungal communities were assessed via ITS1 amplicon sequencing from surface samples collected at seven time points across three cabin locations (comprehensive cabin, plant cabin, and solid waste treatment cabin) [17].
• Antibiotic Resistance Genes: Monitoring included quantification of antibiotic resistance genes (ARGs) due to their implications for crew health in confined environments [11].

This multifaceted environmental monitoring revealed that personnel exchange significantly altered bacterial community diversity, with most airborne bacteria originating from crew members and plants [11]. The plant cabin demonstrated remarkable stability in fungal communities despite crew rotations, highlighting the balancing effect of plant growth on the BLSS microbiome [17].

Integrated Analysis: The Gut-Brain Axis in Confinement

Identification of Potential Psychobiotics

The correlation analysis between psychological scores and microbial abundance data identified four potential psychobiotic bacteria that demonstrated significant associations with mood maintenance:

• Bacteroides uniformis
• Roseburia inulinivorans
• Eubacterium rectale
• Faecalibacterium prausnitzii [12]

These microorganisms were characterized as "potential psychobiotics" — live organisms that, when administered in adequate amounts, confer mental health benefits [12] [18]. Their relative abundance patterns correlated with positive mood outcomes across the isolation period.

Mechanistic Pathways of Microbiota-Mood Interaction

Multi-omics analyses revealed that these psychobiotics influenced mood through three primary mechanistic pathways:

• Short-Chain Fatty Acid (SCFA) Production: Fermentation of dietary fibers by these bacteria produced butyric and propionic acids, which exert neuroactive effects [12].
• Amino Acid Metabolism Regulation: The psychobiotics modulated pathways for aspartic acid, glutamic acid, and tryptophan, including conversion of glutamic acid to gamma-aminobutyric acid (GABA) and tryptophan to serotonin, kynurenic acid, or tryptamine [12].
• Specialized Pathway Regulation: Additional modulation occurred through taurine and cortisol metabolism pathways, potentially affecting stress response systems [12].

Table 2: Potential Psychobiotics Identified in Lunar Palace 365 Mission and Their Mechanisms

* Bacterial Species*	Primary Functions	Associated Metabolic Pathways	Mood-Related Effects
Bacteroides uniformis	Butyrate production, immune modulation	TNF-α and IL-10 regulation, dopamine transporter correlation	Anxiety reduction, mood stabilization
Roseburia inulinivorans	Butyrate and lactate production	Propionate and propanol production from Firmicutes substrates	Depression-like behavior alleviation
Eubacterium rectale	Butyrate, acetate, hydrogen, and lactate production	TLR4/MyD88/NF-κB axis suppression	Anti-inflammatory effects on nervous system
Faecalibacterium prausnitzii	Microbial balance maintenance	Short-chain fatty acid production	Overall mood improvement

Figure 1: Gut-Brain Axis Pathways in Lunar Palace 365 Mission. This diagram illustrates the mechanisms through which potential psychobiotics identified in the mission influence mood through multiple biological pathways.

Experimental Validation of Psychobiotic Efficacy

Animal Model Verification

Following the observational findings from the Lunar Palace 365 mission, researchers conducted rigorous animal experiments to validate the causal relationship between the identified bacteria and mood regulation [12] [18]. The experimental approach included:

• Chronic Unpredictable Mild Stress (CUMS) Model: Rats were subjected to variable, low-intensity stressors to induce anxiety and depression-like behaviors analogous to those potentially occurring in confined environments [18].
• Psychobiotic Administration: CUMS-induced rats received interventions with single strains or mixtures of the identified potential psychobiotics (Bacteroides uniformis, Roseburia inulinivorans, Eubacterium rectale) [18].
• Behavioral Assessment: Treated animals underwent standardized behavioral tests including Forced Swimming Test (FST), Open Field Test (OFT), and Elevated Plus Maze (EPM) to quantify anxiety and depression-like behaviors [18].

Combination Therapy Synergy

The research notably advanced beyond single-strain approaches by investigating multi-strain psychobiotic combinations, recognizing that microbial communities function through complex ecological interactions [18]. The combination therapy demonstrated superior outcomes through several synergistic mechanisms:

• Gut Barrier Integrity: The psychobiotic mixture significantly reduced serum diamine oxidase (DAO) levels (Padj = 0.001), indicating improved gut barrier function and reduced permeability [18].
• Inflammatory Modulation: Treated animals showed decreased pro-inflammatory factors in serum, creating a less neuroinflammatory environment [18].
• Neuroendocrine Regulation: The combination lowered hypothalamic-pituitary-adrenal (HPA) axis activation, reducing cortisol secretion and normalizing the stress response system [18].
• Neurotransmitter Precursor Availability: Treatment increased levels of DL-kynurenine in brain tissue while reducing histamine, creating a more favorable neurochemical environment [18].

Table 3: Experimental Validation of Psychobiotic Efficacy in CUMS Rat Model

Parameter Measured	CUMS-Induced Changes	Psychobiotic Combination Effects	Statistical Significance
Forced Swim Test Immobility	Increased immobility time (Δ = 21.06)	Significant reduction (Δ = -31.04 to -45.55)	Padj < 0.001
Open Field Test Activity	Reduced central entries (Δ = -14.42) and time (Δ = -12.50)	Increased central time (Δ = 12.83)	Padj = 0.0101
Elevated Plus Maze Performance	Fewer open arm entries (Δ = -13.75)	Increased entries (Δ = 11.92) and time (Δ = 12.17)	Padj = 0.0173-0.0201
Serum DAO Levels	Increased (Δ = 0.93 ng/mL)	Significant reduction (Δ = 1.54 ng/mL)	Padj = 0.001
Cortisol Secretion	Elevated	Normalized HPA axis activity	Padj = 0.007

The Scientist's Toolkit: Essential Research Materials

Core Analytical Technologies

The comprehensive monitoring conducted in the Lunar Palace 365 mission relied on sophisticated research tools and methodologies:

• Metagenomic Sequencing Platform: Enabled comprehensive profiling of bacterial and fungal communities through 16S rRNA and ITS1 region sequencing, with downstream bioinformatics analysis for taxonomic classification [12] [17].
• Metaproteomic Analysis: Provided functional insights by characterizing the protein complement of microbial communities, linking taxonomic information to biological activities [12].
• Metabolomic Profiling: Identified and quantified small molecule metabolites including short-chain fatty acids, neurotransmitters, and their precursors, completing the functional picture of microbial activity [12].
• Quantitative PCR (qPCR) Systems: Enabled absolute quantification of specific bacterial taxa, fungal populations, and mycotoxin-related genes to assess potential biohazards in the closed environment [11] [17].

Psychological Assessment Instruments

• Standardized Psychological Batteries: Validated instruments including Symptom Checklist-90 (SCL-90) and Profile of Mood States (POMS) provided quantitative, reproducible metrics for psychological state monitoring [12].
• Behavioral Coding Systems: The Facial Action Coding System (FACS) and wireless interaction monitoring devices enabled objective quantification of nonverbal behaviors and social dynamics [19].

Figure 2: Integrated Research Methodology for Crew Monitoring. This workflow illustrates the comprehensive approach combining multi-omics technologies with psychological assessment to elucidate the gut-brain axis in confined environments.

The psychological and physiological monitoring conducted during the Lunar Palace 365 mission represents a paradigm shift in how we approach human health in confined environments. The research demonstrated that:

• Integrated Monitoring is Essential: Isolated psychological or physiological assessments provide incomplete pictures; their integration reveals crucial gut-brain axis interactions [12].
• Microbiome Stability Correlates with Mental Health: The identification of specific psychobiotics and their mechanistic pathways provides tangible targets for countermeasures against anxiety and depression in extreme environments [12] [18].
• Personalized Approaches are Necessary: Significant individual variability in both psychological responses and microbial ecology underscores the need for personalized rather than one-size-fits-all health maintenance strategies [12].
• Plant Integration Benefits System Stability: The stabilizing effect of plant cabins on fungal communities suggests that BLSS design incorporating biological elements provides more than just nutritional benefits—it contributes to overall environmental and potentially psychological stability [17].

These findings provide a robust scientific foundation for future mission planning, suggesting that microbiome monitoring and targeted modulation could become standard components of crew health maintenance during long-duration space missions. The successful validation of psychobiotic efficacy in animal models paves the way for potential clinical applications in space medicine, potentially offering novel, non-pharmacological approaches to maintaining crew mental health on future missions to the Moon, Mars, and beyond.

Advanced Stability Monitoring Methods in a Closed Ecological System

Within the context of long-term material closure validation, the "Lunar Palace 365" experiment provided a unique opportunity to study microbial succession in a ground-based Bioregenerative Life Support System (BLSS). Understanding the dynamics of airborne microbial communities and antibiotic resistance genes (ARGs) in space life support systems is critically important, as potential pathogens and antibiotic resistance pose a significant health risk to crew that could lead to mission failure [4]. The Lunar Palace 1 (LP1) facility functions as a biosphere that regenerates oxygen, water, and food, allowing humans to survive in a confined space for extended periods [4]. This 370-day mission, which involved two shifts of crew members, represented an ideal setting to investigate how microbial communities respond to prolonged confinement and changes in human occupancy, thereby testing the system's ecological stability and safety [4].

This guide objectively compares the sequencing technologies and methodological approaches used to track bacterial population dynamics throughout this unprecedented experiment, providing researchers with crucial data for selecting appropriate platforms for long-term microbial monitoring.

Experimental Framework of the Lunar Palace 365 Study

The Lunar Palace 365 project was launched on May 10, 2017, by the Institute of Environmental Biology and Life Support Technology at Beihang University. A total of eight volunteers were divided into two groups (G1 and G2), each containing two females and two males, who spent a total of 370 days in the LP1 facility [4]. The project was divided into three distinct phases: the first phase lasted 60 days with crew G1, the second phase lasted 200 days with crew G2, and the third phase lasted 110 days with the return of crew G1 [4]. This crew rotation design enabled researchers to investigate the impact of human presence on microbial community succession.

Researchers collected 34 air dust samples from three key locations within the LP1 system: the plant cabins (I and II), comprehensive cabin, and solid waste treatment cabin [4]. Sampling was executed using high-efficiency particulate absorbing (HEPA) filters to ensure consistent uptake of microbial particles. To enable the sample biomass to meet sequencing requirements, ambient air was sampled continuously over discrete time intervals across the different mission phases and crew shifts [4].

Analytical Approaches for Comprehensive Microbiome Characterization

The study employed a multi-faceted analytical approach to thoroughly characterize the microbial communities:

• DNA Extraction and Amplification: Microbial DNA was extracted from collected air dust samples, followed by amplification of target genes for subsequent sequencing.
• Multi-Platform Sequencing: The analysis incorporated amplicon sequencing, shot-gun sequencing, and quantitative PCR (qPCR) to assess different aspects of the microbiome [4].
• Community and Functional Analysis: Researchers evaluated microbial diversity, species composition, functional potential, and antibiotic resistance profiles to gain a comprehensive understanding of community dynamics [4].
• Viability Assessment: While not used in the Lunar Palace study specifically, research in analogous closed environments has utilized propidium monoazide (PMA) treatment to differentiate viable/intact microbial populations from dead cells, providing a more accurate picture of the active community [20].

Comparative Performance of Sequencing Technologies

The choice of sequencing platform significantly impacts the resolution and accuracy of microbial community analysis. The table below compares the primary technologies relevant to long-term microbiome studies.

Table 1: Comparison of Sequencing Platforms for Microbiome Analysis

Platform	Read Length	Key Applications	Strengths	Limitations
Illumina	Short-read (100-400 bp) [21]	16S rRNA gene sequencing (V3-V4, V4 regions) [21]; Metagenome sequencing [22]	High accuracy; Cost-effective for diversity studies [22]	Limited taxonomic resolution due to short reads [21]
PacBio (Sequel IIe)	Long-read (Full-length 16S rRNA) [21]	Full-length 16S rRNA sequencing; Metagenome-assembled genomes (MAGs) [23]	High accuracy (>99.9%) with circular consensus sequencing; Superior species-level identification [21]	Higher input DNA requirements; Lower throughput
Oxford Nanopore (MinION)	Long-read (Full-length 16S rRNA) [21]	Full-length 16S rRNA sequencing; Metagenome sequencing; Real-time analysis [22]	Rapid turnaround time; Detection of a broader range of taxa compared to Illumina [22]	Higher inherent error rates requiring specialized analysis [21]
Shotgun Metagenomics (Various platforms)	Varies by platform	Functional potential analysis; MAG generation; ARG profiling [4]	Provides insights into functional genes and pathways; Unbiased community profiling [22]	Higher cost; Complex data analysis; Requires greater sequencing depth

Platform Selection for Taxonomic Resolution

For long-term microbial dynamics studies, taxonomic resolution is paramount. Recent comparative evaluations demonstrate that Oxford Nanopore Technologies (ONT) and PacBio provide comparable bacterial diversity assessments, with PacBio showing slightly higher efficiency in detecting low-abundance taxa [21]. Despite differences in raw sequencing accuracy, ONT produces results that closely match those of PacBio, suggesting that its inherent sequencing errors do not significantly affect the interpretation of well-represented taxa in a community [21]. Notably, a study analyzing mouse gut microbiota found that ONT captured a broader range of taxa compared to Illumina's 16S rRNA sequencing [22].

For closed system monitoring where specific pathogens or functional taxa must be tracked, long-read technologies offer significant advantages. Research in analogous closed habitats has identified differential microbial communities on various surface materials, with Actinobacteria, Firmicutes, and Proteobacteria dominating on linoleum, dry wall, and particle board surfaces, while members of Firmicutes and Enterobacteriaceae were more prevalent on glass/metal surfaces [20]. Such material-specific colonization patterns are crucial for designing microbial monitoring protocols in closed habitats.

Advancing Assembly Quality with Long-Read Technologies

The application of long-read sequencing technologies has dramatically improved the contiguity of metagenome-assembled genomes (MAGs). In a landmark study, researchers applied nanopore sequencing and a specialized workflow called Lathe to assemble closed bacterial genomes from complex microbiomes [23]. When tested on a synthetic mixture of 12 bacterial species, this approach successfully assembled seven genomes into single contigs and three more into four or fewer contigs [23]. This represents a substantial improvement over short-read assembly, which typically produces highly fragmented genomes.

The assembly contiguity achieved with long-read technologies is particularly valuable for investigating the role of repeat elements in microbial adaptation and function [23]. In the context of long-term missions, understanding how microorganisms adapt through genomic changes is essential for predicting and managing microbial evolution in closed systems.

Methodological Considerations for Accurate Microbiome Analysis

DNA Extraction and Library Construction Standards

Variability in DNA extraction and library construction methods represents a significant source of bias in microbiome studies. Comprehensive validation studies have compared a wide range of commercial kits for sequencing library construction, identifying those that provide the highest agreement with known "ground truth" microbial compositions [24]. These studies have established that protocol selection significantly impacts the observed microbial community profile, with certain kits introducing substantial GC bias that can overrepresent either low-GC or high-GC genomes depending on the protocol [24].

Standardized protocols for DNA extraction and library construction have been validated for both intra-laboratory precision and inter-laboratory reproducibility [24]. The adoption of such standardized methods is particularly crucial for long-term studies like Lunar Palace 365, where methodological consistency across sampling time points is essential for detecting true temporal changes rather than technical artifacts.

Addressing Compositionality and Sampling Depth Biases

Microbiome data generated by high-throughput sequencing is inherently compositional, meaning that measurements represent relative proportions rather than absolute abundances [25]. This compositionality presents significant challenges for data interpretation, as changes in the abundance of one taxon necessarily affect the apparent abundances of all others.

Table 2: Approaches for Handling Microbiome Data Challenges

Approach Type	Examples	Key Principles	Best Use Cases
Relative Transformations	Total-Sum Scaling (TSS) [26]	Normalizes data to relative proportions	Preliminary analysis; Stable-density communities
Compositional Transformations	Centered Log-Ratio (CLR), Additive Log-Ratio (ALR) [26]	Applies log-ratios to address data compositionality	Datasets without microbial load data
Quantitative Approaches	Microbial load scaling; Quantitative profiling [25]	Incorporates experimental microbial load data to transform to absolute counts	Scenarios with varying microbial loads; Dysbiosis studies
Rarefaction	Sequencing depth downsizing [25]	Randomly subsets data to even sequencing depth	Comparing richness across samples; Low-density communities

Benchmarking studies have demonstrated that quantitative approaches, which incorporate experimental determination of microbial loads, significantly outperform computational strategies designed to mitigate data compositionality and sparsity [25]. These methods not only improve the identification of true positive associations but also reduce false positive detection, making them particularly valuable for analyzing scenarios of low microbial load dysbiosis as observed in inflammatory pathologies [25].

Essential Research Reagents and Tools

The following table details key reagents and materials essential for implementing robust microbiome sequencing workflows in closed system studies.

Table 3: Research Reagent Solutions for Microbiome Sequencing

Reagent/Material	Function	Application Notes
HEPA Filters (Xiaomi Air Purifier 2) [4]	Collection of air dust samples for microbiome analysis	Enables continuous sampling to achieve sufficient biomass for sequencing [4]
Quick-DNA Fecal/Soil Microbe Microprep Kit (Zymo Research) [21]	DNA extraction from complex environmental samples	Effective for challenging samples; used in comparative platform studies [21]
Propidium Monoazide (PMA) [20]	Differentiation of viable/intact cells from free DNA/damaged cells	Critical for accurate assessment of active microbial communities in built environments [20]
SMRTbell Prep Kit 3.0 (PacBio) [21]	Library preparation for PacBio long-read sequencing	Enables full-length 16S rRNA gene sequencing for high taxonomic resolution [21]
Native Barcoding Kit 96 (Oxford Nanopore) [21]	Library preparation for nanopore sequencing	Facilitates multiplexing of samples for efficient long-read sequencing [21]
MetaPolyzyme Enzyme Cocktail [23]	Enzymatic lysis of difficult-to-lyse bacterial cells	Improves DNA extraction efficiency from Gram-positive bacteria in complex samples [23]
ZymoBIOMICS Gut Microbiome Standard (Zymo Research) [24]	Mock community for quality control and method validation	Essential for verifying extraction and sequencing performance across batches [24]

Workflow Visualization of Closed Habitat Microbiome Analysis

The following diagram illustrates the integrated workflow for microbiome analysis in closed habitat studies, from sample collection through data interpretation:

Microbiome Analysis Workflow in Closed Habitats

Key Findings from the Lunar Palace 365 Microbiome Study

Analysis of the airborne microbial communities throughout the 370-day Lunar Palace 365 experiment yielded several critical insights relevant to long-term closed habitat management:

• Human Influence on Microbial Succession: The study found that personnel exchange led to significant differences in bacterial community diversity. Source tracking analysis revealed that most bacteria in the air derived from the cabin crew and plants, demonstrating that human presence had the strongest effect on the succession of microbial diversity in the BLSS [4].

• Distinct Microbial Profile: The bacterial community diversity in the LP1 system was higher than that in a controlled environment but lower than that in an open environment, creating a unique microbial profile specific to the closed habitat [4].

• Antibiotic Resistance Considerations: While crew changes significantly influenced microbial diversity, no substantial differences in microbial function or antibiotic resistance were observed across crew shifts [4]. This finding suggests that functional profiles may remain stable despite compositional changes, an important consideration for risk assessment in long-duration missions.

• Material Selection Impacts: Research in analogous closed habitats has demonstrated that surface material selection significantly influences microbial community structure, with linoleum, dry wall, and particle board surfaces supporting more complex communities compared to metal and glass surfaces [20]. This highlights the importance of material selection in habitat design for microbial control.

The Lunar Palace 365 experiment demonstrated that integrated sequencing approaches successfully tracked bacterial population dynamics over an extended period in a closed habitat, providing valuable insights for future space exploration missions. The findings highlight that microbial diversity in BLSS is heavily influenced by changes in crew and is unique from other open and controlled environments [4].

