Recycling nutrients from organic waste for growing higher plants in the Micro Ecological Life Support System Alternative (MELiSSA) loop during long-term space missions.

Space agencies are developing Bioregenerative Life Support Systems (BLSS) in view of upcoming long-term crewed space missions. Most of these BLSS plan to include various crops to produce different types of foods, clean water, and O2 while capturing CO2 from the atmosphere. However, growing these plants will require the appropriate addition of nutrients in forms that are available. As shipping fertilizers from Earth would be too costly, it will be necessary to use waste-derived nutrients. Using the example of the MELiSSA (Micro-Ecological Life Support System Alternative) loop of the European Space Agency, this paper reviews what should be considered so that nutrients recycled from waste streams could be used by plants grown in a hydroponic system. Whereas substantial research has been conducted on nitrogen and phosphorus recovery from human urine, much work remains to be done on recovering nutrients from other liquid and solid organic waste. It is essential to continue to study ways to efficiently remove sodium and chloride from urine and other organic waste to prevent the spread of these elements to the rest of the MELiSSA loop. A full nitrogen balance at habitat level will have to be achieved; on one hand, sufficient N2 will be needed to maintain atmospheric pressure at a proper level and on the other, enough mineral nitrogen will have to be provided to the plants to ensure biomass production. From a plant nutrition point of view, we will need to evaluate whether the flux of nutrients reaching the hydroponic system will enable the production of nutrient solutions able to sustain a wide variety of crops. We will also have to assess the nutrient use efficiency of these crops and how that efficiency might be increased. Techniques and sensors will have to be developed to grow the plants, considering low levels or the total absence of gravity, the limited volume available to plant growth systems, variations in plant needs, the recycling of nutrient solutions, and eventually the ultimate disposal of waste that can no longer be used.

Closed ecosystems extract energy through self-organized nutrient cycles.

Our planet is a self-sustaining ecosystem powered by light energy from the sun, but roughly closed to matter. Many ecosystems on Earth are also approximately closed to matter and recycle nutrients by self-organizing stable nutrient cycles, e.g., microbial mats, lakes, open ocean gyres. However, existing ecological models do not exhibit the self-organization and dynamical stability widely observed in such planetary-scale ecosystems. Here, we advance a conceptual model that explains the self-organization, stability, and emergent features of closed microbial ecosystems. Our model incorporates the bioenergetics of metabolism into an ecological framework. By studying this model, we uncover a crucial thermodynamic feedback loop that enables metabolically diverse communities to almost always stabilize nutrient cycles. Surprisingly, highly diverse communities self-organize to extract [Formula: see text]10[Formula: see text] of the maximum extractable energy, or [Formula: see text]100 fold more than randomized communities. Further, with increasing diversity, distinct ecosystems show strongly correlated fluxes through nutrient cycles. However, as the driving force from light increases, the fluxes of nutrient cycles become more variable and species-dependent. Our results highlight that self-organization promotes the efficiency and stability of complex ecosystems at extracting energy from the environment, even in the absence of any centralized coordination.

Design of a modular controlled unit for the study of bioprocesses: Towards solutions for Bioregenerative Life Support Systems in space.

Space exploration beyond the Low Earth Orbit requires the establishment of Bioregenerative Life Support Systems (BLSSs), which, through bioprocesses for primary resource recycling, ensure crew survival. However, the introduction of new organisms in confined space habitats must be carefully evaluated in advance to avoid unforeseen events that could compromise the mission. In this work, we have designed and built an experimental chamber, named Growing/Rearing Module (GRM), completely isolated and equipped with micro-environmental monitoring and control systems. This unit is specially intended for the study of single bioprocesses, which can be composed to design functional BLSSs. GRM can be implemented with specific devices for the biological system under study and the control of environmental parameters such as temperature, humidity, lighting and if required, pressure of gaseous components. GRM was validated in experiments of both microgreen cultivation, as a source of fresh food for astronauts, and rearing of the decomposer insect Hermetia illucens for bioconversion of organic waste. During the study of each bioprocess, the environmental and biological data were recorded, allowing to make preliminary assessments of the system efficiency. The GRM, as a completely confined environment, represents the first self-consistent unit that allows to fine-tune the optimal parameters for the operability of different bioprocesses. Furthermore, the upgradability according to the mission needs and the functional integrability of modules differently equipped are the keys to GRM versatility, representing a valuable tool for BLSSs' design.

The MaMBA facility as a testbed for bioregenerative life support systems.

The Moon and Mars Base Analog (MaMBA) is a concept for an extraterrestrial habitat developed at the Center of Applied Space Technology and Microgravity (ZARM) in Bremen, Germany. The long-term goal of the associated project is to create a technologically functioning prototype for a base on the Moon and on Mars. One key aspect of developing such a prototype base is the integration of a bioregenerative life support system (BLSS) and its testing under realistic conditions. A long-duration mission to Mars, in particular, will require BLSS with a reliability that can hardly be reached without extensive testing, starting well in advance of the mission. Standards exist for comparing the capabilities of various BLSS, which strongly focus on technological aspects. These, we argue, should be complemented with the use of facilities that enable investigations and optimization of BLSS prototypes with regard to their requirements on logistics, training, recovery from failure and contamination, and other constraints imposed when humans are in the loop. Such facilities, however, are lacking. The purpose of this paper is to present the MaMBA facility and its potential usages that may help close this gap. We describe how a BLSS (or parts of a BLSS) can be integrated into the current existing mock-up at the ZARM for relatively low-cost investigations of human factors affecting the BLSS. The MaMBA facility is available through collaborations as a test platform for characterizing, benchmarking, and testing BLSS under nominal and off-nominal conditions.

Possible risks for the functioning of cyclic processes in the experimental model of a closed ecosystem.

The purpose of the present study is to consider a number of possible risks that may emerge when processed human wastes are involved into mass exchange processes as fertilizers for plants cultivated in the experimental model of the closed ecosystem (CEEM). The problems relating to the disruption of cycling processes in closed ecosystems can be tentatively divided into two groups: the problems that can be rather easily overcome and the chronic problems. Addition of plant inedible biomass to the soil-like substrate (SLS) can result in a decrease in plant productivity because of allelopathic interactions and enhanced growth of microorganisms. The 30% decrease in wheat productivity by the end of long-duration experiments in the CEEM, with plants grown on quasi-non-renewed solutions based on liquid products prepared by physicochemical mineralization of human wastes, was caused by lower resistance of the plants affected by toxicants accumulated in the solution because of incomplete mineralization of the wastes. The reason for the differences between the macronutrient inflows and outflows was that the donor of human wastes followed a European-type diet while the system produced only part of the plant-based diet. Moreover, macronutrients were partly sorbed in rooting substrates and became unavailable to plants: the substrates in the system retained about 50% of the Ca and 20% ÷ 25% of the Mg, Na, and P inflows over one cycle. These problems are temporary and can be minimized in the foreseeable future.

Water recycle system in an artificial closed ecosystem - Lunar Palace 1: Treatment performance and microbial evolution.

Water recycle systems have important implications to realize material circulation in biological regeneration life support systems, which is of significance for long-term space missions and future planetary base. Based on membrane biological activated carbon reactor (MBAR) technologies, the 'Lunar Palace 365' experiment established various treatment processes for condensate wastewater, domestic wastewater, urine, and used nutrient solutions. The 370-day operation data showed the CODMn index of purified condensate wastewater decreased to 0.74 ± 0.15 mg/L, which met the standards for drinking water quality. The average removal rate of organic contaminants in domestic wastewater by the MBAR was 85.7% ± 10.2%, and this MBAR also had a stable nitrification performance with effluent NO3--N concentrations fluctuating from 145.57 mg/L to 328.59 mg/L. Moreover, the purification of urine achieved the conversion of urea-N to NH4+-N and thus the partial recovery of nitrogen. 16S rDNA sequencing results revealed the evolution of microbial diversity and composition during the long-term operation. Meiothermus, Rhodanobacter, and Ochrobactrum were the dominant microorganisms in various MBARs.

Review of research into bioregenerative life support system(s) which can support humans living in space.