These results can be used to help develop safe, enclosed BLSS that meet the requirements of human survival and habitation in outer space. Furthermore, the methodologies refined during this study enhance our understanding of indoor air microbial communities and contribute to strategies for effectively maintaining safe working and living environments, including areas dedicated to plant growth [4]. As we advance toward long-duration space missions, the lessons learned from microbial monitoring in the Lunar Palace 365 experiment will be instrumental in designing habitats that maintain microbial homeostasis and minimize risks to crew health and system functionality.

Airborne Microbial Community Analysis via Metagenomic Sequencing

The Bioregenerative Life Support System (BLSS) is a critical technology for long-term human survival in space exploration, creating a closed artificial ecosystem where oxygen, water, and food are recycled using biotechnology and engineering control technology [10]. Within these systems, understanding the dynamics of airborne microbial communities is paramount for crew health and system stability, as microorganisms can pose significant health risks through potential pathogens and antibiotic resistance genes (ARGs) that could lead to mission failure [11] [27]. The "Lunar Palace 365" experiment, a 370-day ground-based mission conducted in the "Lunar Palace 1" facility, provided an unprecedented opportunity to study these microbial dynamics in a high-closure, integrated environment with crew shifts [11] [10]. This mission achieved a remarkable 98.2% material closure degree, demonstrating excellent stability in recycling materials crucial for human survival [10].

Metagenomic sequencing has emerged as a powerful tool for comprehensive airborne microbiome analysis, enabling the detection of a wide range of microorganisms without prior targeting of specific pathogens. This approach is particularly valuable in closed systems like the Lunar Palace, where identifying potential pathogens and ARGs is essential for maintaining crew health during long-duration missions [11]. Unlike traditional culture methods that may miss uncultivable organisms, metagenomic approaches provide a complete profile of microbial communities and their functional potential, including virulence factors and antibiotic resistance mechanisms [28] [29].

Metagenomic Sequencing Methodology for Airborne Microbiomes

Sample Collection and Processing

Airborne microbial community analysis begins with effective sample collection, which is particularly challenging due to the low biomass nature of air samples [30]. In the Lunar Palace 365 experiment, air dust samples were collected from different areas and time points using high-efficiency particulate absorbing (HEPA) filters [11]. For optimal results, researchers should consider:

• Sampling Equipment: Coriolis μ sampler (250 L/min flow rate) and Andersen One-Stage Viable Particle Sampler (28.3 L/min flow rate) have been successfully employed in microbial air sampling [31].
• Sampling Duration: Typical sampling times range from 10-15 minutes, balancing sufficient biomass collection with practical operational constraints [31].
• Storage Conditions: Samples should be transported in cold chains and stored at -20°C until processing to preserve nucleic acid integrity [31].

For DNA extraction from low-biomass air samples, the QIAamp DNA Microbiome Kit has demonstrated superior performance compared to alternative methods, recovering significantly more bacterial reads (up to 80 times higher in comparative studies) [32]. This kit effectively lyses both gram-positive and gram-negative bacteria while minimizing host DNA contamination, a crucial factor for obtaining sufficient microbial DNA for sequencing [32].

Library Preparation and Sequencing

Following DNA extraction, libraries are prepared using kits such as the QIAseq Ultralow Input Library Kit, which is specifically designed for minimal input material [29]. Quality control of extracted DNA and prepared libraries is essential, with assessment methods including:

• Qubit Fluorometer for DNA concentration measurement
• Agarose Gel Electrophoresis for quality verification
• Agilent 2100 Bioanalyzer for library quality assessment [29]

Sequencing is typically performed on Illumina platforms (MiSeq, HiSeq 2500, or NovaSeq 6000) in paired-end 250 bases (PE250) mode, generating approximately 50,000-100,000 reads per sample for amplicon sequencing and significantly higher volumes (6-9 GB per sample) for metagenomic sequencing [33]. For samples with high host DNA contamination or complex microbiota, sequencing output may need to increase to 30-300 GB per sample to ensure sufficient microbial genome coverage [33].

Bioinformatics Analysis

The bioinformatics workflow for airborne metagenomic data involves multiple processing steps:

• Quality Control: Adapters, low-quality sequences, and short reads are removed from raw data
• Host DNA Removal: Human sequences are eliminated using reference databases (hg38) with tools like SNAP [29]
• Metagenomic Assembly: Co-assembly of multiple samples significantly improves genome fraction recovery and reduces assembly errors compared to individual assembly [30]
• Taxonomic Classification: Processed reads are aligned against microbial genome databases using alignment tools like Burrows-Wheeler Alignment [29]
• Functional Annotation: ARGs and virulence factor genes (VFGs) are identified using specialized databases

For amplicon sequencing data, QIIME 2 has emerged as the next-generation analysis pipeline, providing a reproducible, interactive, and efficient platform for microbiome analysis [33]. The shift from operational taxonomic units (OTUs) to amplicon sequence variants (ASVs) provides higher resolution in differentiating microbial sequences [33].

Table 1: Key Bioinformatics Tools for Airborne Metagenomic Analysis

Analysis Step	Recommended Tools	Key Features
Quality Control	Trimmomatic, FastQC	Removes adapters, low-quality bases
Host DNA Removal	SNAP, BWA	Uses reference genomes to filter host sequences
Assembly	MEGAHIT, SPAdes	Individual or co-assembly approaches
Taxonomic Classification	Kraken2, MetaPhlAn	Species-level identification
ARG Identification	ARG-ANNOT, CARD	Database-specific antibiotic resistance gene detection

Comparative Performance Analysis of Metagenomic Sequencing

Methodological Comparisons

Metagenomic next-generation sequencing (mNGS) demonstrates distinct advantages and limitations compared to conventional microbiological methods. A comprehensive study of 368 febrile patients with suspected infections revealed that mNGS exhibited significantly higher sensitivity (58.01% vs. 21.65%, p < 0.001) but lower specificity (85.40% vs. 99.27%, p < 0.001) compared to traditional culture methods [29]. The negative predictive value of mNGS was also superior (54.67% vs. 42.9%), making it particularly valuable for ruling out infections [29].

In clinical settings, mNGS has proven especially effective for diagnosing central nervous system (CNS) infections, with an overall sensitivity of 63.1%, specificity of 99.6%, and accuracy of 92.9% across 4,828 samples tested over a 7-year period [34]. Notably, when compared directly to CSF detection methods, mNGS sensitivity increased to 86%, and it identified 21.8% of infections that were missed by all other diagnostic methods [34].

For airborne microbiome analysis, mNGS outperforms amplicon sequencing in providing higher taxonomic resolution (species- or strain-level) and functional information, including ARG and VFG profiles [28] [33]. However, amplicon sequencing remains more cost-effective ($20-50 per sample) and suitable for large-scale studies, while metagenomic sequencing costs range from $100-300 per sample for library construction and sequencing alone [33].

Table 2: Performance Comparison of Microbial Detection Methods

Parameter	Metagenomic Sequencing	16S Amplicon Sequencing	Traditional Culture
Sensitivity	58.01%-86% [29] [34]	Limited to amplified taxa	21.65% [29]
Turnaround Time	2-5 days [34]	1-2 days	1-5 days [29]
Taxonomic Resolution	Species/strain level [33]	Genus level [33]	Species level
Functional Information	Comprehensive (ARGs, VFGs) [28]	Limited	Antibiotic susceptibility
Cost per Sample	$100-300 [33]	$20-50 [33]	$10-50

Technical Advancements: Co-Assembly Approach

A significant technical advancement in airborne metagenomics is the co-assembly approach, which pools sequencing reads from multiple samples before assembly. This method has demonstrated superior performance compared to individual assembly, achieving a higher genome fraction (4.94% ± 2.64% vs. 4.83% ± 2.71%), lower duplication ratio (1.09 ± 0.06 vs. 1.23 ± 0.20), and fewer misassemblies (277.67 ± 107.15 vs. 410.67 ± 257.66) [30].

Co-assembly also produces longer contigs, with one study reporting 762,369 contigs ≥500 bp totaling 555.79 million bp, compared to 455,333 contigs totaling 334.31 million bp with individual assembly [30]. These longer contigs facilitate more reliable identification of mobile genetic elements and functional genes, including ARGs associated with transposable elements.

The benefits of co-assembly increase with sequencing depth, plateauing at approximately 30 million reads, suggesting this as an optimal threshold for airborne metagenomic studies [30]. This approach is particularly valuable for detecting low-abundance ARGs that might be missed in individual assemblies.

Airborne Microbial Communities in Lunar Palace 365

Microbial Community Dynamics

Analysis of airborne microbiomes during the Lunar Palace 365 mission revealed that bacterial community diversity in the BLSS was higher than in controlled environments but lower than in open environments [11] [27]. Crew shifts represented the most significant factor influencing bacterial community succession, with source tracking analysis indicating that most airborne bacteria originated from cabin crew and plants [11].

Interestingly, despite changes in microbial diversity associated with crew shifts, no significant differences in microbial function or antibiotic resistance were observed throughout the mission [11]. This stability suggests that the BLSS maintained a functional homeostasis despite compositional shifts in the microbial community, possibly due to the system's robust design that incorporated biological components like plants for air regeneration.

The presence of plants in the BLSS contributed to the airborne microbial community while simultaneously supporting atmospheric regeneration through photosynthesis. The system maintained excellent gas balance, with CO2 concentration between 246 and 4131 ppm and O2 at approximately 20.95% throughout the 370-day mission [10].

Pathogens and Antibiotic Resistance Genes

While the Lunar Palace 365 study did not report significant pathogen detection, other studies of confined environments have identified potential pathogens in air samples. In hospital environments, airborne samples have contained potentially pathogenic bacteria including Bacillus spp., B. cereus, Clostridium spp., Enterococcus gallinarum, and Staphylococcus spp. [31].

A particularly relevant study from composting facilities—another environment with high biological activity—identified enhanced airborne human-pathogenic antibiotic-resistant bacteria (HPARB) carrying increased abundance and diversity of ARGs and VFGs compared to those in compost samples [28]. The core antibiotic resistome in air samples represented 18.58% of overall ARG subtypes but contributed to 86.31% of ARG abundance, with enriched core ARGs (including mexF, tetW, and vanS) observed at 2.16- to 13.36-times higher levels in air compared to compost [28].

Notably, Mycobacterium tuberculosis was prevalent in air samples and carried more ARG (6) and VFG (130) subtypes than those in compost [28]. The risk score for airborne antimicrobial resistance (AMR) in the composting facility was significantly higher than that in hospital and urban environments, highlighting the potential for AMR spread through airborne routes [28].

Visualization of Experimental Workflows

Airborne Metagenomic Analysis Workflow

Airborne Metagenomic Analysis Workflow: This diagram illustrates the comprehensive process from sample collection to results interpretation, highlighting key methodological options at each stage.

Bioinformatics Pipeline for Metagenomic Data

Bioinformatics Pipeline: This visualization outlines the sequential steps in processing metagenomic data, highlighting essential computational tools and databases at each analysis stage.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Essential Research Reagents and Materials for Airborne Metagenomic Analysis

Category	Product/Kit	Specific Application	Key Features
Sample Collection	Coriolis μ Sampler	High-volume air sampling	250 L/min flow rate; collects into PBS [31]
Sample Collection	Andersen One-Stage Viable Sampler	Viable particle collection	28.3 L/min; uses TSA or blood agar plates [31]
DNA Extraction	QIAamp DNA Microbiome Kit	Optimal for low-biomass air samples	Lyses gram-positive/negative bacteria; reduces host DNA [32]
Library Preparation	QIAseq Ultralow Input Library Kit	Library prep from minimal input	Designed for low DNA concentrations [29]
Sequencing	Illumina MiSeq Reagent Kits	Moderate-throughput sequencing	PE250 mode; ideal for amplicon studies [33]
Sequencing	Illumina NovaSeq Reagent Kits	High-throughput metagenomics	6-9 GB/sample for metagenomic sequencing [33]
Bioinformatics	QIIME 2 Platform	Amplicon data analysis	Reproducible, interactive microbiome analysis [33]
Bioinformatics	SNAP Software	Host DNA removal	Filters human sequences using hg38 database [29]

Metagenomic sequencing represents a powerful methodology for comprehensive airborne microbial community analysis in closed ecological systems like the Lunar Palace 365. The technique offers significant advantages over traditional culture and amplicon sequencing approaches, particularly through its ability to provide species-level taxonomic resolution and functional insights into antibiotic resistance genes and virulence factors.

The co-assembly bioinformatics approach has emerged as a particularly valuable advancement, significantly improving genome recovery while reducing errors compared to individual sample assembly [30]. This method enables more reliable detection of mobile genetic elements that facilitate the spread of antibiotic resistance—a crucial consideration for long-duration space missions where antibiotic options may be limited.

Findings from the Lunar Palace 365 experiment demonstrate that crew presence represents the primary driver of airborne microbial community succession in closed systems [11]. This insight highlights the importance of continuous microbial monitoring and establishes a foundation for developing effective mitigation strategies to maintain crew health during extended space missions.

As bioregenerative life support systems evolve toward greater closure and autonomy, metagenomic sequencing will play an increasingly vital role in verifying system safety and stability. The methodologies and insights generated from terrestrial experiments like Lunar Palace 365 provide critical groundwork for future long-duration lunar and Martian missions, where understanding and controlling airborne microbial communities will be essential for crew health and mission success.

Gut Microbiota-Psychological Health Correlation Studies

The gut-brain axis represents a complex, bidirectional communication network linking the gastrointestinal tract with the central nervous system. This interaction is fundamentally mediated by the gut microbiota, the diverse community of trillions of microorganisms residing in the human intestine. These microbes influence brain function and behavior through neural, endocrine, and immune pathways, including the production of neurotransmitters, regulation of the hypothalamic-pituitary-adrenal (HPA) axis, and modulation of systemic inflammation [35] [36]. Dysbiosis, or an imbalance in this microbial community, has been increasingly associated with a spectrum of mental disorders, including anxiety, depression, bipolar disorder, and schizophrenia [37] [36].

Research into these correlations is advancing novel microbiota-targeted interventions, such as specific probiotics, prebiotics, and dietary strategies, offering promising avenues for managing psychological health [38]. The "Lunar Palace 365" mission, a groundbreaking 370-day ground-based experiment, provides a critical context for this research. It demonstrated the feasibility of a Bioregenerative Life Support System (BLSS), achieving a 98.2% closure degree in recycling materials crucial for human survival [10]. This high-closure environment underscores the importance of understanding and managing the gut microbiome as an integral component of long-term human health in isolated, confined environments, mirroring the conditions expected in future lunar bases. The mission's success in maintaining environmental stability offers a unique framework for studying the gut-brain axis under rigorously controlled conditions [10].

Key Gut Microbiota Alterations in Psychological Disorders

Clinical studies consistently reveal specific changes in the abundance of certain gut bacterial taxa in individuals with mental health disorders compared to healthy controls. These alterations are summarized in the table below.

Table 1: Gut Microbiota Alterations Associated with Psychological Disorders

Mental Disorder	Increased Taxa	Decreased Taxa	Study Details (Participants)
Anxiety	Lactobacillales, Sellimonas, Streptococcus, Fusobacteria [37]	Firmicutes/Bacteroidetes ratio, Faecalibacterium spp., Lachnospira, Butyricicoccus [37]	Prospective study of 129 patients with ulcerative colitis and 49 non-UC patients with depression/anxiety [37]
Depression (MDD)	Akkermansia, Clostridiumsensustricto_1, Prevotella, Klebsiella, Firmicutes (phylum) [37]	Dialister, Fusicatenibacter, Lachnospira, Coprococcus spp., Bacteroidetes (phylum) [37]	Cross-sectional study: 24 patients with current depressive episode (CDE) and 16 HCs; 60 MDD patients and 60 HCs [37]
Bipolar Disorder (BD)	Clostridiaceae, Collinsella, Flavonifractor, Actinobacteria (phylum) [37]	Faecalibacterium, Ruminococcaceae [37]	Cross-sectional study: 115 BD patients and 64 HCs; 113 BD patients and 77 HCs [37]
Schizophrenia	Haemophilus, Veillonella, Lachnospiraceae, Collinsella, Lactobacillus, Proteobacteria [37]	Coprococcus, Ruminococcus, Roseburia, Adlercreutzia, Anaerostipes, Faecalibacterium [37]	Cross-sectional study: 42 acute schizophrenia patients, 40 in remission, and 44 HCs; 48 schizophrenia patients and 48 NCs [37]

A large-scale TwinsUK cohort study employed a novel pyramid-layer design to control for genetic and environmental confounders. This robust approach identified the genus Parabacteroides as being associated with the diagnosis of mental disorders, suggesting its potential role as a modifier in mental health [39]. Furthermore, research from the Houston Methodist team has advanced the pursuit of biomarkers, using sophisticated data analytics to narrow down candidate bacterial taxa associated with treatment-resistant anxiety and depression from 10,000 to 43 pro-inflammatory strains [40]. This reduction in gut microbial diversity is a recurring theme in psychiatric conditions, observed across different patient populations regardless of geography or lifestyle [40].

Experimental Protocols in Microbiota-Gut-Brain Axis Research

Microbiome Analysis Techniques

The field relies on culture-independent molecular techniques to profile the gut microbiome.

• 16S Ribosomal RNA (rRNA) Gene Sequencing: This is the most commonly used method. It involves extracting DNA from fecal samples, amplifying the highly conserved 16S rRNA gene using polymerase chain reaction (PCR) with universal primers, and then sequencing the amplified genes. This allows for the identification of different bacteria present in the sample to the genus or species level [35].
• Shotgun Metagenomics: A more comprehensive but expensive technique where all the extracted DNA in a sample is sequenced. This not only identifies which bacteria are present but also allows for an assessment of their functional potential by analyzing the entire set of genes present in the microbiome [35].
• Metabolomic Profiling: This involves the large-scale identification and quantification of small molecules, or metabolites, in biological samples like blood plasma or feces. It is crucial for understanding the functional output of the gut microbiome, including molecules like short-chain fatty acids (SCFAs) that are involved in gut-brain signaling [41].

Study Designs for Controlling Confounders

A significant challenge in the field is accounting for factors that influence the microbiome, such as diet, genetics, and medication.

• Co-twin Control Design: The TwinsUK study utilized a pyramid-layer design comparing monozygotic (MZ) and dizygotic (DZ) twins discordant for mental disorders. This design is powerful because MZ twins share nearly 100% of their genetic material, while DZ twins share about 50%. By comparing a twin with a disorder to their healthy co-twin, researchers can control for shared genetics, early-life environmental exposures, and many lifestyle factors, allowing for a clearer identification of microbiome changes associated with the disorder itself [39].
• Longitudinal Clinical Studies: Research like that conducted at Houston Methodist involves collecting microbiome data from patients at multiple time points during treatment. This approach helps determine whether changes in the gut bacterial population precede symptomatic improvement or vice versa, providing insights into causality and treatment response [40].

Intervention and Manipulation Studies

• Probiotic/Prebiotic Interventions: These are randomized controlled trials where participants are administered specific live beneficial bacteria (probiotics) or substrates that selectively enhance the growth of beneficial bacteria (prebiotics). The effects on mental health outcomes (e.g., depression and anxiety scores) are then monitored [38].
• Germ-Free and Antibiotic-Treated Animal Models: Germ-free (GF) animals, born and raised in sterile environments, are used to establish proof-of-principle for the microbiome's role in stress responsivity and behavior. Microbiome depletion using antibiotics provides a less extreme, clinically relevant model for studying the consequences of microbial disturbance [35].
• Fecal Microbiota Transplantation (FMT): This involves transferring fecal matter from a human donor to a recipient, effectively transferring the donor's microbial community. FMT from healthy donors to animal models or patients has been used to investigate the ability of the microbiota to transfer a health or disease phenotype [35].

Efficacy of Microbiota-Targeted Interventions

Interventions aimed at modulating the gut microbiome show potential for improving mental health. The table below compares the efficacy of different approaches based on current research.

Table 2: Efficacy of Microbiota-Targeted Interventions for Mental Health

Intervention Type	Example Strains/Substances	Reported Outcomes	Level of Evidence
Probiotics	Lactobacillus helveticus R0052 & Bifidobacterium longum R0175 (CEREBIOME); Multistrain (B. longum, B. bifidum, B. lactis, L. acidophilus) [38]	Improved clinical symptoms of depression; Reduction in anxiety symptoms in Generalized Anxiety Disorder [38]	Promising results in pilot studies and some RCTs; not yet supported as stand-alone therapy [38]
Prebiotics	Galactooligosaccharides (GOS) [38]	Reduction in preclinical anxiety in healthy females; No effect on depressive symptoms in MDD cohort [38]	Limited and inconclusive data; efficacy may depend on host factors and baseline microbiota [38]
Synbiotics	Combinations of probiotics and prebiotics [38]	Improvements in depression, stress, and anxiety in specific populations (e.g., patients with coronary artery disease) [38]	Emerging evidence; positive outcomes in recent studies but more research needed [38]
Whole Dietary Interventions	High intake of vegetables, fruits, and dietary fiber; Polyphenols; Omega-3 fatty acids [37] [38]	Positive association with mental health; Reduction in depressive symptoms; Proposed restoration of a eubiotic state [37] [38]	Extensive observational data; benefits partially mediated by gut microbiota [38]
Fecal Microbiota Transplant (FMT)	Transfer of entire microbial consortium from healthy donor [38] [35]	Reversal of adverse behaviors in animal models; Significant improvements in fatigue in IBD patients with comorbid depression [38]	Early stages for psychiatric disorders; insufficient data to support clinical use; safety risks remain [38]

It is important to note that the efficacy of these interventions can be highly variable. Strain specificity, dosage, study duration, and individual host factors such as baseline gut microbiota composition and habitual diet are critical determinants of success [38]. For instance, a combined dietary and probiotic approach was found to have a greater reducing effect on anxiety symptoms than each intervention alone, highlighting the potential of multi-faceted strategies [38].

Signaling Pathways and Mechanisms of Action

The gut microbiota influences brain function and mental health through multiple, interconnected biological pathways. The following diagram summarizes the primary mechanisms involved in the microbiota-gut-brain axis.

Figure 1: Key Signaling Pathways of the Microbiota-Gut-Brain Axis

The diagram illustrates four primary communication routes:

• The Neural Pathway: Gut bacteria can produce or influence neurotransmitters like GABA, serotonin, dopamine, and noradrenaline [35] [36]. These signals can directly stimulate the vagus nerve, which serves as a direct neural connection from the gut to the brain [37] [39].
• The Endocrine Pathway: The gut microbiome is integral to the development and function of the hypothalamic-pituitary-adrenal (HPA) axis, the body's central stress response system [35]. Dysbiosis can lead to HPA axis dysregulation, altering cortisol levels and impacting stress responsiveness and mood [39]. Microbes also modulate the metabolism of tryptophan, a precursor for serotonin, influencing its availability for brain function [37] [35].
• The Immune Pathway: Dysbiosis can trigger an immune response, increasing the production of pro-inflammatory cytokines [36] [40]. This systemic inflammation can increase the permeability of the blood-brain barrier (BBB), allowing inflammatory molecules to enter the brain and influence neural function [36]. Furthermore, gut bacteria produce short-chain fatty acids (SCFAs) like butyrate, propionate, and acetate through the fermentation of dietary fiber. SCFAs have systemic anti-inflammatory effects and are crucial for maintaining the integrity of the BBB [37] [39].

The Scientist's Toolkit: Key Reagents and Materials

Table 3: Essential Research Reagents and Materials for Gut-Brain Axis Studies

Item/Reagent	Function/Application	Key Details
16S rRNA Primers	Amplification of the bacterial 16S rRNA gene for taxonomic identification via sequencing.	Target highly conserved regions of the gene; allows for classification of bacteria present in a sample [35].
Probiotic Strains	Live microorganisms studied for their beneficial effects on host mental health when administered.	Common strains include Lactobacillus (e.g., L. helveticus) and Bifidobacterium (e.g., B. longum); often used in multi-strain formulations [37] [38].
Prebiotic Substances	Non-digestible food ingredients that selectively stimulate the growth of beneficial gut bacteria.	Examples include Galactooligosaccharides (GOS), dietary fibers, and alpha-lactalbumin [37] [38].
Germ-Free (GF) Mouse Models	Animals born and raised in sterile isolators to be completely free of all microorganisms.	Used as a "blank slate" to prove the microbiome's causal role in behavior and brain function [35].
Fecal Collection Kits	Standardized kits for the collection, stabilization, and transport of stool samples from human subjects or animal models.	Essential for ensuring the stability of microbial DNA/RNA for downstream sequencing analysis [35].
Shotgun Metagenomics Kits	Reagents for library preparation and whole-genome sequencing of all DNA in a sample.	Allows for both taxonomic profiling and functional analysis of the gut microbiome [35].
Metabolomics Kits	Kits for the extraction and analysis of small molecules (metabolites) from plasma, serum, or fecal samples.	Used to quantify microbial metabolites like Short-Chain Fatty Acids (SCFAs) and neurotransmitters [41].

The correlation between gut microbiota and psychological health is a robust finding, supported by a growing body of evidence detailing specific microbial alterations in psychiatric disorders and the efficacy of targeted interventions. The experimental paradigms and tools, ranging from twin studies to advanced sequencing technologies, are providing increasingly sophisticated insights into the mechanisms of the gut-brain axis. The context of long-term, closed ecological systems, as exemplified by the Lunar Palace 365 mission, highlights the profound importance of understanding and managing this relationship for human health in extreme environments [10].

Future research must focus on translating these findings into clinical practice. This will require larger, well-defined clinical cohorts, longitudinal studies to establish causality, and a deeper understanding of how individual factors like genetics, diet, and medication history influence the personal efficacy of microbiome-targeted therapies [38] [40] [41]. The development of microbiome-informed biomarkers holds significant promise for deciphering the biological basis of clinical heterogeneity in depression and other disorders, ultimately paving the way for precision medicine approaches and novel therapeutic development in psychiatry [41]. As the field moves forward, integrating multi-omics data and accounting for the complex, community-level behaviors of microbes within the gut ecosystem will be crucial for unlocking the full potential of the microbiota-gut-brain axis [42].

Water Purification System Performance and Validation Methods

The pursuit of long-term human survival in closed ecosystems, such as those required for lunar bases or deep space exploration, demands water purification systems of unparalleled efficiency and reliability. The Bioregenerative Life Support System (BLSS), as tested in the groundbreaking "Lunar Palace 365" experiment, represents the pinnacle of this technology, aiming for near-complete material closure. In this context, the performance and rigorous validation of water recycling systems are not merely an engineering challenge but a fundamental prerequisite for life support. This guide objectively compares the performance of various water purification technologies, from space-grade bioreactors to terrestrial point-of-use filters, by examining supporting experimental data. The analysis is framed within the validation methods pioneered in the Lunar Palace 365 mission, which achieved a 98.2% system closure with 100% water recycling, providing a critical benchmark for long-term material closure validation [10].

Performance Comparison of Water Purification Technologies

The following tables compare the performance of various water purification systems, from advanced space-grade bioreactors to commercial terrestrial filters, based on experimental data from controlled studies.

Advanced and Biological System Performance

Table 1: Performance of advanced institutional and biological water purification systems.

System Type	Key Contaminant Removal	Experimental Performance	Context & Validation
Gradient Nanocomposite RO Membrane [43]	Salt ions	Water permeance: 21.34 L h⁻¹ m⁻² bar⁻¹; Salt rejection: 96.08%	Lab-scale RO desalination; 35.8% energy saving in filtration process.
Membrane Biological Activated Carbon Reactor (MBAR) - Condensate Water [6]	Organic contaminants (CODMn)	Effluent CODMn: 0.74 ± 0.15 mg/L (met drinking standards)	"Lunar Palace 365" experiment; 370-day operation treating humidity condensate.
MBAR - Domestic Wastewater [6]	Organic contaminants, Nitrogen	Organic removal: 85.7% ± 10.2%; Stable nitrification performance	"Lunar Palace 365" experiment; treatment of sanitary wastewater.
Slow Sand Filtration [44]	Turbidity, Fecal Coliform	Turbidity removal: 83.51% ± 8.75% (dry season); Effective coliform removal	Evaluation of a municipal-scale plant in Shire Indassilassie, Ethiopia.

Point-of-Use Filter Performance

Table 2: Performance comparison of commercial point-of-use water filters after standard testing protocols.

Filter Name / Type	Heavy Metal Removal (Sample)	Inorganic Non-Metal Removal (Sample)	General Contaminant Removal
ZeroWater 5-Stage [45]	Chromium 6: 99%Lead: 99%Copper: 99.9%	PFOA/PFOS: 95.1%Fluoride: 99%Chlorine: 99%	Rated capacity: 15 gallons. Tested at pH 6.5 & 8.5.
Brita 2-Stage Standard [45]	Chromium 6: 93%Lead: 85%Copper: 93%	PFOA/PFOS: 0%Fluoride: 3%Chlorine: 95%	Rated capacity: 20 gallons. Tested at pH 6.5 & 8.5.
Ceramic Candle Filters (e.g., CCK) [46]	Not Specified	Fluoride: ~97% reduction (from 0.385 ppm to 0.010 ppm)	Tested over 6 months; significant reduction of fluoride.
Pot Ceramic Filter (PCF) [47]	Turbidity: 98-99%Total Dissolved Solids: 9-18%	E. coli: 4-5 LRV (Log Reduction Value)	Long-term (14-month) lab evaluation with spiked water.

Detailed Experimental Protocols and Validation Methodologies

Rigorous, standardized protocols are essential for generating comparable performance data and validating systems for critical applications like long-duration space missions.

Lunar Palace 365 MBAR Validation Protocol

The water recycle system in the Lunar Palace 365 experiment established separate, dedicated processes for different wastewater streams, including condensate, domestic wastewater, urine, and used nutrient solutions from hydroponics [6]. The validation of the Membrane Biological Activated Carbon Reactor (MBAR) technology followed an integrated workflow.

Core Methodology Steps: [6]

• System Design: Implementation of dedicated MBARs for different wastewater streams (CW-MBAR for condensate, DW-MBAR for domestic wastewater, etc.).
• Operational Parameters: Long-term operation (370 days) under closed-loop conditions with real crew members, monitoring hydraulic retention times and filtration rates.
• Performance Sampling: Periodic collection of influent and effluent water samples from each MBAR unit.
• Analytical Methods:
  • Organic Contamination: Measured by the CODMn index (Permanganate Index) to confirm drinking water standards were met.
  • Nitrogen Transformation: Analysis of NO3-N, NH4-N, and other species to track nitrification performance and urea hydrolysis.
  • Microbial Community Analysis: 16S rDNA sequencing to reveal the evolution of functional microbiota (e.g., Meiothermus, Rhodanobacter) responsible for system stability.

Filtration Plant and Point-of-Use Filter Evaluation

Microscopic Evaluation Technique (MET) for Filtration Plants: [48] This protocol evaluates a plant's ability to remove pathogen-sized particles.

• Sample Collection: A minimum of 300 gallons each of raw (influent) and finished (effluent) water is collected.
• Particle Analysis: Samples are analyzed microscopically for the presence and quantity of specific particulate groups, most critically Giardia-sized and larger particles.
• Performance Rating: Systems are rated based on the levels found in the effluent:
  • Excellent: Essentially all Giardia-sized debris is removed.
  • Good: Giardia-sized particles remain at "few" levels in 300 gallons.
  • Moderate/Questionable: Giardia-sized particles are at "moderate" levels.
  • Poor: Giardia-sized debris is in the "moderate to many" range, indicating vulnerability.

Point-of-Use Filter Testing (NSF/ANSI Standard): [45] [46]

• Water Preparation: Water samples are prepared with specific concentrations of contaminants (metals, inorganic non-metals) in accordance with NSF/ANSI standards. For contaminants outside the standard, concentrations of 10 times the EPA drinking water limit may be used.
• Filtration: The prepared water is passed through the filter at a controlled rate (e.g., 1 liter every 90 minutes) until the filter's rated capacity is exhausted.
• Analysis: Effluent water samples are analyzed using techniques like spectrophotometry or mass spectrometry to determine the remaining concentration of each contaminant [45] [46].
• Calculation: The percentage reduction for each contaminant is calculated from the initial and final concentrations.

The Scientist's Toolkit: Key Research Reagents and Materials

Table 3: Essential materials and reagents for advanced water purification research and validation.

Item Category	Specific Examples	Primary Function in Research & Validation
Membrane Materials	Graphene Oxide (GO) laminates, Cellulose support layers, Polyamide Thin Films [43]	Form the selective barrier for salt rejection and molecular separation in desalination and nanofiltration.
Biological Media	Mixed Microbial Consortia, Biological Activated Carbon [6]	Degrade organic contaminants and facilitate nitrification in bioreactor-based systems like the MBAR.
Analytical Standards	Spectrophotometry Kits (e.g., for Fluoride), COD Test Reagents, Ion Standards for ICP-MS [44] [46]	Quantify specific contaminant concentrations for accurate performance evaluation.
Model Contaminants	Escherichia coli K-12, PFOA/PFOS solutions, Specific salt solutions (NaCl, etc.) [45] [47]	Serve as challenge agents in controlled laboratory experiments to test filter efficacy and log reduction values.

The validation of water purification systems for extreme environments like the Moon relies on a multi-faceted approach, combining cutting-edge materials science with robust biological processes and stringent testing protocols. The Lunar Palace 365 experiment demonstrated that Membrane Biological Activated Carbon Reactors (MBARs) can successfully treat various wastewater streams to potable standards, supporting humans in a 98.2% closed system [10]. While advanced materials like gradient nanocomposite membranes show great promise for energy-efficient desalination [43], terrestrial point-of-use data reveals a wide performance spectrum, particularly for emerging contaminants like PFAS [45]. The continuous and long-term validation methodologies honed in ground-based BLSS facilities like Lunar Palace 1 are indispensable. They provide the critical data and operational confidence required to translate these technologies into reliable life support systems for the future of human space exploration.

Material Degradation Assessment in Controlled Environments

The pursuit of long-duration human space exploration, including the establishment of lunar bases, is contingent on the development of Bioregenerative Life Support Systems (BLSS) [10]. These are closed artificial ecosystems that recycle air, water, and food for crews using biological and physicochemical processes [8]. The long-term reliability of such systems depends not only on biological stability but also on the physical integrity of their constituent materials. Within the sealed environment of a BLSS, material degradation can lead to equipment failure, air quality issues, and potential system breaches, jeopardizing mission success and crew safety [49].

The "Lunar Palace 365" project, a landmark 370-day ground-based mission conducted inside the "Lunar Palace 1" (LP1) facility, serves as a critical test bed for validating these technologies [10]. This mission provides a unique framework for studying material degradation within a high-closure, controlled environment, simulating the conditions of a long-term extraterrestrial habitat. This guide objectively compares the assessment methodologies and findings on material stability from this experiment, providing a protocol for evaluating material performance in analogous closed-loop systems.

Material Degradation in a BLSS: Context from Lunar Palace 365

In any confined environment, particularly one aiming for near-total material closure like the LP1 system which achieved 98.2% closure [10], the degradation of materials presents a multifaceted challenge. Degradation can be defined as the undesirable change in the properties of a material, leading to a loss of performance [49]. Within a BLSS, these processes are driven by a complex interplay of physical, chemical, and biological factors.

The LP1 facility integrated plant cultivation cabins, a human habitat, and waste processing systems [5]. This combination creates a distinctive environment where materials are exposed to constant humidity from plant transpiration and human activity, fluctuating oxygen and carbon dioxide levels, and a diverse microbial community [10] [8]. Understanding degradation characteristics is essential, as the breakdown products can affect the biochemical environment, and material failure can disrupt the delicate balance of the closed ecosystem [49].

The primary goals of material degradation assessment within the Lunar Palace 365 context were to:

• Identify Vulnerable Components: Pinpoint materials and systems most susceptible to degradation, thereby informing future design choices.
• Monitor Degradation Progression: Establish baseline conditions and track changes over the mission's duration to understand degradation kinetics.
• Assess Biosafety and Environmental Impact: Evaluate whether material breakdown posed any risk to crew health or the function of the biological subsystems [8].

Comparative Assessment Methodologies

The assessment of material state in a controlled environment like LP1 relies on a combination of visual inspection, molecular analysis, and chemical characterization. The table below compares the key experimental protocols employed in the Lunar Palace 365 mission and related fields for evaluating material and environmental stability.

Table 1: Comparison of Key Assessment Methodologies for Degradation and Stability

Methodology	Primary Application in LP1	Key Experimental Protocol	Data Outputs
Visual Damage Cataloguing	Qualitative assessment of physical material degradation [50]	Systematic survey using a predefined glossary of damage terms (e.g., 'cracking', 'blooming', 'discoloration'). Documentation via high-resolution photography.	Objective description of degradation phenomena; independent of interpretation of underlying causes [50].
Microbial Community Analysis (16S rRNA sequencing)	Monitoring bacterial population dynamics on surfaces and equipment [8]	Surface sampling with sterile swabs; DNA extraction; PCR amplification of target genes; Illumina MiSeq sequencing; bioinformatic analysis for diversity and composition.	Bacterial community structure; temporal stability; relative abundance of taxa, including potential pathogens [8].
Fungal Community Analysis (ITS sequencing)	Profiling surface mycobiome and assessing mycotoxin potential [5]	Surface sampling with sterile swabs; DNA extraction; PCR amplification of the ITS1 region; Illumina sequencing; qPCR for specific mycotoxin genes (e.g., idh, tri5).	Fungal diversity and community structure; source tracking (e.g., plant vs. human origin); copy number of mycotoxin-related genes [5].
Fourier-Transform Infrared Spectroscopy (FTIR)	Chemical identification of polymers and degradation products (e.g., blooming) [50]	ATR-FTIR allows for non-destructive, on-site material identification. A beam of infrared light is passed through the sample, and the absorption spectrum is used to identify chemical functional groups.	Molecular fingerprint of materials; identification of polymer types and surface degradation products like plasticizers or additives [50].

Key Experimental Data and Findings from Lunar Palace 365

The integrated application of these methodologies during the 370-day mission yielded critical quantitative data on the system's environmental stability and its impact on materials.

Bacterial Population Dynamics and Biosafety

A primary concern was whether the closed environment would lead to the proliferation of harmful microorganisms that could degrade materials or pose a health risk. The bacterial dynamics study revealed a stable microbial community over the long-term operation [8]. Crucially, the research team observed a low abundance of potential pathogens, minimal antibiotic resistance genes, and, most significantly for material integrity, a negligible impact of the isolated bacterial strains on equipment materials [8]. This indicated a favorable biosafety profile and suggested that microbial-induced corrosion or degradation was well-controlled, a finding attributed to the integration of plants and the overall system balance.

Surface Fungal Diversity and Mycotoxin Potential

Fungi are known to cause material degradation and produce mycotoxins. Research within LP1 showed that, unlike other confined habitats, its fungal community was uniquely structured, with plants being the most significant source of surface fungi [5]. While crew turnover did perturb the fungal community, the presence of the plant cabin acted as a stabilizing buffer. Importantly, qPCR analysis targeting key mycotoxin genes found no significant differences in mycotoxin gene copy numbers across different locations or crew shifts [5]. This suggests that the BLSS design, centered on plant integration, did not promote an environment conducive to the accumulation of mycotoxins, which are often associated with material decay and health risks.