To travel beyond the Earth and realize long-term survival in deep space, humans need to construct Bioregenerative Life Support System (BLSS), which reduces the requirement for supplies from the Earth by in situ regenerating oxygen, water and food needed by astronauts, and prevents pollution to extraterrestrial bodies by recycling waste. Since the 1960s, the USSR/Russia, the United States, Europe, Japan, and China carried out a number of studies with abundant achievements in BLSS systematic theories, plants/animals/microorganisms unit technologies, design/construction, and long-term operation/regulation. China's "Lunar Palace 365″ experiment realized Earth-based closed human survival for a year, with a material closure of >98%. However, a lot of research work is still needed to ultimately realize BLSS application in space, especially given the space experiment of BLSS never carried out, and the overall impact of space environment on BLSS unknown. Lunar exploration projects such as lunar village and lunar research station are successively proceeding. Therefore, future BLSS research will focus on lunar probe payload carrying experiments to study mechanisms of small uncrewed closed ecosystem in space and clarify the impact of space environmental conditions on the ecosystem, so as to correct the design and operation parameters of Earth-based BLSS. Such research will provide theoretical and technological support for BLSS application in crewed deep space exploration.

Design and establishment of a large-scale controlled ecological life-support system integrated experimental platform.

A Controlled Ecological Life-Support System (CELSS) can meet the demands of food, oxygen, and water for human, as well as providing psychological benefits during deep space exploration by the continuous materials regeneration. Many key techniques of the platform are needed to explore before applying to the extraterrestrial planets. In this study, a large-scale CELSS integrated experimental platform was designed and constructed to meet the basic life-support material demands of six crew members (max). The platform was composed of four kinds of cabins including Crew Cabin (CC), Plant Cabin (PC), Life-Support Cabin (LSC), Resource Recycling Cabin (RRC) and affiliated facilities. Eight cabins were involved in the platform, i.e., CCs I and II, PCs I, II, III and IV, LSC, and RRC. The platform involved 15 subsystems and covered a plant culture area of 206.6 m2 (a max extensible area of 260 m2) and a total volume of 1340 m3. The joint debuggings and the 4-subject 180-day CELSS integration experiment were carried out successfully. The material closures were 55% (on average) for food (70.8% in highly efficient production period), 100% for atmospheric regeneration, 100% for water regeneration, and 87.7% for recycled solid waste in the 4-subject 180-day integration experiment. It verified that the indicators of the platform meet the technical requirements and realize food regeneration, air regeneration and water regeneration through the integration of physico-chemical technique and biological technique for the long-term survivals of six crew members in the closed cabins.

Immunological Aspects of Isolation and Confinement.

Beyond all doubts, the exploration of outer space is a strategically important and priority sector of the national economy, scientific and technological development of every and particular country, and of all human civilization in general. A number of stress factors, including a prolonged confinement in a limited hermetically sealed space, influence the human body in space on board the spaceship and during the orbital flight. All these factors predominantly negatively affect various functional systems of the organism, in particular, the astronaut's immunity. These ground-based experiments allow to elucidate the effect of confinement in a limited space on both the activation of the immunity and the changes of the immune status in dynamics. Also, due to simulation of one or another emergency situation, such an approach allows the estimation of the influence of an additional psychological stress on the immunity, particularly, in the context of the reserve capacity of the immune system. A sealed chamber seems a convenient site for working out the additional techniques for crew members selection, as well as the countermeasures for negative changes in the astronauts' immune status. In this review we attempted to collect information describing changes in human immunity during isolation experiments with different conditions including short- and long-term experiments in hermetically closed chambers with artificial environment and during Antarctic winter-over.

Optimized crop growth area composition for long duration spaceflight.