Table 2: Summary of Key Stability Findings from the Lunar Palace 365 Mission

Assessment Parameter	Key Finding	Implication for Material Degradation
System Closure Degree	98.2% of crucial materials were recycled and regenerated [10].	Demonstrates exceptional control over the internal environment, minimizing external variables and allowing accurate study of intrinsic degradation.
O₂ and H₂O Recycling	100% recycling of oxygen and water for human use was achieved [10].	Closed water loops maintain constant humidity, a key factor in hydrolytic degradation of polymers and metals.
Bacterial Community	Temporally stable with low pathogen abundance and minimal material impact [8].	Low risk of microbial-influenced corrosion (MIC) and biodegradation of polymers, ensuring longer material service life.
Fungal Community	Plant-driven, stable mycobiome with low and stable mycotoxin potential [5].	Reduces risk of fungal-mediated deterioration of organic materials (e.g., cables, insulation) and associated toxin release.

The Scientist's Toolkit: Essential Research Reagents and Materials

The following table details key reagents, materials, and tools essential for conducting assessments similar to those in the Lunar Palace 365 experiment, particularly for monitoring biological and material stability.

Table 3: Key Research Reagent Solutions for Material and Environmental Assessment

Item	Function/Application	Specific Example from Research Context
Sterile Swab & Salt Solution	Collection of surface microbial samples (bacteria and fungi) for DNA analysis [5].	Swabs stored in 0.85% NaCl solution used for sampling defined surfaces (10x10 cm area) in LP1 cabins [5].
FastDNA Spin Kit	Isolation of high-quality genomic DNA from complex environmental samples [5].	Used for extracting DNA from swab samples prior to PCR amplification and sequencing [5].
ITS1F/ITS2R Primers	PCR amplification of the fungal Internal Transcribed Spacer (ITS) region for metagenomic sequencing [5].	Primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) were used to profile the mycobiome [5].
KOD FX Neo Polymerase	High-fidelity PCR amplification for next-generation sequencing library preparation [5].	Used for robust amplification of the ITS region from low-biomass environmental samples in LP1 [5].
ATR-FTIR Spectrometer	Non-destructive chemical identification of polymers and surface degradation products [50].	Identifies chemical functional groups; used to analyze "blooming" phenomena and classify material composition [50].
Visual Damage Catalogue	Objective, consistent qualitative assessment of material degradation phenomena [50].	A predefined glossary with terms like 'blooming', 'cracking', and 'discoloration' standardizes observations across surveys [50].

Experimental Workflow for an Integrated Assessment

The comprehensive assessment of material degradation and environmental stability requires a structured workflow that integrates the methodologies described above. The following diagram visualizes this multi-stage process, from initial sampling to final interpretation, as implemented in studies like the Lunar Palace 365 mission.

Integrated Assessment Workflow: This workflow outlines the multi-phase process for evaluating material and environmental stability, from initial sampling to final assessment.

The "Lunar Palace 365" experiment demonstrates that a well-designed BLSS can maintain remarkable environmental and biological stability over long durations. The assessment protocols validated within this mission—ranging from molecular analysis of microbial communities to visual cataloguing of material defects—provide a robust framework for material degradation assessment in controlled environments. The key finding for system designers is that the biological components of a BLSS, particularly the integration of plants, contribute significantly to maintaining a stable and safe environment that is not conducive to aggressive material degradation [8] [5]. The data and methodologies presented in this guide offer a benchmark for future research and development aimed at ensuring the long-term viability of habitats for deep space exploration.

Challenges and System Optimization in Prolonged Isolation

Managing Microbial Succession and Antibiotic Resistance Genes (ARGs)

The success of long-duration space missions and extraterrestrial habitation hinges on the creation of robust, self-sustaining life support systems. Among the most critical challenges is managing the complex microbial ecosystems that inevitably coexist with human crews. The Lunar Palace 365 experiment, a 370-day ground-based test inside the Lunar Palace 1 (LP1) bioregenerative life support system (BLSS), provides unparalleled insights into microbial succession dynamics and the persistence of antibiotic resistance genes (ARGs) in a sealed environment [11]. Understanding these patterns is not merely an academic exercise but a fundamental requirement for ensuring crew health and mission success, as imbalances in the microbial community can lead to both infectious disease risks and functional disruptions within the life support system itself.

This guide compares the microbial and ARG profiles observed in the LP1 BLSS against other controlled environments, including the International Space Station (ISS) and spacecraft assembly cleanrooms. The objective data presented herein stem from rigorous, culture-independent molecular analyses, including 16S rRNA amplicon sequencing, shotgun metagenomics, and quantitative PCR (qPCR), providing a comprehensive toolkit for researchers evaluating microbial control strategies for long-term closed-loop systems [11] [51].

Comparative Analysis of Microbial Communities

Microbial communities are the unseen inhabitants of any built environment, and their composition is shaped by the unique constraints of the habitat. The following table summarizes key characteristics of microbial communities across different controlled environments, as revealed by high-throughput sequencing.

Table 1: Comparative Microbial Community Profiles in Closed and Controlled Environments

Environment	Dominant Bacterial Taxa/Features	Dominant Fungal Taxa/Features	Alpha Diversity (Compared to LP1)	Primary Microbial Sources
Lunar Palace 1 (BLSS)	Human- and plant-associated bacteria [11]	Higher abundance of Ascomycota and Basidiomycota [52]	Baseline (Higher than other closed systems) [52]	Crew (human skin, gut), plants [11] [52]
International Space Station (ISS)	Human skin-associated bacteria (e.g., Staphylococcus, Corynebacterium) [51]	Information missing	Lower [52]	Crew (human skin) [51]
Spacecraft Assembly Cleanroom	Bacillus, Acinetobacter (enriched in air) [53]	Information missing	Information missing	Human shedding [53]

Key Experimental Protocols for Microbial Community Analysis

The data in Table 1 were derived from standardized and reproducible experimental protocols. A summary of the core methodologies is provided below.

• Sample Collection: In the Lunar Palace 365 experiment, air dust samples were collected using a high-efficiency particulate absorbing (HEPA) filter from a Xiaomi Air Purifier. Surface samples were collected from various locations, including the plant cabin (PC), comprehensive cabin (CC), and solid waste treatment cabin (SC) [11] [52].
• DNA Extraction and Sequencing: Total genomic DNA was extracted from the collected samples. For bacterial community analysis, the 16S rRNA gene (e.g., V3-V4 region) was amplified and sequenced. For fungal community analysis, the Internal Transcribed Spacer (ITS1 or ITS2) region was targeted. Shotgun metagenomic sequencing was also employed for a gene-centric functional analysis [11] [52] [51].
• Bioinformatic Analysis: Sequencing reads were processed into Amplicon Sequence Variants (ASVs) using pipelines like QIIME 2 or DADA2. Taxonomic assignment was performed against reference databases (e.g., SILVA for bacteria, UNITE for fungi). Diversity metrics (alpha and beta diversity) were calculated to compare community structure within and between samples [52].

The following diagram illustrates the succession dynamics of microbial communities in the LP1 system in response to internal and external drivers.

Figure 1: Microbial succession drivers and outcomes in the Lunar Palace 1 BLSS. Crew changes significantly perturb the community, while plant cultivation promotes stability and diversity.

Comparative Analysis of Antibiotic Resistance Genes

The proliferation and persistence of ARGs represent a significant health risk in closed environments, where antibiotic options are limited and host immunity may be altered. The Lunar Palace 365 experiment and studies of other controlled environments have quantified these risks.

Table 2: Antibiotic Resistance Gene (ARG) Profiles Across Environments

Environment	Dominant ARG Types / Notable Genes	Key Findings & Quantification	Primary Influencing Factors
Lunar Palace 1 (BLSS)	Genes associated with β-lactam, cationic antimicrobial peptide, and vancomycin resistance [11]	No significant difference in ARG abundance observed between crew shifts [11]	Human presence; limited impact from crew rotation [11]
International Space Station (ISS)	ARGs associated with β-lactam, cationic antimicrobial peptide, and vancomycin; multidrug-resistance efflux pumps [51]	Increase in ARG and virulence factors over time; persistence of pathogenic metagenome sequences [51]	Microgravity-induced effects, prolonged microbial accumulation [51]
Spacecraft Assembly Cleanroom	blaTEM-1, tetW, ermB [53]	ermB showed high copy number and was enriched on surfaces compared to air [53]	Ecological niche (surface vs. air); human-derived microorganisms [53]

Key Experimental Protocols for ARG Analysis

The profiling of ARGs relies on sensitive molecular techniques to detect and quantify these genetic elements, even at low abundances.

• Quantitative PCR (qPCR): This method was used for the absolute quantification of specific, high-priority ARGs (e.g., ermB, bla_TEM-1) and mobile genetic elements (MGEs). The abundance of ARGs is often normalized to the 16S rRNA gene copy number to allow for cross-sample comparisons [11] [53].
• High-Throughput qPCR: This technology allows for the simultaneous quantification of hundreds of ARG subtypes in a single sample, providing a broad profile of the "resistome" [54].
• Shotgun Metagenomics: Total DNA is sequenced without targeting specific genes. The resulting sequences can be aligned against specialized ARG databases (e.g., CARD, ARDB) to identify and quantify a vast array of ARGs and associated MGEs, while also providing contextual information on the host bacterial species [51].

The diagram below outlines the experimental workflow for a comprehensive analysis of microbial communities and ARGs.

Figure 2: Integrated workflow for analyzing microbial communities and ARGs. Combining these methods provides a complete picture of the microbiome and resistome.

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful characterization of microbial succession and ARGs depends on a suite of specialized reagents and tools. The following table details key solutions used in the cited studies.

Table 3: Key Research Reagent Solutions for Microbiome and Resistome Analysis

Reagent / Solution	Primary Function in Analysis	Application Example
Propidium Monoazide (PMA)	A viability dye that penetrates membrane-compromised cells and intercalates with DNA, suppressing its amplification. This allows for the selective analysis of intact/viable cells [51].	Differentiating between living and dead microorganisms in ISS surface samples to assess the risk from viable pathogens [51].
High-Fidelity DNA Polymerase	Enzymatic amplification of target genes (e.g., 16S rRNA, ITS) with low error rates for high-quality next-generation sequencing library preparation [11] [52].	Generating amplicons for sequencing the bacteriome and mycobiome of LP1 environmental samples.
16S rRNA Gene Primers (e.g., 515F/806R)	Target-specific oligonucleotides designed to amplify hypervariable regions of the bacterial 16S rRNA gene for taxonomic classification [11].	Profiling the bacterial community composition in air dust samples from the Lunar Palace 1.
ITS Gene Primers (e.g., ITS1F/ITS2)	Target-specific oligonucleotides designed to amplify the fungal Internal Transcribed Spacer (ITS) region for taxonomic classification of fungi [52].	Characterizing the surface fungal diversity and identifying unique ASVs in the LP1.
ARG-Specific qPCR Assays	Pre-designed or custom TaqMan or SYBR Green assays for the sensitive and absolute quantification of specific antibiotic resistance genes (e.g., ermB, tetW) [11] [53].	Tracking the abundance and rebound of specific ARGs like tetX and sul1 during composting or in cleanrooms [53] [54].

The data generated from the Lunar Palace 365 experiment and comparable controlled environments provide a critical evidence base for managing microbial risks in closed systems. Key conclusions for long-term material closure validation include the overarching influence of human occupants on microbial succession, the persistence of ARGs even in the absence of direct antibiotic selection, and the stabilizing role played by integrated biological components like plant modules. The distinctively higher microbial diversity in the LP1 BLSS, compared to the ISS, suggests that systems incorporating multiple biological kingdoms may develop more robust and stable microbial ecologies. For future extraterrestrial habitats, microbial management strategies must move beyond simple sterilization and embrace an ecological perspective that monitors and manages the microbiome as an integral part of the life support system.

The success of long-term human space exploration and extraterrestrial habitation hinges on the development of robust Bioregenerative Life Support Systems (BLSS) that can maintain metabolic balance despite internal and external perturbations. Crew shift transitions represent a significant metabolic disturbance in these closed systems, altering resource consumption patterns and waste production. This review examines findings from the groundbreaking 370-day "Lunar Palace 365" mission, which achieved unprecedented 98.2% material closure while systematically investigating crew rotation impacts. We compare system performance across different crew groups, analyzing gas exchange dynamics, water recycling efficiency, and microbial community succession. Experimental data demonstrate that proper system design and management protocols can effectively buffer crew-induced disturbances, maintaining life support functionality despite significant metabolic perturbations. These findings provide critical validation of long-term material closure for future lunar bases and deep space missions.

Bioregenerative Life Support Systems (BLSS) represent the most advanced approach to maintaining human life in long-duration space missions, creating artificial ecosystems that recycle oxygen, water, and nutrients through biological and physicochemical processes [10]. Unlike earlier life support systems that relied primarily on physicochemical processes, BLSS incorporate higher plants, microorganisms, and technological subsystems to achieve higher degrees of material closure and regeneration [6].

The "Lunar Palace 365" project, conducted in the Lunar Palace 1 (LP1) facility at Beihang University, marked a significant milestone in BLSS development [10]. This 370-day ground-based experiment achieved the highest closure degree (98.2%) reported to date, with 100% recycling of oxygen and water for human use, while maintaining excellent system stability despite multiple crew rotations [10]. The mission architecture specifically investigated the impact of crew shifts on system metabolic balance, recognizing this as a critical operational challenge for long-duration space habitats where crew changes are inevitable.

Table 1: Lunar Palace 1 System Specifications

Parameter	Specification
Total Area	160 m²
Total Volume	500 m³
Cabin Components	2 plant cabins, 1 comprehensive cabin (with bedrooms, living room, bathroom, insect culturing room)
Crew Capacity	4 persons per shift
Mission Duration	370 days
Plant Species Cultivated	35 species
System Closure Degree	98.2%

Experimental Design and Methodologies

Lunar Palace 365 Mission Architecture

The Lunar Palace 365 project employed a structured three-phase experimental design specifically engineered to analyze crew transition effects [10] [17]. Eight volunteers were divided into two groups (Group I and Group II), each containing two females and two males, who inhabited the Lunar Palace 1 facility in a rotational sequence:

• Phase 1 (60 days): Group I initial residence
• Phase 2 (200 days): Group II residence (longest continuous habitation)
• Phase 3 (110 days): Group I returned, replacing Group II

This design enabled direct comparison of system performance under different crew metabolic patterns while maintaining continuous operation, effectively simulating crew rotations anticipated in actual lunar base operations [10].

Metabolic Monitoring Protocols

Comprehensive monitoring systems tracked key metabolic parameters throughout the mission:

Gas Exchange Analysis: O₂ and CO₂ concentrations were continuously monitored using integrated sensor arrays positioned throughout the facility. Fluctuations were tracked against crew activities and shift changes, with specific attention to dark phase dynamics when plant cabins switched from oxygen production to consumption [10].

Water Recycling Performance: Multiple Membrane Biological Activated Carbon Reactors (MBARs) were deployed for separate wastewater streams [6]:

• Condensate wastewater treatment via CW-MBAR
• Domestic wastewater processing via DW-MBAR
• Urine nitrogen recovery via Urine-MBAR
• Nutrient solution purification for hydroponic systems

Water quality parameters (CODₘₙ, nitrogen species, contaminants) were analyzed regularly to assess treatment efficiency and system stability [6].

Microbial Community Tracking: Surface and air samples were collected at seven time points (D58, D90, D123, D156, D216, D310, D330) from three primary locations (comprehensive cabin, plant cabin, solid waste treatment cabin) [17]. Molecular analysis included:

• ITS1 amplicon sequencing for fungal community characterization
• 16S rRNA sequencing for bacterial communities
• qPCR quantification of mycotoxin genes (idh, ver1, nor1, tri5)
• Shotgun metagenomics for functional potential assessment

Data Analysis Framework

Statistical analyses included source tracking to identify microbial origins, differential abundance testing to identify crew-associated changes, and multivariate statistics to correlate microbial shifts with system performance parameters [17] [4]. Metabolic balance was assessed through mass flow calculations comparing input and output streams across crew transition boundaries.

Impact Analysis of Crew Shift Transitions

Gas Exchange Stability During Crew Rotations

The BLSS demonstrated remarkable resilience to crew-induced disturbances in atmospheric balance. Despite significant differences in metabolic patterns between crew groups, the system maintained O₂ and CO₂ concentrations within acceptable ranges throughout all mission phases [10].

Table 2: Gas Concentration Stability Across Crew Transitions

Parameter	Pre-Transition (Group I)	Post-Transition (Group II)	Overall Mission Range
CO₂ Concentration	478-3981 ppm	483-4131 ppm	246-4131 ppm
O₂ Concentration	20.53%-20.91%	20.51%-20.94%	20.4%-21.0%
Daily CO₂ Fluctuation	100-2000 ppm	100-2500 ppm	100-2500 ppm
Disturbance Recovery Time	1-2 days	1-2 days	<3 days

Critical to this stability were active management strategies implemented during crew rotations. Researchers regulated soybean photoperiod and solid waste reactor activity to minimize atmospheric disruptions [10]. The system's robustness was particularly evident in its rapid recovery following transitions, with atmospheric disturbances typically stabilizing within 1-3 days despite the complete exchange of all four crew members.

Water Recycling Performance Resilience

The water recycle system exhibited consistent performance across crew transitions, demonstrating the robustness of MBAR technologies to metabolic variations:

Table 3: Water Treatment Efficiency Across Crew Groups

Treatment Process	Input Wastewater	Treatment Efficiency	Output Quality
Condensate Wastewater (CW-MBAR)	CODMn: 1.35-3.47 mg/L	Stable organic removal	CODMn: 0.74±0.15 mg/L (meets drinking standards)
Domestic Wastewater (DW-MBAR)	Organic concentration: 85.7±10.2% removal	Efficient nitrification	NO₃⁻-N: 145.57-328.59 mg/L
Urine Treatment (Urine-MBAR)	Urea hydrolysis	Nitrogen recovery	Conversion to NH₄⁺-N
Nutrient Solution Recycling	Phytotoxic organics removal	Plant growth maintenance	Reuse in hydroponic systems

The MBAR systems maintained stable microbial communities dominated by Meiothermus, Rhodanobacter, and Ochrobactrum genera, which demonstrated functional resilience despite crew-related perturbations [6]. This biological stability ensured consistent water purification performance across all mission phases, with purified condensate wastewater consistently meeting drinking water standards regardless of crew group composition [6].

Microbial Community Succession Patterns

Crew transitions induced significant but manageable perturbations in the microbial ecosystems:

Fungal Community Dynamics: Source tracking analysis revealed that plants served as the primary source of surface fungi (65.3% contribution), while human-associated fungi represented a smaller proportion (19.8%) [17]. Despite significant differences in fungal community diversity between crew groups (PERMANOVA, p<0.05), the plant cabins demonstrated remarkable stability, showing no significant fluctuations based on occupant changes [17]. Mycotoxin potential remained stable throughout crew rotations, with no significant differences in gene copy numbers for idh, ver1, nor1, or tri5 genes [17].

Bacterial Community Response: Air dust analysis revealed that bacterial community diversity in the LP1 system was higher than in controlled environments but lower than in open environments [4]. Crew changes significantly altered bacterial community composition, with human presence identified as the strongest driver of microbial succession. However, despite compositional changes, metabolic functional profiles remained stable, indicating functional redundancy within the microbial communities [4].

System Response and Stabilization Mechanisms

Diagram: System Response to Crew Transition Disturbances. The BLSS employed multiple stabilization mechanisms to maintain metabolic balance during crew rotations.

Plant Cabin Buffering Capacity

The integration of plant cabins proved crucial for stabilizing system metabolism during crew transitions. Plants functioned as dominant contributors to the surface fungal microbiome (65.3% source contribution) and helped maintain community balance despite human-induced fluctuations [17]. The 35 cultivated plant species demonstrated high production efficiency, fully meeting crew requirements for plant-based food while providing critical buffering capacity for gas exchange disturbances [10].

Plant cabins maintained stable fungal communities regardless of occupant changes, unlike comprehensive cabins which showed significant shifts with crew rotations [17]. This stabilizing effect underscores the importance of balanced ecological design in BLSS, where biological components can mitigate human-induced perturbations.

Microbial Functional Resilience

Despite compositional changes in microbial communities following crew rotations, the system maintained functional stability through several mechanisms:

• Functional Redundancy: Different microbial taxa performed similar metabolic functions, ensuring continuity in waste processing and resource recovery [4] [6].
• Established Biofilms: MBAR systems maintained stable microbial consortia dominated by Meiothermus, Rhodanobacter, and Ochrobactrum that persisted despite crew-related perturbations [6].
• Metabolic Flexibility: Microbial communities demonstrated adaptability to varying input streams resulting from different crew metabolic patterns.

Research Reagents and Methodological Toolkit

Table 4: Essential Research Reagents and Analytical Tools for BLSS Metabolic Studies

Reagent/Kit	Application	Function in Analysis
FastDNA Spin Kits (MP Biomedicals)	DNA isolation from surface and air samples	Extraction of high-quality microbial DNA for community analysis [17]
ITS1F/ITS2R Primers	Fungal community characterization	Amplification of ITS1 region for fungal identification and diversity assessment [17]
Mycotoxin-specific qPCR Primers (idh, ver1, nor1, tri5)	Mycotoxin potential assessment	Quantification of genes involved in mycotoxin production for safety monitoring [17]
HEPA Filters (Xiaomi Air Purifier 2)	Air dust collection	Standardized sampling of airborne microbial communities [4]
16S rRNA Primers	Bacterial community analysis	Characterization of bacterial diversity and composition in air and water systems [4] [6]
Sterile Swab Tubes (0.85% NaCl)	Surface sampling	Standardized collection of surface-associated microorganisms [17]

Comparative Performance Analysis

When compared to other life support system architectures, the Lunar Palace 1 BLSS demonstrated distinct advantages in handling crew transitions:

Versus Physicochemical Systems (e.g., ISS): The BLSS approach showed reduced dependency on consumables and better recovery of nutrients from waste streams [6]. While physicochemical systems demonstrate high reliability, they lack the buffering capacity provided by biological components during metabolic disturbances.

Versus Simpler Closed Systems: The incorporation of multiple biological components (plants, microorganisms) and technological systems (MBARs) created complementary stabilization mechanisms that simpler systems lack. The 98.2% closure degree achieved in Lunar Palace 365 significantly exceeds the performance of most previously reported systems [10].

Microbiome Uniqueness: The LP1 system exhibited significantly different fungal community structures compared to other confined habitats like the International Space Station and inflatable lunar/Mars analog habitat (ILMAH), with higher alpha diversity and distinct composition [17]. This diversity likely contributed to system resilience during crew transitions.

The Lunar Palace 365 mission provides compelling experimental validation that properly designed BLSS can maintain metabolic stability during crew shift transitions, achieving 98.2% material closure over 370 days of continuous operation [10]. Critical to this success were the system's multiple stabilization mechanisms: active gas management, plant-based buffering, resilient water recycling systems, and functionally redundant microbial communities.

Key findings with implications for future lunar base design include:

• Strategic Integration of Biological Components: Plants provide crucial buffering capacity against human-induced disturbances and should be integral to life support architecture [10] [17].

• Modular System Design: Separate processing streams for different waste types (condensate, domestic, urine) enable more stable operation during metabolic perturbations [6].

• Microbial Management: Understanding and managing microbial community dynamics is essential for maintaining system stability through crew rotations [17] [4] [6].

Future research should focus on optimizing crew transition protocols, developing real-time metabolic monitoring systems, and further elucidating plant-microbe-human interactions in closed environments. The experimental evidence from Lunar Palace 365 represents a significant advancement toward sustainable human presence beyond Earth, demonstrating that the metabolic challenges of crew rotations can be successfully managed through ecological design principles.

Psychological Stress Mitigation Through Identified Psychobiotics

The growing prevalence of stress-related mental disorders represents a critical global health challenge, driving research into novel therapeutic approaches that extend beyond conventional pharmacological treatments. Psychobiotics—live microorganisms that confer mental health benefits through the gut-brain axis—have emerged as promising agents for modulating the body's stress response systems [55] [56]. This review systematically evaluates the efficacy of specific psychobiotic strains in mitigating psychological stress, with particular emphasis on their mechanisms of action, experimental protocols, and comparative effectiveness. The findings are contextualized within the framework of long-term material closure validation, drawing insights from the "Lunar Palace 365" experiment research on bacterial community dynamics in confined environments [8].

The gut-brain axis represents a complex bidirectional communication network linking emotional and cognitive centers of the brain with peripheral intestinal functions. This signaling occurs through multiple pathways including neural connections (particularly the vagus nerve), immune mediators, and endocrine routes [56] [57]. Within this framework, psychobiotics influence central nervous system function and behavior through the production of neuroactive compounds, regulation of the hypothalamic-pituitary-adrenal (HPA) axis, and modulation of inflammatory responses [55] [57].

Mechanisms of Action: The Psychobiotic-Gut-Brain Signaling Axis

Psychobiotics exert their stress-modulating effects through multiple interconnected biological pathways. Understanding these mechanisms is essential for appreciating their therapeutic potential and contextualizing experimental findings.

Table 1: Primary Mechanisms of Psychobiotic Action in Stress Mitigation

Mechanism	Biological Process	Key Psychobiotic Metabolites	Physiological Outcome
Neurotransmitter Regulation	Synthesis of GABA, serotonin, dopamine, norepinephrine	GABA, serotonin precursors, dopamine metabolites	Reduced anxiety, improved mood, stress resilience
HPA Axis Modulation	Regulation of cortisol/corticosterone release	Short-chain fatty acids (SCFAs), neuropeptides	Attenuated stress response, normalized cortisol patterns
Immune Function Modulation	Control of inflammatory cytokine production	SCFAs (butyrate, acetate, propionate), anti-inflammatory cytokines	Reduced neuroinflammation, improved gut barrier integrity
Gut Permeability Reduction	Enhancement of intestinal barrier function	SCFAs, tight junction proteins	Prevented "leaky gut," reduced systemic inflammation

The following diagram illustrates the primary signaling pathways through which psychobiotics modulate the stress response:

Comparative Efficacy of Psychobiotic Strains

Systematic analysis of clinical and preclinical studies reveals significant variations in stress-mitigating efficacy across different psychobiotic strains. The table below summarizes quantitative findings from key intervention studies:

Table 2: Comparative Efficacy of Psychobiotic Strains in Stress Reduction

Psychobiotic Strain	Study Duration	Population	Stress Reduction Outcome	Biomarker Changes
Lactobacillus rhamnosus [58]	4 weeks	Healthy volunteers (N=88)	Significant reduction in negative mood (p<0.05)	Reduced cortisol levels [55]
Bifidobacterium longum [55]	4-8 weeks	Mixed clinical and healthy	Improved gut-brain axis regulation	Cortisol modulation, HPA normalization
Lactobacillus plantarum [59] [57]	8 weeks	Mild-to-moderate depression (N=60)	Significant PHQ-9 reduction (19.3±2.9 to 9.0±1.9, p<0.05)	Enhanced neurotransmitter production
Multi-species probiotic [58]	4 weeks	Healthy volunteers	Reduced negative mood after 2 weeks	Not specified
Bifidobacterium breve [57]	4-24 weeks	Various clinical populations	Promising effects on depressive symptoms	SCFA production, inflammatory control

The evidence indicates that multi-strain formulations often demonstrate superior efficacy compared to single-strain interventions [60]. This synergistic effect likely stems from complementary mechanisms of action across different bacterial species, creating a more comprehensive modulation of the gut-brain axis.

Experimental Protocols and Methodologies

Standardized Psychobiotic Intervention Protocol

Robust experimental design is essential for evaluating psychobiotic efficacy. The following workflow visualizes a comprehensive methodology adapted from recent high-quality studies:

Key Methodological Considerations

Participant Selection: Studies typically employ strict inclusion criteria, excluding individuals with recent antibiotic or probiotic use, significant gastrointestinal disorders, or severe psychiatric conditions [58]. Sample sizes are determined through power calculations to ensure adequate statistical power, typically targeting 80-90% power at α=0.05 [58].

Intervention Protocols: Psychobiotic interventions commonly utilize freeze-dried powders containing 1×10^9 to 2.5×10^9 colony-forming units (CFU) per gram, administered daily in sachets dissolved in lukewarm water [58]. Placebo controls consist of identical-looking sachets containing only the carrier materials (e.g., maize starch and maltodextrins) without active bacterial strains.

Outcome Measures: Comprehensive assessment includes both psychological and physiological metrics. Psychological measures encompass validated self-report questionnaires such as the Perceived Stress Scale (PSS), State-Trait Anxiety Inventory (STAI), and Positive and Negative Affect Schedule (PANAS) [58]. Physiological biomarkers include salivary cortisol levels, inflammatory markers (e.g., cytokines), and gut permeability indicators.

Research Reagent Solutions Toolkit

Table 3: Essential Research Materials for Psychobiotic Stress Studies

Reagent/Material	Specifications	Research Function	Example Application
Probiotic Strains	Lactobacillus rhamnosus, Bifidobacterium longum (1×10^9 - 2.5×10^9 CFU/g)	Experimental intervention	Daily supplementation in freeze-dried form [58]
Placebo Materials	Maize starch, maltodextrins	Control condition	Matched for color, taste, and smell to active intervention [58]
Cortisol Assay Kits	Salivary cortisol ELISA	HPA axis activity biomarker	Diurnal cortisol measurement, stress response assessment [55]
Microbiome Analysis	16S rRNA sequencing	Gut microbiota composition	Pre/post-intervention microbial community analysis [57]
Inflammatory Marker Panels	Cytokine ELISA (IL-6, TNF-α, CRP)	Systemic inflammation assessment	Monitoring immune modulation by psychobiotics [56]
Psychological Assessments	Validated questionnaires (PSS, STAI, PANAS)	Subjective stress and mood measurement	Baseline and endpoint psychological evaluation [58]

Implications for Closed Ecological Systems: Lunar Palace 365 Context

The "Lunar Palace 365" experiment provided valuable insights into bacterial community dynamics within closed bioregenerative life support systems (BLSS) [8]. Key findings demonstrated that despite distinct bacterial compositions across different functional areas and crew members, temporal stability was maintained throughout the 370-day experiment [8]. Notably, the system exhibited low abundance of potential pathogens and minimal antibiotic resistance, suggesting favorable biosafety attributes [8].

These findings have significant implications for psychobiotic applications in long-duration space missions. The demonstrated stability of bacterial populations in closed systems supports the feasibility of maintaining beneficial psychobiotic strains throughout extended missions. Furthermore, the observation that plant integration enhances environmental biosafety [8] suggests potential synergies between psychobiotic supplementation and plant-based life support systems for comprehensive crew health maintenance.

This systematic evaluation demonstrates that specific psychobiotic strains, particularly Lactobacillus rhamnosus, Bifidobacterium longum, and Lactobacillus plantarum, exhibit significant potential for mitigating psychological stress through multiple gut-brain axis pathways. The most consistent benefits emerge from multi-strain formulations administered over 4-8 week periods, with measurable improvements in both psychological symptoms and physiological stress biomarkers.

Future research should prioritize standardized methodologies, personalized strain selection based on individual microbiome profiles, and investigation of synergistic effects between psychobiotics and conventional stress management approaches. The integration of psychobiotic interventions within closed ecological systems, as validated by Lunar Palace 365 research, presents a promising avenue for maintaining crew mental health during long-duration space missions while offering novel approaches to stress management in terrestrial populations.

Equipment Failure Scenarios and System Resilience Protocols

The "Lunar Palace 365" experiment, a 370-day ground-based isolation mission conducted in the Lunar Palace 1 (LP1) facility, represents a seminal study in validating long-term material closure for bioregenerative life support systems (BLSS) [10] [1]. This research provides critical experimental data on system resilience when confronting equipment failures and operational disruptions. The LP1 facility, encompassing a 160 m² area with 500 m³ volume, was designed as an artificial closed ecosystem integrating human, plant, animal, and microbial components to achieve high-degree material circulation [11] [5]. Facing the requirements for future lunar bases, the mission specifically tested the system's robustness under various disturbance scenarios, establishing crucial protocols for maintaining life support functionality during equipment-related emergencies [10] [1].

Quantitative Analysis of System Performance Under Disruption

The Lunar Palace 365 mission demonstrated exceptional system closure and maintained core life support functions despite intentional simulated failures. The data reveal a highly resilient system capable of sustaining human life under disrupted conditions.

Table 1: Overall System Performance Metrics During Lunar Palace 365 Mission

Performance Parameter	Normal Operation	During Disturbance	Recovery Post-Disturbance
Mission Duration	370 days total	Included multiple simulated failures	Maintained full duration
System Closure Degree	98.2% [10]	Minimal degradation	Full recovery demonstrated
O₂ Recycling Rate	100% [10]	Maintained	100% maintained
Water Recycling Rate	100% [10]	Maintained	100% maintained
Plant Food Production	Fully met crew needs [10]	Temporary perturbations	Re-established stability
Urine Recovery	99.7% [10]	Maintained	99.7% maintained
Solid Waste Recovery	67% [10]	Maintained	67% maintained

Table 2: Documented Failure Scenarios and System Responses

Failure Scenario	Impact on System	Resilience Protocol	Outcome
Power Failure	Disruption of environmental controls	Biological modulation technologies [1]	Maintained gas balance
Equipment Malfunction	CO₂/O₂ concentration fluctuations	Adjustment of soybean photoperiod and solid waste reactor activity [10]	Rapid minimization of disturbance effects
Crew Shift Changes	Metabolic rate variations affecting gas balance	Active gas management strategies [10]	System stability maintained
Extended Duration (5-day overstay)	Psychological and resource stress	Pre-planned contingency protocols [1]	Successful mission completion

Experimental Protocols for Resilience Validation

Mission Architecture and Crew Rotation

The 370-day Lunar Palace 365 experiment was strategically divided into three phases with crew rotations specifically designed to test system resilience to human metabolic variations [10] [11]. The protocol implementation followed this structure:

• Phase 1: Group I (4 crew members) inhabited the facility for 60 days [10]
• Phase 2: Group II (4 different crew members) occupied the facility for a record-breaking 200 days [10]
• Phase 3: Group I re-entered the cabin, replacing Group II, for the final 110 days [10]

This rotational approach enabled researchers to study how the BLSS maintained stability during inevitable crew changes in long-term missions, particularly examining disturbances to O₂ and CO₂ balance caused by variations in human metabolic rates [10]. The experimental design recognized that gas balance cannot be achieved by biological self-organization alone and requires active human intervention, learning from previous failures such as the Biosphere 2 project which was suspended due to gas imbalance [10].

Biological Modulation Technologies

The core resilience protocol tested in Lunar Palace 365 centered on biological modulation technologies to maintain system stability during equipment failures and other disruptions [1]. The key methodologies included:

• Plant Photoperiod Adjustment: Regulating the light cycles of soybean cultivation to manipulate photosynthetic activity for O₂ production and CO₂ consumption [10]
• Solid Waste Reactor Activity Control: Modifying the operation of solid waste bioreactors to manage CO₂ emission rates [10]
• Microbial Community Management: Leveraging defined microbial consortia in water recycling systems to maintain treatment performance during operational perturbations [6]

These biological modulation approaches proved highly effective, with the system demonstrating "strong robustness" and the capability to "quickly minimize the effects of disturbances" [10]. This represented a significant advancement over previous BLSS implementations that relied more heavily on energy-intensive mechanical systems for environmental control.

Water Recycling System Resilience

The water recycling system in Lunar Palace 1 employed multiple Membrane Biological Activated Carbon Reactors (MBARs) to process different wastewater streams, providing inherent resilience through functional redundancy [6]. The experimental protocol included:

• Separate Treatment Trains: Independent MBAR systems for condensate wastewater, domestic wastewater, urine, and used nutrient solutions [6]
• Microbial Community Monitoring: Regular 16S rDNA sequencing to track the evolution of microbial diversity and composition in various MBARs during long-term operation [6]
• Performance Validation: Comprehensive water quality testing to verify treatment efficacy, with results showing purified condensate wastewater achieving a CODMn index of 0.74 ± 0.15 mg/L, meeting drinking water standards [6]

This approach demonstrated stable organic contaminant removal (85.7% ± 10.2% for domestic wastewater) and nitrification performance despite the extended duration and simulated failure scenarios [6]. The system maintained dominant microbial populations including Meiothermus, Rhodanobacter, and Ochrobactrum throughout the mission, indicating ecological stability [6].

System Interrelationships and Resilience Protocols

The following diagram illustrates the integrated relationship between failure scenarios and the biological resilience protocols that maintained system stability in the Lunar Palace 365 experiment:

Integrated Failure Response in Lunar Palace 365

Research Reagent Solutions for BLSS Resilience Testing

The following essential materials and reagents were critical for implementing and monitoring the resilience protocols in the Lunar Palace 365 experiment:

Table 3: Key Research Reagents and Materials for BLSS Resilience Experiments

Reagent/Material	Application Function	Experimental Role
MBAR Systems	Membrane Biological Activated Carbon Reactors	Wastewater treatment and nutrient recovery [6]
16S rDNA Sequencing Kits	Microbial community analysis	Tracking evolution of functional microbiota in water systems [6]
FastDNA Spin Kits	DNA extraction from environmental samples	Microbial monitoring of air and surfaces [5]
ITS1F/ITS2R Primers	Fungal community analysis	Surface mycobiome characterization [5]
qPCR Reagents	Antibiotic resistance gene quantification	Monitoring ARG distribution in enclosed environment [61]
35 Plant Species	Bioregenerative components	Oxygen production, food supply, and CO₂ consumption [10]
Yellow Mealworms	Animal protein production	Waste conversion and nutritional supplementation [10]

The Lunar Palace 365 experiment represents a significant milestone in demonstrating the resilience of bioregenerative life support systems against equipment failures and operational disruptions. Through intentional simulation of power failures, equipment malfunctions, and crew rotations, the project validated biological modulation technologies as effective countermeasures against system destabilization. The achieved 98.2% system closure degree under these challenging conditions provides crucial validation data for designing future lunar habitats. Particularly noteworthy is the demonstration that biological systems can be actively managed to maintain gas balance and resource recycling when mechanical systems are compromised. These findings substantially advance our understanding of long-term material closure in controlled ecological systems and provide essential protocols for ensuring human survival in future deep space exploration missions.

Plant Cultivation Optimization for Continuous Life Support

The pursuit of long-term human presence on the Moon necessitates the development of robust Bioregenerative Life Support Systems (BLSS), where higher plant cultivation serves as the core functional unit for providing oxygen, fresh food, and water recycling [62]. The "Lunar Palace 365" experiment, a landmark 370-day closed-ground test, was a critical step in validating the material closure and long-term viability of such systems [6] [4]. A central challenge within a BLSS is optimizing plant cultivation to achieve maximum productivity and sustainability while using in-situ resources. This guide compares the performance of two key optimization approaches tested within the Lunar Palace research context: the integration of earthworms to improve soil substrates and the deployment of advanced water recycling technologies for hydroponic systems.

Comparative Performance Analysis of Optimization Approaches

The following table summarizes the quantitative performance data for the two primary plant cultivation optimization strategies investigated.