This paper presents an optimized composition of crop growth areas for biological life support systems with respect to nutrition and equivalent system mass. For this purpose, crop growth area compositions from literature are compared with compositions derived from an optimization algorithm. The optimization algorithm uses literature data for crop growth rates and crop nutrient content to minimize the required crop growth area required to supply all nutrients for a human. The algorithm derives the required crop growth area per crew member under different dietary boundary conditions and the resulting nutrient supply is compared to reported diets from crops for spaceflight. The primary goal of this optimization is to find the minimal area required to supply all relevant macronutrients. The minimal area for the exact desired composition of macronutrients (carbohydrates, fats and proteins) was 106.86 m² using chard, lettuce, peanut, bell pepper, snap beans and spinach. If a deviation in the macronutrient composition is allowed the required area can be reduced to 39.88 m² of wheat and white potatoes. Since the variety of crops is a relevant factor for long term food supply, a limit of the maximum growth area per crop was introduced to derive a diet with more variety, which resulted in a minimal area of 57.04 m² using drybean, rice, snap beans, sweet potato, wheat and white potato. Based on this result, a further manual adjustment of the crop growth areas was performed to also introduce lettuce and tomato in the crops provided and adjust the remaining crop compositions to receive better macro- and micronutrient conformity while maintaining a crop growth area of 57 m². One major result of this analysis is that soybeans are not the most favorable crop with regard to protein and fat productivity and the focus of NASA crop selection for full nutrient supply on soybean results in exceedingly large required crop areas of 164.15 m². The resulting crop growth areas from both the optimization and literature are then analyzed as plant growth chambers (PGC) in the Life Support Trade-Off Tool (LiSTOT) of the institute of astronautic from the Technical University of Munich (TUM). LiSTOT calculates the impact of the PGC on an ISS based environmental control and life support system (ECLSS) using averaged steady state values for the plants from literature. Based on this result LiSTOT scales the physical chemical systems and calculates the resulting equivalent system mass (ESM) of the different cases. This approach allows the consideration of not only the PGC ESM, but also the impacts the PGC has on other ECLSS systems and their ESM. The ESM values for PGC were updated to assume LEDs instead of high pressure sodium lamps resulting in a new logistic mass of the PGC of 1.28 kg/(y m²) and a lower specific system mass of 87.7 kg/m². The mass balance analysis of carbon within the overall ECLSS lead to a reduction of the plant growth area to 50.6 m² and the break-even time with the ISS ECLSS was calculated to 87.2 years. With more optimistic assumptions for the LED and using urine as nutrient supply this time can be reduced to 14.6 years. The analysis also showed that the derived crop composition is not only favorable regarding nutrient supply but also with regard to the ESM and break-even time compared to previously reported crop compositions. Only the PGC with only wheat and white potatoes has a lower ESM but also provides a less balanced nutrient supply. This PGC is downscaled to 37.55 m² to achieve carbon balance and a break-even time of 38.4 years or 10.3 years with the optimistic assumptions.

Large and interacting effects of temperature and nutrient addition on stratified microbial ecosystems in a small, replicated, and liquid-dominated Winogradsky column approach.

Aquatic ecosystems are often stratified, with cyanobacteria in oxic layers and phototrophic sulfur bacteria in anoxic zones. Changes in stratification caused by the global environmental change are an ongoing concern. Increasing understanding of how such aerobic and anaerobic microbial communities, and associated abiotic conditions, respond to multifarious environmental changes is an important endeavor in microbial ecology. Insights can come from observational and experimental studies of naturally occurring stratified aquatic ecosystems, theoretical models of ecological processes, and experimental studies of replicated microbial communities in the laboratory. Here, we demonstrate a laboratory-based approach with small, replicated, and liquid-dominated Winogradsky columns, with distinct oxic/anoxic strata in a highly replicable manner. Our objective was to apply simultaneous global change scenarios (temperature, nutrient addition) on this micro-ecosystem to report how the microbial communities (full-length 16S rRNA gene seq.) and the abiotic conditions (O2 , H2 S, TOC) of the oxic/anoxic layer responded to these environmental changes. The composition of the strongly stratified microbial communities was greatly affected by temperature and by the interaction of temperature and nutrient addition, demonstrating the need of investigating global change treatments simultaneously. Especially phototrophic sulfur bacteria dominated the water column at higher temperatures and may indicate the presence of alternative stable states. We show that the establishment of such a micro-ecosystem has the potential to test global change scenarios in stratified eutrophic limnic systems.

Plant biology for space exploration - Building on the past, preparing for the future.

A review of past insights of space experiments with plants outlines basic space and gravity effects as well as gene expression. Efforts to grow plants in space gradually incorporated basic question on plant productivity, stress response and cultivation. The prospect of extended space missions as well as colonization of the Moon and Mars require better understanding and therefore research efforts on biomass productivity, substrate and water relations, atmospheric composition, pressure and temperature and substrate and volume (growth space) requirements. The essential combination of using plants not only for food production but also for regeneration of waste, and recycling of carbon and oxygen production requires integration of complex biological and engineering aspects. We combine a historical account of plant space research with considerations for future research on plant cultivation, selection, and productivity based on space-related environmental conditions.

Operation overview of a biological waste treatment system during the 4-crew 180-day integrated experiment in the controlled ecological life support system (CELSS).