Table 1: Performance Comparison of Cultivation Optimization Approaches

Optimization Approach	Key Performance Metrics	Experimental Results	Comparative Efficacy
Earthworm-Enhanced Substrate [62]	Wheat Growth Parameters:- Plant production- Growth metrics	Achieved approximately 80% of growth parameters compared to the positive control (vermiculite with nutrient solution) [62].	Effectively supports "seed-to-seed" cultivation using in-situ resources, though not yet matching ideal growth media.
	Substrate Physical Properties:- Bulk density- Hydraulic conductivity	22.4% reduction in bulk density; 14% increase in hydraulic conductivity with 45 earthworms [62].	Significantly mitigates lunar soil simulant compaction, improving root access to water and oxygen.
	Substrate Chemical Properties:- Salinity- Soil Organic Matter (SOM)	Successive mitigation of salinity; 24.9% increase in SOM content with 45 earthworms [62].	Effectively addresses salinization from solid waste and enhances soil fertility.
Membrane Biological Activated Carbon Reactor (MBAR) [6]	Water Purification Performance:- Condensate Wastewater (CODMn)	Effluent CODMn of 0.74 ± 0.15 mg/L, meeting drinking water standards [6].	Provides high-quality process water and potable water from wastewater streams.
	Nutrient Solution Recycling:- Organic contaminant removal	85.7% ± 10.2% removal rate of organic contaminants from used nutrient solutions [6].	Enables high-rate recycling of nutrient solutions for hydroponic systems, closing the water loop.
	Nitrogen Recovery:- Nitrification performance	Effluent NO³⁻-N concentrations from domestic wastewater treatment fluctuated from 145.57 mg/L to 328.59 mg/L [6].	Facilitates the recovery and conversion of nitrogen for use as a plant nutrient.

Detailed Experimental Protocols

Earthworm-Mediated Soil Improvement Protocol

This experiment was designed to test the complementary effects of earthworms on a substrate of lunar soil simulant and organic solid waste [62].

• Experimental Groups: The study established five distinct groups:
  • Positive Control (V): Wheat cultivated in vermiculite with nutrient solution, representing ideal conditions.
  • Negative Control (LS): Wheat cultivated in pure lunar soil simulant.
  • Treatment Groups: Lunar soil simulant amended with solid waste and varying earthworm abundances: 15 (LS+15ew), 30 (LS+30ew), and 45 (LS+45ew) individuals [62].
• Cultivation System: A whole-life-span wheat cultivation ("from seed to seed") was conducted. Organic solid waste, a simulated byproduct of BLSS (from plant residues and human feces), was placed in diagonal-corner feeding zones for the earthworms to gradually digest, transfer, and mix with the lunar soil simulant [62].
• Key Measurements:
  • Substrate Physical Properties: Bulk density and hydraulic conductivity were measured to assess compaction.
  • Substrate Chemical Properties: pH, electrical conductivity (EC), salinity, total nitrogen, soil organic matter (SOM), and humus content were analyzed post-harvest (day 65).
  • Plant Parameters: Morphological, agronomic, and physiological parameters of the wheat were measured and compared against the control groups [62].

Water Recycling and Nutrient Management Protocol

This protocol outlines the operation of the water recycle system during the "Lunar Palace 365" experiment [6].

• System Design: The water recycle system employed separate Membrane Biological Activated Carbon Reactors (MBARs) to treat four distinct wastewater streams: condensate wastewater, domestic wastewater, urine, and used nutrient solutions from hydroponics [6].
• Operational Parameters: The system was operated continuously for 370 days. The MBARs were inoculated with an enriched but undefined, mixed microbial community. Key operational data, including contaminant concentrations in influent and effluent, were monitored throughout the experiment [6].
• Key Measurements:
  • Water Quality: The Chemical Oxygen Demand (CODMn) index was used to track the removal of organic contaminants.
  • Nitrogen Cycle Management: The concentration of nitrate-nitrogen (NO³⁻-N) in the effluent was measured to assess nitrification performance. The hydrolysis of urea in urine to ammonium-nitrogen (NH⁴⁺-N) was also monitored to evaluate nitrogen recovery [6].
  • Microbial Analysis: 16S rDNA sequencing was performed on samples from the MBARs to reveal the evolution of microbial diversity and identify dominant microorganisms, such as Meiothermus, Rhodanobacter, and Ochrobactrum [6].

System Workflows and Functional Relationships

The following diagram illustrates the complementary roles of the two optimization approaches within a closed-loop BLSS, highlighting their contributions to material closure.

The Scientist's Toolkit: Key Research Reagents and Materials

For researchers aiming to replicate or build upon these experiments, the following table details the essential materials and their functions.

Table 2: Essential Research Materials for BLSS Plant Cultivation Studies

Material/Reagent	Function in Research	Specific Application Example
Lunar Soil Simulant	Physically and chemically mimics real lunar regolith for experimental cultivation studies [62].	Serves as the base, in-situ resource for plant growth substrates, allowing testing of improvement techniques [62].
Earthworms (Eisenia fetida)	Acts as a "soil engineer" to digest solid waste, form soil aggregates, and mitigate substrate compaction [62].	Integrated into treatment groups to vermicompost solid waste and improve the structure of lunar soil simulant [62].
Organic Solid Waste	Simulates the inevitable organic byproduct (from crew and plants) within a BLSS, used as a soil amendment [62].	Amended to lunar soil simulant to provide organic matter, with and without earthworms, to test fertility improvements [62].
Membrane Biological Activated Carbon Reactor (MBAR)	A bioreactor for wastewater treatment, combining biodegradation with physical filtration and adsorption [6].	Used to reclaim water from condensate, domestic wastewater, and used nutrient solutions, achieving high purity levels [6].
Vermiculite	A sterile, high-porosity mineral used as an optimal growth medium control in plant experiments [62].	Served as the positive control substrate to benchmark the performance of the experimental lunar soil simulant mixtures [62].
Specific Microbial Consortia	Undefined, mixed microbial communities drive the breakdown of pollutants and nutrients in biological reactors [6].	Dominant microorganisms like Meiothermus and Rhodanobacter were identified as key to MBAR function in closed systems [6].

Validation Metrics and Comparative Analysis with Terrestrial Stability Methods

This guide provides a comparative analysis of biosafety performance within Bioregenerative Life Support Systems (BLSS), with a specific focus on findings from the ground-breaking 370-day "Lunar Palace 365" mission. The assessment quantitatively evaluates pathogen abundance, antibiotic resistance potential, and material degradation impacts, benchmarking these results against other controlled environments. Data demonstrate that the integration of plant cultivation modules within Lunar Palace 1 (LP1) significantly enhanced biosafety by suppressing potential pathogens and maintaining material integrity, achieving a 98.2% system closure with minimal biosafety risks. These outcomes provide critical validation for long-term material closure in confined habitats essential for future lunar bases and deep space exploration.

Bioregenerative Life Support Systems (BLSS) are advanced closed artificial ecosystems designed to sustain human life during long-duration space missions by recycling oxygen, water, and food through biological processes [10]. The "Lunar Palace 365" experiment, conducted over 370 days in the Lunar Palace 1 (LP1) facility, represented a landmark mission to develop technologies for maintaining system stability under long-term operational conditions and crew shifts [10]. A critical component of BLSS viability is biosafety assessment, which ensures the health of crew members by monitoring and controlling hazardous biological agents. In closed systems, even minor imbalances in microbial communities can lead to significant risks, including the proliferation of opportunistic pathogens, the emergence of antibiotic resistance, and the biodegradation of system components [11]. This guide objectively compares the biosafety performance of the LP1 system against other environments, analyzing experimental data on pathogen abundance and material compatibility to validate its suitability for long-term human habitation in extraterrestrial environments.

Comparative Biosafety Performance Metrics

The biosafety assessment of the Lunar Palace 1 (LP1) system during the 370-day "Lunar Palace 365" mission focused on three primary metrics: bacterial community dynamics, the abundance of potential pathogens, and the prevalence of antibiotic resistance genes (ARGs). The following tables summarize the quantitative findings and compare them with data from other controlled environments.

Table 1: Bacterial Community Dynamics and Pathogen Abundance in Different Environments

Environment / System	Bacterial Diversity (Shannon Index)	Dominant Bacterial Phyla	Key Potential Pathogens Detected	Relative Abundance of Potential Pathogens
Lunar Palace 1 (BLSS)	Moderate [11]	Proteobacteria, Firmicutes, Actinobacteria [63]	Acinetobacter, Staphylococcus [63]	Low [63] [8]
International Space Station (ISS)	Information Missing	Proteobacteria, Firmicutes, Actinobacteria [11]	Staphylococcus aureus [11]	Information Missing
Terrestrial Controlled (Non-BLSS)	Lower than BLSS [11]	Information Missing	Information Missing	Information Missing
Terrestrial Open Environment	Higher than BLSS [11]	Information Missing	Information Missing	Information Missing

Table 2: Antibiotic Resistance Gene (ARG) Profile and Material Impact Assessment

Environment / System	Key Antibiotic Resistance Genes Detected	Abundance of ARGs	Impact on Equipment & Materials
Lunar Palace 1 (BLSS)	Tet(K) [11]	Minimal [63] [8]	Negligible material degradation [63]
International Space Station (ISS)	Information Missing	Increased levels reported [11]	Information Missing
Multifunctional Sports/Educational Facilities	Tet(K) [11]	Information Missing	Information Missing

Analysis of Comparative Data

The data reveal that the LP1 system maintained a stable bacterial community with a low abundance of potential pathogens such as Acinetobacter and Staphylococcus [63]. Crucially, the abundance of Antibiotic Resistance Genes (ARGs), such as those conferring tetracycline resistance [e.g., tet(K)], was found to be minimal [11]. This favorable biosafety profile is attributed to the integration of plant modules within the BLSS, which contributed to a more balanced and resilient microbial ecosystem [63] [8]. Furthermore, isolated bacterial strains showed a negligible impact on equipment materials, indicating no significant threat of biodegradation to system integrity over the mission's duration [63].

Experimental Protocols for Biosafety Assessment

The biosafety findings in the LP1 system were derived from rigorous, standardized experimental protocols. The following workflows detail the key methodologies employed for microbial and material analysis.

Microbial Community and ARG Analysis

The assessment of airborne and surface-associated microbiomes in the LP1 system involved a multi-step molecular biology workflow.

Protocol Details:

• Sample Collection: Air dust was systematically collected from designated locations within the LP1 habitat (Plant Cabins, Comprehensive Cabin) using high-efficiency particulate air (HEPA) filters at different mission phases [11].
• DNA Extraction: Total genomic DNA was extracted from the collected air dust samples to facilitate subsequent molecular analyses [11].
• Sequencing & Quantification: The extracted DNA was subjected to 16S rRNA gene amplicon sequencing to profile bacterial community composition and diversity. Shotgun metagenomic sequencing was employed to access the full genetic content, including functional genes and ARGs. Quantitative PCR (qPCR) was used to determine the absolute abundance of bacterial loads and specific ARGs [11].
• Data Analysis: Sequencing data were processed to determine microbial diversity and temporal dynamics. Metagenomic data were analyzed for antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Source tracking analysis was performed to identify the primary origins (e.g., crew, plants) of the airborne microbiota [11].

Material Compatibility Assessment

The potential for microbial degradation of system components was evaluated through a targeted materials testing protocol.

Protocol Details:

• Strain Isolation & Culture: Bacterial strains were isolated from various surfaces and the atmosphere within the LP1 system and cultured to obtain pure isolates for testing [63].
• Material Exposure Test: Key polymer and metal materials used in the construction of LP1 components were exposed to the isolated bacterial strains under controlled laboratory conditions that simulated the BLSS environment [63].
• Compatibility Evaluation: The exposed materials were analyzed for signs of biodegradation, including visual corrosion, biofilm formation, mass loss, and surface degradation using techniques such as scanning electron microscopy (SEM). The study concluded that the impact of isolated bacterial strains on equipment materials was negligible, indicating favorable material compatibility [63].

The Scientist's Toolkit: Research Reagent Solutions

The following table details key reagents, materials, and equipment essential for conducting biosafety assessments in a closed BLSS environment, as utilized in the Lunar Palace 365 research.

Table 3: Essential Research Reagents and Materials for BLSS Biosafety Assessment

Item	Function in Biosafety Assessment
HEPA Filters	Collection of airborne microbial particles and dust for community and ARG analysis [11].
DNA Extraction Kits	Isolation of high-quality genomic DNA from diverse sample types (air, surface, biological) for downstream molecular analysis [11].
16S rRNA Primers	Amplification of specific bacterial gene regions for profiling microbial community composition and diversity via sequencing [11].
qPCR Master Mixes	Quantification of absolute abundance of total bacteria and specific antibiotic resistance genes (ARGs) [11].
Shotgun Metagenomic Sequencing Kits	Comprehensive analysis of all genetic material in a sample, enabling functional profiling and detection of ARGs and virulence factors [11].
Selective Culture Media	Isolation and cultivation of specific bacterial groups, including potential pathogens, from complex environmental samples [63].
High-Efficiency Particulate Air (HEPA) Filtration System	Maintenance of sterile air supply and containment of aerosols within the closed laboratory environment [64] [65].
Class II Biological Safety Cabinet (BSC)	Primary containment barrier providing a sterile work environment and protecting the user from exposure to aerosols [64] [65] [66].
Autoclave	Sterilization and decontamination of all biohazardous waste and laboratory equipment prior to disposal or reuse, a fundamental biosafety practice [64] [65].
Appropriate Disinfectants (e.g., 1:10 Bleach)	Routine decontamination of work surfaces and immediate treatment of spills to inactivate viable pathogenic materials [64].

The comprehensive biosafety data from the Lunar Palace 365 mission demonstrates that a well-designed BLSS can effectively maintain a low-risk microbial environment conducive to long-term human habitation. The low abundance of potential pathogens, minimal presence of antibiotic resistance genes, and negligible material biodegradation collectively validate the robustness of the LP1 system's ecological balance. These findings are pivotal for the future design and operation of long-duration space missions, underscoring that integrating biological components like plants is not merely for life support but is a critical strategy for ensuring overall system biosafety and sustainability.

Bioregenerative Life Support Systems (BLSS) represent the most advanced approach to sustaining human life in long-duration space missions, utilizing biological processes to recycle air, water, and waste while producing food. This comparative analysis examines the ground-based Lunar Palace 1 (LP1) facility, validated through the 370-day "Lunar Palace 365" mission, against the International Space Station's (ISS)-based physico-chemical systems and other relevant closed environmental systems. The core thesis centers on how the Lunar Palace 365 experiment provides critical validation data for achieving long-term material closure, a prerequisite for future deep space exploration and lunar habitation [10].

Lunar Palace 1 (LP1)

LP1 is an integrated, ground-based BLSS facility designed to achieve a high degree of material closure through biological regeneration. The "Lunar Palace 365" mission, a 370-day closed isolation experiment, was conducted to test the system's robustness under long-term operation and crew shifts [10]. The system comprises two plant cabins and one comprehensive cabin, hosting a total volume of 500 m³ [67] [10]. Its core technological approach is bioregeneration, relying on higher plants and microorganisms to regenerate air, produce food, recycle water, and process waste, creating an artificial, Earth-like ecosystem [10].

International Space Station (ISS) ECLSS

The ISS Environmental Control and Life Support System (ECLSS) is primarily based on physico-chemical processes. A key recent addition is the Advanced Closed Loop System (ACLS), a technology demonstrator developed by the European Space Agency. The ACLS rack recycles carbon dioxide from the cabin air via a unique amine-based adsorbent. The concentrated CO₂ is then processed in a Sabatier reactor to create water and methane. The water is subsequently electrolyzed to produce oxygen for the crew [68]. This system exemplifies a hybrid approach, moving beyond open-loop configurations but still reliant on engineering processes rather than biological ones.

Other Closed Systems

Other systems provide valuable historical context and technological benchmarks:

• BIOS-3 (Russia): An early ground-based closed ecological system.
• Biosphere 2 (USA): A large-scale terrestrial experiment that demonstrated the profound challenges of maintaining gas balance in a closed ecological system, ultimately failing due to an irreversible drop in O₂ and a rise in CO₂ [10].
• Mars 500: A ground-based simulation focusing on the psychological and physiological aspects of a 520-day Mars mission, but lacking a functional BLSS [11].

Quantitative Performance Comparison

The table below summarizes key performance metrics for LP1 and the ISS's ACLS, highlighting their fundamental differences in approach and capability.

Table 1: Performance Metrics of LP1 and ISS Life Support Systems

Performance Metric	Lunar Palace 1 (LP1)	ISS Advanced Closed Loop System (ACLS)
System Type	Bioregenerative (BLSS)	Physico-Chemical
Mission Duration Validated	370 days ("Lunar Palace 365") [10]	≥1 year of operation planned over 2 years [68]
O₂ Regeneration	100% from plants [10]	Produced via electrolysis of recycled and supplied water [68]
Water Recovery	100% for human use; potable water meets standards [10]	Recycles ~50% of CO₂ into water for O₂ production [68]
CO₂ Processing	Removed and converted by plants; O₂ produced via photosynthesis [10]	Concentrated via amine scrubber and processed via Sabatier reactor [68]
Food Production	Fully met crew's plant-based food needs [10]	Not applicable; all food is supplied from Earth
Waste Recycling	67% of solid waste; 99.7% of urine recovered [10]	Not a primary function; methane from Sabatier reactor is vented [68]
Overall Material Closure	98.2% [10]	Partial; focuses on O₂ recovery and CO₂ removal, requires resupply

Detailed Experimental Protocols in Lunar Palace 365

The validation of LP1 relied on a multi-faceted experimental protocol during the "Lunar Palace 365" mission, designed to assess everything from system-level material flows to crew microbiome interactions.

System-Level Material Flow and Stability Monitoring

The core protocol for validating material closure involved continuous monitoring and balancing of mass flows [10].

• Gas Dynamics: Concentrations of O₂ and CO₂ were monitored continuously throughout the 370-day mission. The system maintained CO₂ between 246 and 4131 ppm, demonstrating robust stability despite daily photosynthetic fluctuations and crew shift changes [10].
• Water Recycling: All water for human use, including drinking and irrigation, was regenerated within the system. The purity of potable water was confirmed to meet condensate water standards [10].
• Waste Processing: The recovery of nitrogen and other nutrients from urine (99.7%) and the bioconversion of solid waste (67%) into useful products like soil-like substrate were quantitatively tracked [10].

Multi-Omics Analysis of Gut Microbiota

A significant protocol within the mission investigated the microbiota-gut-brain axis, linking system environment to crew health.

• Sample Collection: 103 sets of psychological survey data and corresponding fecal samples were collected from crew members at defined intervals after a 28-day adaptation period post-entry [67].
• Molecular Analysis: The fecal samples underwent metagenomic sequencing (103 samples), metaproteomic analysis (90 samples), and metabolomic analysis (56 samples) to characterize the gut microbiome at the genetic, protein, and metabolic levels [67].
• Data Correlation: Abundance data for microbial species was correlated with psychological factor scores (from the SCL-90 and POMS surveys) to identify mood-associated bacteria. The stationarity of these time-series data was confirmed using autocorrelation function (ACF) analysis [67].

Diagram 1: Multi-omics workflow for psychobiotic identification.

Airborne Microbiome and Antibiotic Resistance Gene (ARG) Monitoring

This protocol assessed the environmental microbiome dynamics within the closed habitat.

• Air Dust Sampling: 34 air dust samples were collected from different locations and at different time points across the three mission phases using HEPA filters [11].
• DNA Sequencing and qPCR: Samples were analyzed via 16S rRNA amplicon sequencing, shotgun metagenomic sequencing, and quantitative PCR (qPCR) to profile the bacterial community and quantify absolute abundances of bacteria and ARGs [11].
• Source Tracking: Computational source tracking analysis was performed to determine the origin of airborne bacteria (e.g., human, plant) [11].

Analysis of Key Differentiators

Degree of Closure and Self-Sufficiency

The most significant differentiator is the overall material closure. LP1 achieved a 98.2% closure for materials crucial for human survival, demonstrating near-complete self-sufficiency [10]. In contrast, while the ISS's ACLS improves resource efficiency by recycling CO₂ and saving 400 liters of water annually, it remains a partially closed system that depends on Earth resupply for food, hydrogen, and other consumables [68]. LP1's integration of food production is a transformative capability absent in current orbital systems.

Functional Mechanisms: Biological vs. Physico-Chemical

The core technologies define the capabilities and complexities of each system.

• LP1's Biological Pathways: LP1's functionality is driven by photosynthesis and microbial action. The system's robustness was tested through active interventions, such as adjusting soybean photoperiod and solid waste reactor activity, proving its ability to recover from internal disturbances [10]. Furthermore, the mission uncovered specific psychobiotic gut microbiota (e.g., Bacteroides uniformis, Faecalibisterium prausnitzii) that contribute to crew mental health by producing neurotransmitters and modulating the immune system, a benefit unique to biologically-rich environments [67].

Diagram 2: Psychobiotic mood regulation pathways in LP1.

• ISS's Physico-Chemical Pathways: The ACLS operates on a series of engineered reactions: adsorption, Sabatier reaction, and electrolysis. While highly reliable and compact, this process is inherently linear and vital resources are lost (e.g., methane is vented overboard, explaining why only 50% of the recovered CO₂ is ultimately converted to oxygen) [68].

System Stability and Disturbance Response

LP1 demonstrated remarkable resilience, a critical finding for long-term missions. The system was able to quickly minimize the effects of major disturbances, such as crew shift changes and power failures, through biological self-organization and targeted human intervention [10]. This contrasts with the experience of Biosphere 2, where gas imbalances became irreversible and led to mission failure [10]. The ISS systems, while mechanically robust, do not possess this capacity for biological self-recovery.

Impact of Crew on the System Environment

In LP1, the crew is an integral component of the ecosystem. Research showed that personnel exchange led to significant differences in the airborne bacterial community diversity, with most bacteria deriving from the crew and plants [11]. This highlights a dynamic interaction between crew and habitat. On the ISS, the environment is more static and defined by its machinery, with the human impact being more of a logistical and contaminant challenge than an integral ecological variable.

The Scientist's Toolkit: Key Research Reagents and Materials

The following table details essential reagents, materials, and tools used in the featured "Lunar Palace 365" research, providing a reference for replicating such closed-system studies.

Table 2: Key Research Reagents and Materials from Lunar Palace 365 Studies

Item Name	Function / Application	Experimental Context
HEPA Filter (Xiaomi Air Purifier)	Collection of airborne microbial particles and dust for metagenomic studies.	Airborne microbiome sampling [11]
Symptom Checklist-90 (SCL-90)	Standardized psychological questionnaire to assess mood and psychiatric symptoms.	Crew psychological assessment [67]
Profile of Mood States (POMS)	Psychological rating scale to measure transient, distinct mood states.	Crew psychological assessment [67]
Metagenomic Sequencing	Comprehensive profiling of all genetic material in a sample (feces, air).	Gut and airborne microbiome analysis [67] [11]
Metaproteomic Analysis	Large-scale study of proteins in a sample, providing functional data.	Analysis of protein expression in gut microbiota [67]
Metabolomic Analysis	Comprehensive analysis of unique chemical fingerprints from cellular processes.	Identification of metabolites (e.g., SCFAs) in gut microbiota [67]
qPCR (Quantitative PCR)	Quantifies the absolute abundance of specific bacteria or genes (e.g., ARGs).	Quantification of bacteria and antibiotic resistance genes in air [11]
Chronic Unpredictable Mild Stress (CUMS) Model	A rodent model for inducing depression- and anxiety-like behaviors.	Validation of psychobiotic effects on mood in animals [67]

The comparative analysis reveals a fundamental divergence in philosophy and capability between the LP1 and ISS life support paradigms. The ISS's ACLS and similar physico-chemical systems offer proven, reliable technologies for near-Earth orbit, focusing on incremental closure of specific loops (e.g., oxygen and water). In contrast, the Lunar Palace 1 system, as validated by the "Lunar Palace 365" mission, demonstrates the feasibility of a comprehensive bioregenerative approach, achieving near-total material closure (98.2%), integrated food production, and inherent system resilience. The LP1 experiment provides the most robust validation to date for using BLSS as the foundation for long-duration lunar bases and future Mars missions, while also generating unexpected insights into the critical role of the gut-brain axis in crew health. Future development should focus on hybridizing the reliability of physico-chemical systems with the comprehensive closure and autonomy of bioregenerative systems.

Validating Stability-Indicating Analytical Methods (SIAMs) in BLSS

The integration of Stability-Indicating Analytical Methods (SIAMs) into Bioregenerative Life Support Systems (BLSS) is crucial for ensuring the long-term stability and safety of pharmaceuticals in space exploration. This review examines the validation of SIAMs within the context of the groundbreaking Lunar Palace 365 experiment, a 370-day ground-based mission that achieved 98.2% material closure. We compare the performance of various chromatographic techniques, provide detailed experimental protocols for forced degradation studies, and outline a comprehensive framework for analytical method validation in closed ecological systems essential for supporting human survival in extraterrestrial environments.

Stability-Indicating Analytical Methods (SIAMs) are validated quantitative procedures that measure the active pharmaceutical ingredient (API) without interference from degradation products, impurities, or excipients [69]. In the context of a Bioregenerative Life Support System (BLSS), where resupply is impossible and resource recycling is critical, pharmaceutical stability becomes a paramount concern for crew health. The Lunar Palace 365 mission demonstrated unprecedented system closure, recycling 100% of oxygen and water, with 98.2% of materials crucial for human survival being regenerated within the system [10]. This achievement highlights both the feasibility and the necessity of developing specialized SIAMs that can function reliably in such closed environments, where unique stress factors including microgravity, increased radiation, and confined microbial ecosystems may accelerate drug degradation.

The International Council for Harmonisation (ICH) guidelines mandate that analytical methods must be properly validated and suitable for detection and quantification of degradation products and impurities [69]. These methods must demonstrate specificity, accuracy, precision, and robustness to provide reliable data throughout extended space missions. As BLSS technology advances toward implementation in lunar bases and Martian exploration, establishing validated SIAMs ensures that pharmaceuticals remain safe, efficacious, and stable throughout their intended shelf life, despite the unique environmental challenges of space habitats [10].

Analytical Techniques for Stability-Indicating Methods

Chromatographic Methods Comparison

Various chromatographic techniques have been employed as stability-indicating assays, each with distinct advantages and limitations for pharmaceutical analysis in confined environments. The selection of an appropriate method depends on the physicochemical properties of the drug substance, including pKa, log P, solubility, polarity, and volatility [69].

Table 1: Comparison of Chromatographic Techniques for Stability-Indicating Assays

Technique	Advantages	Limitations	BLSS Applicability
HPLC [69] [70]	High resolution, versatility, minimal sample preparation, applicable to diverse compounds	Requires significant solvent consumption, method development can be time-consuming	Excellent for comprehensive monitoring of multiple pharmaceuticals; solvent recycling should be considered
GC [69]	Excellent for volatile compounds, high separation efficiency	Limited to volatile and thermally stable compounds, derivatization often required	Suitable for monitoring volatile degradation products; energy-intensive for BLSS
HPTLC [69]	Parallel analysis of multiple samples, minimal solvent consumption	Lower resolution compared to HPLC, limited quantification accuracy	Ideal for resource-limited environments; minimal solvent waste advantageous for BLSS
CE [69]	High efficiency, minimal sample and solvent volumes	Lower sensitivity compared to HPLC, more complex method development	Promising for BLSS due to minimal resource consumption; requires technical expertise
SFC [69]	Fast analysis, environmentally friendly (CO2-based)	Limited method knowledge, requires specialized equipment	Excellent green alternative for BLSS; utilizes recycled CO2 from system

Hyphenated Techniques

Hyphenated systems combining chromatographic separation with spectroscopic detection provide powerful tools for parallel quantitative and qualitative analysis of drug substances and impurities [69]. These include:

• HPLC-DAD: Enables peak purity assessment by collecting full UV spectra [71]
• LC-MS: Provides structural information for impurity identification [69] [70]
• GC-MS: Ideal for volatile degradation products [69]
• LC-NMR: Powerful for definitive structural elucidation [69]

For BLSS applications, HPLC-UV remains the dominant technique for stability-indicating methods due to its robustness, precision, and compatibility with most small-molecule drugs [70]. The wide linear response range exceeding five orders of magnitude and peak area precision of 0.1–0.5% RSD make it ideal for quality control applications in resource-limited environments [70].

Stability-Indicating Method Development Framework

Method Development Workflow

The development of a validated SIAM follows a systematic approach that ensures the method can reliably detect and quantify changes in API concentration while separating degradation products. The following workflow illustrates this development process:

Key Development Considerations

The traditional five-step approach to HPLC method development proposed by Snyder and associates provides a logical framework for creating stability-indicating methods [70]:

• Define method type: For SIAMs, this is typically a quantitative procedure for API and impurities determination that must separate all key components [70].

• Gather sample and analyte information: Understanding physicochemical properties (pKa, logP, λmax) and potential degradation pathways informs column and mobile phase selection [70] [71].

• Initial method development: Performing "scouting" runs with broad gradient methods (e.g., 5-100% organic modifier in 10 minutes) provides preliminary separation data [70].

• Method fine-tuning and optimization: Selectivity tuning through manipulation of mobile phase composition, pH, temperature, and gradient profile achieves resolution of critical peak pairs [70].

• Method validation: Comprehensive validation following ICH guidelines confirms the method meets all required performance characteristics [72].

For BLSS applications, method development should prioritize robustness and resource efficiency, considering the limited availability of solvents and reagents in closed systems. Reversed-phase HPLC with UV detection is particularly suitable for space applications due to its predictability, reliability, and compatibility with most pharmaceutical compounds [70].

Experimental Protocols for Forced Degradation Studies

Forced Degradation Protocol

Forced degradation studies are performed under conditions more severe than accelerated stability conditions to identify potential degradation products and validate the stability-indicating nature of the analytical method [73]. The following protocol outlines a systematic approach:

• Sample Preparation: Prepare drug substance and drug product solutions at appropriate concentrations (typically 1 mg/mL) in suitable solvents [70].

• Stress Conditions Application:

  • Acid Hydrolysis: Expose to 0.1-1M HCl at elevated temperatures (40-70°C) for several hours to days [74] [73]
  • Base Hydrolysis: Expose to 0.1-1M NaOH at elevated temperatures (40-70°C) for several hours to days [74] [73]
  • Oxidative Stress: Treat with 0.1-3% hydrogen peroxide at room temperature for several hours to days [74] [73]
  • Thermal Stress: Expose solid drug and drug product to dry heat (50-70°C) for extended periods [74]
  • Photolytic Stress: Expose to UV and visible light per ICH Q1B option 2 conditions [73]

• Degradation Extent Monitoring: Aim for approximately 5-20% degradation to avoid secondary degradation products while generating sufficient impurities for detection [71]. Studies show significant degradation under various stress conditions, such as 90.33% under alkaline stress, 70.60% under acidic stress, and 33.95% under oxidative stress at 70°C for tonabersat [74].

• Sample Analysis: Analyze stressed samples using the developed chromatographic method and compare with untreated controls [73].

Method Validation Parameters

SIAMs must be validated according to ICH guidelines to demonstrate they are suitable for their intended purpose [72]. The following table outlines key validation parameters and typical acceptance criteria:

Table 2: Method Validation Parameters and Acceptance Criteria for SIAMs

Validation Parameter	Evaluation Procedure	Acceptance Criteria	BLSS Considerations
Specificity [69] [71]	Resolution between API and nearest impurity	Resolution factor ≥ 2.0	Critical in BLSS with limited medical options
Accuracy [74] [72]	Recovery of spiked API from placebo	98-102% recovery	Essential for dosage precision in remote settings
Precision [74] [72]	Repeatability (n=6) and intermediate precision	%RSD ≤ 2.0%	Ensures method reliability with different operators
Linearity [74] [72]	Minimum of 5 concentration levels	R² ≥ 0.999	Verifies detector response across expected range
Range [72]	From LOQ to 120-150% of test concentration	Covers specification limits	Must encompass stability changes over long missions
Robustness [69] [70]	Deliberate variations in method parameters	System suitability still met	Crucial for method transfer between Earth and BLSS
LOD/LOQ [74]	Signal-to-noise ratio of 3:1 and 10:1	LOQ typically 5 μg/mL	Must detect low-level degradants in closed systems

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of SIAMs in a BLSS environment requires careful selection of research reagents and materials that ensure method reliability while considering resource constraints.

Table 3: Essential Research Reagent Solutions for SIAM Development and Validation

Reagent/Material	Function in SIAM	BLSS-Specific Considerations
C18 Chromatographic Columns [74] [70]	Separation of APIs from degradants using hydrophobic interactions	Longevity and robustness critical due to limited replacement options
MS-Compatible Mobile Phase Additives (e.g., formic acid, ammonium acetate) [70]	pH adjustment and ion pairing while maintaining MS compatibility	Prioritize volatile additives suitable for potential LC-MS use
HPLC-Grade Solvents (acetonitrile, methanol) [69] [70]	Mobile phase components for analyte elution	Recycling or regeneration systems may be necessary in BLSS
Reference Standards (API and known impurities) [71]	Method development and qualification	Limited storage space necessitates careful selection and prioritization
Forced Degradation Reagents (HCl, NaOH, H₂O₂) [74] [73]	Generation of degradation products for method validation	Minimum quantities sufficient for validation studies
Color Reference Solutions [75]	Visual assessment of solution coloration	Digital spectrophotometry preferred over subjective visual assessment

Lunar Palace 365: A Case Study in Closed-System Validation

The Lunar Palace 365 mission provides a groundbreaking case study for implementing analytical methods in a high-closure BLSS. This 370-day mission achieved remarkable system stability with 100% recycling of oxygen and water, and 98.2% closure of materials crucial for human survival [10]. Within this environment, several factors directly impact pharmaceutical stability and SIAM validation:

Environmental Stability and Monitoring

The BLSS demonstrated excellent environmental stability despite crew shift changes and system disturbances. Key environmental parameters were maintained within narrow ranges [10]:

• CO₂ concentration: 246-4131 ppm (average 1484 ± 612 ppm)
• O₂ concentration: 19.5-20.9% (average 20.3 ± 0.3%)
• Temperature: 21.7-24.6°C (average 23.5 ± 0.6°C)
• Relative humidity: 46.8-73.9% (average 62.8 ± 5.3%)

This remarkable environmental control provides a stable baseline for pharmaceutical storage, potentially simplifying stability challenges compared to more variable environments.

Microbial Dynamics and Pharmaceutical Stability

Microbiome analysis during Lunar Palace 365 revealed that human presence significantly influenced bacterial community diversity in the air dust, with most bacteria deriving from cabin crew and plants [11]. While this study focused on air microbiomes, the findings highlight the importance of considering microbial impacts on pharmaceutical stability in closed systems, including:

• Potential for microbial degradation of pharmaceuticals
• Changes in degradation pathways due to unique microbial ecosystems
• Need for sterility maintenance in parenteral products

The demonstrated robustness of the BLSS in maintaining environmental stability despite crew changes [10] provides confidence that well-designed SIAMs can maintain performance throughout long-duration missions with multiple crew rotations.

The validation of Stability-Indicating Analytical Methods within Bioregenerative Life Support Systems represents a critical intersection of pharmaceutical analysis and space life support technology. The Lunar Palace 365 mission has demonstrated the feasibility of maintaining long-term environmental stability in closed systems, achieving 98.2% material closure while supporting crewed operations over 370 days [10]. This achievement provides a foundation for implementing robust SIAMs that can ensure pharmaceutical stability throughout extended space missions.

Future developments in this field should focus on:

• Miniaturized analytical systems that reduce solvent and consumable requirements
• Multi-analyte methods that simultaneously monitor multiple pharmaceuticals in BLSS
• Automated monitoring systems that provide real-time stability data
• Advanced detection techniques including MS and NMR for definitive impurity identification
• Integration with BLSS resource recycling to minimize waste generation

As humanity prepares for long-duration lunar missions and eventual Martian exploration, the lessons from Lunar Palace 365 combined with robust SIAM validation protocols will ensure that pharmaceutical products remain safe, stable, and efficacious throughout the journey, supporting crew health and mission success in the final frontier.

Gut Microbiota as Biomarkers for System-Wide Stability Assessment

Within the context of long-term space exploration, the stability of engineered biological systems is paramount for mission success. The "Lunar Palace 365" experiment, a 370-day isolation study conducted within the Lunar Palace 1 (LP1) facility, a closed manned Bioregenerative Life Support System (BLSS), provides an unprecedented opportunity to validate long-term material closure and assess system-wide stability through a novel lens: the human gut microbiome [67]. In this isolated environment, where crew members experienced controlled, constant conditions with minimal microbial exchange, the gut microbiota emerges as a highly sensitive biomarker ecosystem, reflecting both the physiological and psychological status of the crew [67] [11].

The core premise is that a healthy, stable gut microbiome is characterized by greater microbial diversity, a balanced abundance of beneficial bacteria, and functional resilience—the ability to resist and recover from perturbations [76]. In a closed environment, where external influences are minimized, monitoring the dynamics of this internal microbial community offers a powerful, non-invasive means to assess the overall stability of the host-microbe system. Recent research has demonstrated that the gut microbiome's stability is body-site specific, with the stool microbiome being relatively stable compared to other sites, and is heavily influenced by host factors [77]. Disruptions to this stability, known as dysbiosis, can have cascading effects on host health through various gut-organ axes, including the gut-brain axis [76]. This review will objectively compare the performance of specific gut microbial taxa and analytical methodologies as biomarkers for system-wide stability, drawing on experimental data from the Lunar Palace 365 mission and related studies to provide a framework for future mission planning and therapeutic development.

The Lunar Palace 365 Experiment: A Benchmark for Stability Research

The Lunar Palace 365 experiment serves as an ideal ground-based analog for studying microbiome dynamics in long-duration spaceflight. This multicrew, closed experiment was conducted in the LP1 facility, which comprises two plant cabins and one comprehensive cabin [67]. Key features that make it exceptional for biomarker discovery include:

• High Closure and Controlled Environment: The BLSS facility had almost no material exchange with the outside world, largely avoiding microbial contamination, with constant environmental conditions and strictly monitored microbiome levels on air, water, and material surfaces [67] [11].

• Standardized Living Conditions: Crew members followed fixed schedules, consumed identical food sources from the system, and maintained documented health status, minimizing confounding variables [67].

• Comprehensive Longitudinal Sampling: The collection of 103 paired psychological assessments and fecal samples over the 370-day mission enabled robust correlation analysis between microbial and psychological parameters [67].

This controlled setting allowed researchers to distinguish intrinsic microbiome dynamics from externally driven changes, establishing a benchmark for evaluating gut microbiota as stability biomarkers during prolonged confinement.

Key Gut Microbiota Biomarkers and Their Stability Signatures

Psychobiotic Candidates as Psychological Stability Biomarkers

Analysis of the gut microbiome-psychological factor correlation in the Lunar Palace 365 experiment identified four potential psychobiotics—live organisms that confer mental health benefits—that serve as promising biomarkers for psychological stability in closed environments [67] [78].

Table 1: Potential Psychobiotics Identified in Lunar Palace 365 as Stability Biomarkers

Bacterial Species	Primary Function	Correlation with Psychological Health	Relevance to System Stability
Bacteroides uniformis	Dietary fiber fermentation; metabolic regulation	Positive mood regulation	Core species in healthy adult gut; indicates functional stability [67] [79]
Roseburia inulinivorans	Butyrate production; anti-inflammatory effects	Stress resilience	Butyrate production supports gut barrier integrity; key for system homeostasis [67]
Eubacterium rectale	Short-chain fatty acid production	Mood improvement	Reduced abundance indicates dysbiosis; marker of system perturbation [67]
Faecalibacterium prausnitzii	Microbial balance maintenance; anti-inflammatory properties	Positive mood correlation	Health-associated species; decreased in multiple disease states; key stability indicator [67] [76]

These four bacterial species demonstrated significant associations with mood scores and represent robust biomarkers for monitoring psychological stability in closed environments. Their functional roles in maintaining gut barrier integrity, producing beneficial metabolites, and regulating inflammatory responses make them particularly valuable for assessing overall system health [67].