Waste management and treatment is vital to health care and material circulation, especially in the Controlled Ecological Life Support System (CELSS) with finite resources for long-duration manned space missions. A closed ecological-cycle integrated 4-crew 180-day experiment platform was established to investigate the key technologies such as effective cultivation of higher plant, water treatment and recycling, waste management and treatment. In this study, generated waste during the integrated experiment was classified as renewable and non-renewable waste. The renewable waste including all crew feces and part of inedible plant biomass were treated in a biological system where the aerobic composting technology was utilized. The performance in relation to degradation effect, phytotoxicity and nutrient evaluation was examined during the continuous 180 days. The long-term operation results displayed that 96.26 kg feces and 74.4 kg wheat straw were treated, and 90.6 kg compost product was discharged in nine batches. The microbial community variation was analyzed and Firmicutes, Actinobacteria and Proteobacteria enriched in the compost. The phytotoxicity of compost was examined by seed germination index (GI) and GI of Chinese cabbage ranged from 88% to 132% for all batches. Compared to grown in vermiculite only, the lettuce yield increased 19% when grown in a mixture of vermiculite and processed compost. The summary of this work will be helpful to facilitate future applications of aerobic composting technology as the bio-based waste treatment technology in CELSS.

Biology and crop production in Space environments: Challenges and opportunities.

Long-term manned space-exploration missions and the permanence of human colonies on orbital stations or planetary habitats will require the regeneration of resources onboard or in-situ. Bioregenerative Life Support Systems (BLSSs) are artificial environments where different compartments, involving both living organisms and physical-chemical processes, are integrated to achieve a safe, self-regulating, and chemically balanced Earth-like environment to support human life. Higher plants are key elements of such systems and Space greenhouses represent the producers' compartment. Growing plants in Space requires the knowledge of their growth responses not only to all environmental factors acting on Earth, but also to specific Space constraints such as altered gravity, ionizing radiations and confined volume. Moreover, cultivation techniques need to be adjusted considering such limitations. The type and intensity of environmental factors to be taken into account depend on the mission scenarios. Here, we summarize constraints and opportunities of cultivating higher plants in Space to regenerate resources and produce fresh food onboard. Both biological and agro-technological issues are considered briefly going through experiments both ground-based on Earth and in Space.

Aquatic invertebrate protein sources for long-duration space travel.

During the summer of 2020, NASA returned to launching astronauts to the International Space Station (ISS) from American soil. By 2024, NASA's mission is to return to the Moon, and by 2028 create a sustainable presence. Long duration missions come with obstacles, especially when trying to create a sustainable environment in a location where "living off the land" is impossible. Some resources on the Moon can be recovered or resupplied; however, many resources such as those needed for sustaining life must be recycled or grown to support humans. To achieve sustainability, food and water must be grown and recycled using elements found within the habitat. NASA's current work focuses on food resupply and growing plants as supplemental nutrient content. This paper examines the possibility for using aquaculture systems to purify water while growing nutrient-rich species as food sources, which aquatic food sources would be ideal for a habitat environment, and which species might provide an ideal test case for future studies aboard ISS. The aquatic species should be rapidly grown with high protein content and low launch mass requirements. Although there are numerous challenges and unknown technology gaps for maintaining aquaculture systems in reduced gravity environments, the benefit of employing such systems would be of great advantage towards creating a sustainable presence beyond Earth's orbit for sustainable aquaculture.

Deep Physical-Chemical Purification of Gas Medium in Artificial Ecosystems.

The results of experiments on application of a newly developed facility for oxidation of volatile organic compounds on a platinum catalyst are presented. The feasibility of using this method in artificial ecosystems as a whole and in mass exchange of closed biological-technical life support systems in particular is shown. The possibility of deep purification of gas emitted from the reactor of physical-chemical processing of organic wastes is demonstrated. Wheat growing experiment on using the facility for oxidation of volatile organic compounds in a sealed chamber was performed. No adverse effect of probable toxic oxidation products on wheat plants during a 4-day experiment was determined.

The effect of supplementation of the soil-like substrate with wheat straw mineralized to different degrees on wheat productivity in closed ecosystems.