Taxonomic and Functional Diversity as Resilience Biomarkers

Beyond specific taxa, broader ecosystem properties of the gut microbiome serve as valuable biomarkers for system resilience:

• Diversity Indices: Microbial richness and evenness provide a primary indicator of ecosystem health and stability. In the LP1 environment, crew members' gut microbiota was dominated by Bacteroidetes, Firmicutes, and Proteobacteria at the phylum level, consistent with healthy adult populations [67] [79]. However, significant individual variations in composition were observed, highlighting the personalized nature of microbial ecosystems while still maintaining core functions [67].

• Functional Redundancy: The gut microbiome exhibits functional stability despite taxonomic variability. Different microbial species can perform similar metabolic functions, creating resilience against perturbations [79]. This principle was demonstrated in the LP1, where crew members maintained core metabolic functions despite individual variations in microbial composition [67].

• Temporal Stability: The gut microbiome's resistance to fluctuation over time serves as a key stability biomarker. Studies have shown that microbiome samples from the same individual, even when separated by years, tend to be more similar than samples from different individuals, suggesting an individual-specific "microbial fingerprint" that maintains stability over time [80] [77].

Analytical Frameworks for Assessing Microbiome Stability

Multi-Omics Approaches for Comprehensive Biomarker Validation

The Lunar Palace 365 research employed a comprehensive multi-omics strategy to validate gut microbiota as stability biomarkers, providing multiple layers of evidence for their biomarker utility.

Table 2: Multi-Omics Approaches for Microbiome Stability Assessment in Lunar Palace 365

Methodology	Samples Analyzed	Key Insights	Application in Stability Assessment
Metagenomics	103 fecal samples	Identified potential psychobiotics at species level; revealed taxonomic composition and functional potential	Determines baseline microbial community structure and genetic capacity for stability
Metaproteomics	90 fecal samples	Confirmed active microbial functions; identified expressed proteins in metabolic pathways	Assesses functional activity rather than potential; verifies actual metabolic operations
Metabolomics	56 fecal samples	Detected microbial metabolites (SCFAs, neurotransmitters); connected microbial activity to host physiology	Provides direct evidence of microbial functional output and host-microbe interactions
16S rRNA Sequencing	Used in related confined space studies [81]	Profiled microbial community structure at genus level; tracked changes over time	Cost-effective longitudinal monitoring of community shifts during confinement

This integrated approach enabled researchers to move beyond mere correlation to establish mechanistic links between microbial presence and host physiological status, strengthening the validation of these microbial signatures as robust stability biomarkers [67].

Ecological Stability Metrics and Mathematical Modeling

The field of gut microbiome stability assessment has developed two complementary approaches for quantifying stability, both relevant to closed environment studies:

• Observational Stability Measures: Based on statistical analysis of microbiome changes over time, typically measured as beta diversity between temporal samples. This approach has revealed that individuals exhibit varying degrees of temporal variability in their microbiome, with greater stability considered a marker of ecosystem health [80].

• Mathematical Modeling Approaches: Based on ecological principles and parameterized using longitudinal microbiome data. These include:

  • Local (Asymptotic) Stability: Characterizes system behavior near equilibrium after small perturbations [80]
  • Structural Stability: System's ability to maintain dynamic behavior under smooth parameter changes [80]
  • Robustness: Proportion of species loss required to trigger secondary extinctions [80]

A recent meta-analysis comparing these approaches found substantial correlation between them, supporting their complementary use in assessing microbiome stability in closed environments [80].

Mechanistic Pathways Linking Gut Microbiota to System Stability

The Gut-Brain Axis in Closed Environments

The Lunar Palace 365 research revealed that gut microbiota influences psychological stability through several key biochemical pathways, with the identified psychobiotics acting through multiple mechanisms to support mood regulation [67].

Diagram 1: Gut-Brain Axis Signaling Pathways in Closed Environments

This multi-pathway approach demonstrates how gut microbiota serves as an effective biomarker for psychological stability, with specific microbial functions directly contributing to neurological homeostasis through distinct but interconnected mechanisms [67].

Microbial Community Dynamics in Confined Spaces

Longitudinal profiling of microbiome stability across body sites reveals that the stool microbiome demonstrates greater stability compared to skin and nasal microbiomes, making it particularly valuable for long-duration monitoring in closed environments [77]. This stability arises from the complex ecological interactions within the microbial community and its established relationship with the host.

Studies of confined space operations have demonstrated that closed environments induce structural adjustments to the microbial community, including enrichment of Firmicutes and reduction of Bacteroides, leading to dynamic reprogramming of host metabolites and gradual activation of inflammation markers [81]. These measurable changes in microbial architecture provide early warning biomarkers for system instability before clinical symptoms manifest.

Experimental Protocols for Biomarker Validation

Lunar Palace 365 Sampling and Multi-Omics Workflow

The validation of gut microbiota as stability biomarkers requires rigorous experimental methodologies. The Lunar Palace 365 study implemented a comprehensive protocol:

Diagram 2: Experimental Workflow for Microbiome Biomarker Validation

This comprehensive protocol ensured that identified biomarkers were not merely correlational but linked to mechanistic pathways validating their role in system stability [67].

Stability Assessment Methodologies

For assessing microbiome stability, two primary methodological frameworks have emerged:

• Longitudinal Sampling and Beta Diversity Analysis: This involves collecting samples from the same individuals over time and calculating within-individual similarity using distance metrics like Bray-Curtis dissimilarity. Lower temporal variability indicates higher stability [80] [77].

• Mathematical Modeling of Community Dynamics: Using time-series data to parameterize models of microbial dynamics, typically based on generalized Lotka-Volterra equations, which can predict stability properties including resistance to perturbation and recovery capacity [80].

A meta-analysis of 9 interventional and time series studies encompassing 3,512 gut microbiome profiles found correlation between these approaches, supporting their combined use for robust stability assessment [80].

The Scientist's Toolkit: Essential Research Reagents and Platforms

Implementing gut microbiota stability assessment requires specific research tools and platforms. The following table details key solutions used in foundational studies:

Table 3: Essential Research Reagent Solutions for Microbiome Stability Studies

Research Solution	Specific Application	Function in Stability Assessment
16S rRNA Gene Sequencing	Taxonomic profiling of microbial communities	Measures diversity and compositional stability over time; cost-effective for longitudinal studies [81]
Shotgun Metagenomics	Comprehensive gene content analysis	Assesses functional potential and tracks specific bacterial strains as stability biomarkers [67] [79]
LC-MS/MS (Liquid Chromatography-Mass Spectrometry)	Metabolite detection and quantification	Identifies and quantifies microbial metabolites (SCFAs, neurotransmitters) linking microbiota to host physiology [67] [81]
Olink Targeted Proteomics	Inflammatory biomarker profiling	Measures host inflammatory response to microbial changes; connects dysbiosis to host physiology [81]
gnotobiotic Animal Models	Mechanistic pathway validation	Isolates causal relationships between specific microbes and host outcomes in controlled systems [67]
Computational Modeling Platforms	Ecological stability analysis	Parameterizes models to predict stability properties and identify tipping points [80]

These tools enable researchers to move from observational correlations to mechanistic understanding of how gut microbiota serves as a biomarker for system-wide stability.

Comparative Performance of Microbiome Biomarkers Versus Traditional Approaches

When evaluating gut microbiota as stability biomarkers against conventional assessment methods, several advantages emerge:

• Early Detection Capability: Microbial community shifts often precede clinical symptoms, providing an early warning system for system instability [76] [81].

• Multi-System Integration: Gut microbiota simultaneously reflects psychological, immunological, and metabolic status through different axes of interaction, providing a more holistic stability assessment than single-system biomarkers [67] [76].

• Non-Invasive Monitoring: Fecal sampling provides a non-invasive method for repeated measurements, essential for long-duration missions where frequent blood draws or other invasive procedures are impractical [67].

• Functional Redundancy: The core functional stability of the microbiome, despite taxonomic variations between individuals, provides a robust framework for developing generalized countermeasures that can be personalized as needed [79].

However, challenges remain in standardizing assessment protocols and accounting for significant inter-individual variation in baseline microbiome composition, necessitating personalized approaches to stability monitoring [80] [77].

The Lunar Palace 365 experiment has demonstrated that gut microbiota serves as a highly sensitive, multi-faceted biomarker system for assessing stability in long-term closed environments. The identification of specific psychobiotics and their mechanistic pathways provides a foundation for developing microbiota-based countermeasures for mission-critical stability maintenance [67] [78].

Future research directions should focus on:

• Developing standardized stability metrics that integrate both taxonomic and functional features
• Validating personalized stability baselines that account for inter-individual variation
• Designing targeted interventions (prebiotics, probiotics, dietary adjustments) to enhance microbial resilience
• Integrating microbiome stability metrics with other physiological and psychological monitoring systems

As we advance toward long-duration space missions to the Moon and Mars, the gut microbiome will play an increasingly important role not just in maintaining crew health, but in providing critical biomarkers for assessing the stability of the entire human-life support system complex. The lessons from Lunar Palace 365 provide an essential reference for future applications of microbiome monitoring in extreme environments and for neuropsychiatric treatments on Earth [67].

The quest to ensure the stability and efficacy of pharmaceuticals in harsh, isolated environments is paramount for the success of long-duration space missions. This challenge has catalyzed the development of advanced testing methodologies within ground-based space analog environments, which simulate the unique stressors of spaceflight. These analogs provide a critical testing ground not only for preparing for extraterrestrial missions but also for refining pharmaceutical stability testing on Earth. The research conducted within the Lunar Palace 365 experiment offers a seminal case study in this domain. This year-long mission, conducted within the ground-based Bioregenerative Life Support System (BLSS) "Lunar Palace 1" (LP1), was designed to create a closed, self-sustaining ecosystem integrating plant cultivation, human habitation, and waste recycling [8] [63]. The project's core thesis was validating the long-term stability of a closed artificial ecosystem, providing an unparalleled platform to study the complex interplay of environmental factors on material, including pharmaceutical, stability in a controlled yet realistic setting [8]. This guide objectively compares the experimental approaches and data generated within this space analog context against traditional terrestrial pharmaceutical stability testing protocols.

Spaceflight Environment vs. Terrestrial Stability Testing

The stability of a drug product, encompassing both its active pharmaceutical ingredient (API) potency and the formation of degradation impurities, is systematically assessed on Earth through guidelines like the ICH Q1A(R2). These tests focus on environmental factors such as temperature, humidity, and light (UV/visible) [82]. The spaceflight environment, however, introduces a more complex matrix of stressors that are not fully addressed by conventional protocols.

Table 1: Comparison of Key Stressors in Terrestrial vs. Spaceflight Stability Testing

Stress Factor	Terrestrial (ICH Guidelines)	Spaceflight Environment	Space Analog Simulation
Radiation	Ultraviolet/Visible light (ICH Q1B)	Galactic Cosmic Radiation (GCR), Solar Particle Events (SPE), ionizing radiation	Ground-based radiation facilities (e.g., NASA Space Radiation Laboratory) [83]
Gravity	1G	Microgravity (µG)	Not typically simulated; effects studied on ISS [82]
Vibration	Not typically stressed	Excessive launch and operational vibration	Vibration tables simulating launch profiles [82]
Atmosphere	Ambient air, controlled O₂ & humidity	Hard vacuum, variable O₂/CO₂, closed-loop atmosphere	Closed chambers with controlled atmospheric composition [8]
Psychological & Microbial	Not considered	Isolation, confinement; unique microbial communities	Isolated, confined environments (HERA, LP1) with crew [8] [84]

As delineated in Table 1, space analogs like LP1 and radiation testing facilities are employed to simulate these spaceflight-specific stressors on Earth. The LP1 analog, in particular, provides a holistic environment to study the combined effects of a closed atmosphere and a distinct microbiome on material stability over long durations [8] [63].

Experimental Protocols in Space Analog Pharmaceutical Research

Simulating Space Radiation for Accelerated Drug Stability Studies

A cornerstone of space analog pharmaceutical research is the ground-based simulation of space radiation and its long-term effects. A pivotal 2025 study exposed four solid oral medications—Acetaminophen, Amoxicillin, Ibuprofen, and Promethazine—to simulated Galactic Cosmic Radiation (GCRSim) at the NASA Space Radiation Laboratory [83].

Key Experimental Protocol:

• Irradiation Groups: Medications from identical manufacturing lots were assigned to four groups: a non-irradiated control group, a traveling control group, irradiation group I (0.5 Gy GCRSim), and irradiation group II (1.0 Gy GCRSim). The doses were selected to represent cumulative exposure expected during a Mars mission.
• Dosimetry: Thermoluminescence dosimeters (TLDs) were used to precisely measure the radiation dose absorbed by each drug sample, ensuring accuracy despite noted slight deviations from the target dose [83].
• Long-Term Storage and Analysis: Following irradiation, the drug products were stored for 34 months under controlled room temperature conditions, as defined by the United States Pharmacopeia (USP). They were analyzed at 2, 18, and 34 months for:
  • API Content: Using stability-indicating high-performance liquid chromatography (HPLC) or tandem mass spectrometry (UPLC-MS/MS) methods to quantify the potency of the active ingredient [83] [85].
  • Degradation Impurities: HPLC analysis was used to identify and quantify known and unknown degradation products against USP standards [83].
  • Dissolution Rate: Physical stability was assessed by measuring the drug's dissolution profile, a critical factor for bioavailability [83].

This protocol's logic is encapsulated in the workflow below:

Holistic Stability Assessment in a Closed Ecosystem (Lunar Palace 365)

Beyond targeted radiation studies, the Lunar Palace 365 project provided a platform for a more comprehensive stability assessment. While the provided search results focus on the microbial ecology within the LP1 system [8] [5] [63], the methodologies establish a protocol for monitoring the biological and chemical environment that directly impacts material and pharmaceutical stability.

Key Experimental Protocol:

• Surface Sampling: Sterile swabs, moistened with 0.85% NaCl solution, were used to sample defined surface areas (10 cm x 10 cm) across different functional cabins (Comprehensive Cabin, Plant Cabin, Solid Waste Treatment Cabin) at multiple time points over the 370-day mission [5].
• DNA Extraction and Molecular Analysis: Genomic DNA was extracted from the samples. The stability and evolution of the microbial environment were assessed via:
  • 16S rRNA Amplicon Sequencing: To characterize the dynamics and structure of the bacterial community, including the abundance of potential pathogens and antibiotic resistance genes [8] [63].
  • ITS Amplicon Sequencing: To profile the fungal community (mycobiome) [5].
  • Quantitative PCR (qPCR): Used to detect and quantify specific genes, including mycotoxin-producing genes (e.g., idh, ver1, nor1, tri5), providing a direct measure of biosafety risk and biochemical stability in the environment [5].
• Biosafety Correlation: The resulting microbial data was correlated with the system's operational parameters, notably the presence of plant cabins, to assess their role in maintaining a stable and safe environment [8] [5].

Comparative Data: Space Analog vs. Traditional Findings

The experimental protocols executed in space analog environments have yielded quantitative data that can be directly compared with traditional stability assumptions.

Table 2: Comparative Drug Stability Data from Spaceflight and Analog Studies

Drug Product / Parameter	Traditional Assumption / Control Data	Space Analog / Spaceflight Exposure Data	Source
Acetaminophen, Amoxicillin, Ibuprofen, Promethazine (API Content)	USP Acceptance Criteria: 90-110% potency. Non-irradiated controls remained within specification.	GCRSim (0.5 & 1.0 Gy) + 34-month storage: All samples met USP potency criteria (90-110%). No consistent radiation-dependent effect on API loss observed.	[83]
Various Drugs in LEO (API Content)	Assumption of stable degradation rate under controlled conditions.	Medications stored on ISS for up to 2.4 years showed a ~1.5x increase in degradation rate compared to terrestrial controls, though potency remained within 10% of controls.	[85]
Biosafety: Pathogen Abundance (Lunar Palace 1)	Unknown or variable in a closed habitat.	Low abundance of potential pathogens and minimal antibiotic resistance genes observed throughout the 370-day mission.	[8] [63]
Biosafety: Mycotoxin Potential (Lunar Palace 1)	Potential for accumulation in closed spaces.	No significant differences in mycotoxin gene copy numbers detected across different mission phases and locations.	[5]

The data in Table 2 challenges the primary concern that simulated space radiation would accelerate drug degradation. The 2025 GCRSim study concluded that exposure to ionizing radiation in the range of a Mars mission does not facilitate the degradation of the solid oral drugs tested [83]. This finding is significant for long-duration mission planning. Furthermore, the stable and safe microbial environment demonstrated in the LP1 analog underscores the importance of integrated biological systems, like plant cabins, in maintaining biosafety and, by extension, a less stressful environment for stored pharmaceuticals [8] [5].

The Scientist's Toolkit: Key Research Reagents and Materials

The experiments cited rely on a specific set of reagents, analytical tools, and facilities to generate high-fidelity data.

Table 3: Essential Research Tools for Space Analog Pharmaceutical Stability

Tool / Material	Function in Research	Example Use Case
NASA Space Radiation Lab (NSRL)	Ground-based facility that simulates galactic cosmic radiation using particle accelerators for controlled irradiation studies.	Exposing pharmaceutical solid dosage forms to precise doses of GCRSim [83].
Stability-Indicating HPLC/UPLC-MS/MS	High-performance and ultra-performance liquid chromatography coupled with mass spectrometry for precise quantification of API and degradation impurities.	Analyzing potency of Acetaminophen and impurities in Amoxicillin post-irradiation [83] [85].
Thermoluminescence Dosimeters (TLDs)	Passive radiation dosimeters that measure the exact radiation dose absorbed by a material during exposure.	Verifying the actual radiation dose delivered to drug samples in NSRL beam [83].
FastDNA Spin Kit (or equivalent)	Kit for efficient extraction of high-quality genomic DNA from complex environmental samples.	Isating DNA from surface swabs in Lunar Palace 1 for subsequent microbiome analysis [5].
ITS1F/ITS2R & 16S rRNA Primers	Primers for amplifying fungal (Internal Transcribed Spacer) and bacterial (16S ribosomal RNA) genes for community profiling via sequencing.	Characterizing surface fungal and bacterial diversity and dynamics in the LP1 habitat [5] [63].
qPCR Assays for Mycotoxin Genes	Quantitative PCR with primers specific to genes involved in mycotoxin synthesis (e.g., tri5, ver1) to assess biosafety risk.	Quantifying the potential for aflatoxin and trichothecene production in the closed environment [5].

The research conducted in space analogs provides robust, data-driven insights that refine our understanding of pharmaceutical stability. The key conclusion for mission planners and pharmaceutical scientists is that for solid oral dosage forms, the cumulative ionizing radiation of a long-duration mission may pose a lower-than-expected risk to drug potency [83]. This allows for a reallocation of resources towards more critical challenges, such as optimizing drug repackaging to protect against the greater threat of humidity and oxygen [85] [82].

The relationship between this space analog research and terrestrial applications is symbiotic. The rigorous, predictive testing protocols developed for space—such as using ground-based radiation as an accelerated stability model—can inform better testing strategies on Earth, especially for products requiring heightened assurance of stability under extreme conditions. Conversely, the foundational principles of ICH guidelines provide the baseline from which space analog testing protocols are built. The holistic approach validated in the Lunar Palace 365 experiment, which demonstrates that integrating biological systems like plants can create a more stable and safer environment [8] [5], offers a powerful model for designing closed-loop systems on Earth and for ensuring the success of future interplanetary exploration.

Conclusion

The Lunar Palace 365 experiment provides unprecedented validation of long-term material closure in a complex integrated ecosystem, achieving 98.2% closure over 370 days with minimal pathogen risk and stable microbial communities. Key takeaways include the critical role of plant integration for system biosafety, the identification of specific psychobiotics for mental health maintenance, and robust methodologies for monitoring material and biological stability. These findings have profound implications for pharmaceutical stability testing, suggesting that BLSS-derived approaches could enhance predictive modeling for drug shelf-life and storage conditions. Future research should focus on translating these space analog methodologies to terrestrial applications, particularly for stability testing of biologics, complex formulations, and microbiome-based therapeutics, ultimately advancing both space exploration capabilities and pharmaceutical quality assurance.

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