Successful incorporation of soil-like substrate (SLS) into biotechnical life support systems is often complicated by the necessity to maintain the balance between flows of mineral elements taken up from the substrate by growing plants and mineral elements added to the SLS as components of mineralized plant inedible biomass. An imbalance between these two flows can be caused by the addition of recalcitrant plant waste such as wheat straw. The purpose of this study was to determine whether the availability of essential nutrients to be taken up by the roots of the wheat plants grown on the SLS could be enhanced by supplementing the SLS with the products derived from wheat straw subjected to different levels of physicochemical mineralization in the aqueous solution of hydrogen peroxide. Different degrees of straw mineralization were achieved by using different ratios of the aqueous solution of hydrogen peroxide to straw. The study showed that supplementation of the SLS with insufficiently oxidized products of physicochemical mineralization of straw resulted in a decrease in the grain yields. The inhibitory effect of the straw subjected to physicochemical oxidation increased with a decrease in the degree to which the straw had been oxidized. Only supplementation with the straw mineralized to the highest possible degree did not inhibit plant growth and development, and the crop yield in that treatment was higher than in the other treatments.

Effect of solid waste fermentation substrate on wheat (Triticum aestivum L.) growth in closed artificial ecosystem.

Bioregenerative Life Support System (BLSS) is a closed artificial ecosystem and could provide oxygen, food, water and other substrates for long-term deep space survival. The treatment and recycle of the solid waste are crucial and rate-limiting steps in BLSS, and it's reported that the solid waste such as the inedible plants and human feces could be fermented aerobically and then reused as fertilizer for growing plants in BLSS, which may be an effective way to improve the solid waste recycling rate. However, the recycling performance and the effect on the system need to be evaluated. In this study, the fermented and decomposed solid waste product from the 365d BLSS experiment with human involved in Lunar Palace 1 was utilized, and was added to the Hoagland nutrient solution as a supplementary fertilizer in the weight proportion of 5% and 10%, respectively, for the cultivation of wheat (Group-5% and Group-10%). Then, the effects on wheat germination, morphology, photosynthesis, biomass, the conductivity of the cultured substrates and microorganisms were detected and compared with those of the CK group cultured using only Hoagland nutrient solution. The results showed that this planting method had no inhibitory effect on the wheat germination, root length and yield, and might even promote the vegetative growth of wheat in terms of Vigor index, plant height, leaf area and net photosynthesis rate to some extent. The added solid waste fermentation substrate as well as the planting environment in Lunar Palace 1 both had significant influences on the rhizosphere microorganisms of wheat. The bacteria diversity was more abundant than fungi at phylum level, and the relative abundance varied along with the wheat growth period. The relative abundance of the cellulose degrading microorganisms including Actinobacteria and Ascomycota increased in Group-5% and Group-10% compared with CK group along with the growth of wheat. Moreover, the proper reuse of the fermentation substrate could reduce the use of inorganic salts by 9.8%-11.9% and save 40L•m - 2 of water for wheat cultivation. This research has considerable application significance in future deep space exploration.

Wheat Cultivation and Nutrient Control for the 180-day CELSS Integrated Experiment.

This research aimed to select the well-adapted wheat cultivar and to explore an optimum nutrient control pattern for wheat cultivation in the 180-day integrated experiment of controlled ecological life support system (CELSS). In the experiment, six wheat cultivars from different areas of China were preselected and cultivated in four separate recirculating hydroponic systems (HySy), nutrients in which could be controlled and recycled according the values of pH, electrical conductivity (EC) and dissolved oxygen (DO). Wheat covered an area of 111.3 m2 and had been planted in 17 batches with a 15-day time interval to realize stable regeneration of oxygen, water and food during the 180-day duration in the closed cabin. The results indicated that different cultivars displayed different adaptabilities to the controlled environment. Wt04 had a stronger adaptability with the highest yield (12.82 g DM m-2 d-1) and edible radiation use efficiency (RUE) (0.28 g DM mol-1) whereas Wt06 adapted this environment poorly because of its excessive vegetative growth. For the morphological characters, wheat plants tended to dwarf in the CELSS environment compared with the field. An innovative controlling pattern was established for nutrient supplement. Through the real-time monitoring of pH, EC and DO of the nutrient solution and the periodical detection of the contents of nutrient elements, the nutrient solution could be controlled and recycled continuously without being renewed under a suitable state for wheat plants growth during the 180-day integrated experiment.