Introduction

Establishing a life-sustaining system for extraterrestrial planets is a significant challenge in humanity’s quest for space exploration. The vast distances, high costs, and environmental differences from Earth necessitate the development of self-sufficient technologies for food and oxygen production in spacecrafts and colonies. The harsh extraterrestrial environments, characterized by intense radiation, distinct atmospheres, and scarce water resources, demand innovative approaches to harnessing finite cosmic resources for sustainable, recycling-oriented ecosystems (Fig. 1a).

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Concept of this study. (a) Conceptual diagram of the research. (b) Introduction of the Ecosphere and Biosealed created in this study, along with a mention of the prior study, Biosphere 2.

Pioneering efforts like Biosphere 2 (Allen, 1993, Fig. 1b, left panel1,2,3) have demonstrated the complexities of creating and maintaining such ecosystems in enclosed spaces. While Biosphere 2 provided invaluable data on ecosystem sustainability and human well-being in closed environments, it also revealed challenges related to structural flaws, altered soil microbial communities, oxygen depletion, food shortages, and interpersonal tensions. These challenges underscore the need for refined, smaller-scale ecosystem transfer concepts for sustainable space colonization and highlight the difficulties in maintaining long-term ecological balance.

In this study, we address these challenges by introducing the novel “Ecosphere” and “Biosealed” systems, compact, natural, closed-loop ecosystems designed to replicate Earth’s ecosystems within customizable containers. Our research focuses on plant growth and survival, the role of microorganisms, and cultivation using simulated extraterrestrial soil. We aim to elucidate the mechanisms and challenges of sustaining life in a closed environment and to identify potential solutions (Fig. 1b, right panel). Notably, our study investigates plant cultivation in simulated extraterrestrial soils from the Luna and Ryugu asteroid, exploring the presence and impact of microorganisms4,5,6,7. This approach is crucial for understanding the feasibility of growing plants in extraterrestrial environments, a critical step towards human space colonization8.

Bringing components of natural systems together with humans is an indispensable and important item for space migration to different celestial bodies, especially for a longer-term migration. Since the whole terrestrial ecosystem cannot be transported to another planet, a systematic comprehension of each ecosystem component, such as microbe colonies in different cultures with appropriate humidity conditions, should be made to establish the mini-(core)biomes necessary for space migration, rather than comprehension of individual species9,10.

This study aims to identify essential ecosystem components which can be used as non-physio-chemical life-supporting systems for supporting life in space by cultivating a minimum set of ecosystems. Our research goes beyond replicating previous closed-system experiments. We introduce novel design elements, such as incorporating groundwater layers to address moisture deficiency, a common challenge in enclosed environments. Additionally, we employ metagenomic analysis to quantify and characterize microbial communities in both our custom ecosystems and simulated space soils. This comprehensive approach allows us to gain deeper insights into the complex interactions between plants, microbes, and their environment, paving the way for the development of more robust and sustainable life-support systems for future space exploration.

Natural circulation in a sealed space by Ecosphere 1

Inspired by Biosphere 2, we developed a sealed container named “Ecosphere 1”, with enhanced airtightness. This container is made of glass and is sealed with melted rubber or silicone for the lid. It is designed to be more compact than its predecessors, aiming to identify challenges in a basic closed ecological system by emulating a natural environment.

To investigate the characteristics of natural circulation in the closed space of Ecosphere 1, we enclosed nutrient-rich soil collected from a natural environment, and seeds of clover, a leguminous plant with nodules, into the Ecosphere 1. As a result, mainly clover growth was observed. Emphasizing the role of microbes in maintaining a stable natural ecosystem, experiments were conducted using soil containing naturally derived microbes. Finally, Ecosphere 1 was placed outdoors on a wooden board at a height of about 1 m above the ground to avoid heat from asphalt and concrete. The natural cycles inside it were then observed for 4 years, reflecting the seasonal changes in Japan.

Our analysis revealed the coexistence of plants and Cyanobacteria, establishing diurnal oxygen supply and natural circulation through their activity. We also observed the decomposition process of withered clover stems and leaves. Mass conservation tests further confirmed the absence of flaws in the Ecosphere 1 structure (Supplementary Fig. 1a, b, Tables 1, 2). These results suggest the viability of sustaining a unique ecosystem in a confined space and underscore the integrity of the Ecosphere 1 design.

Remarkably, Ecosphere 1 adapted to the diverse external environment of Japan’s four seasons, exhibiting its own natural cycles (Fig. 2a). In the first spring, only soil, water, and seeds were introduced into Ecosphere 1, and it was placed in a sunny location. Within 2–3 days, the clover seeds germinated. As summer approached and internal temperatures rose, most plants inhabiting the surface perished due to the heat retention of the sealed glass container. However, decomposers breaking down the dead plants were identified (Supplementary Fig. 2a). Furthermore, Cyanobacteria proliferated, covering both the surface and the ground (Fig. 2b). Their photosynthesis likely provided oxygen, possibly sustaining aerobic bacteria (Supplementary Fig. 2 b,c). In autumn, new shoots emerged from presumed rhizomes beneath the soil, while Cyanobacteria receded into the earth (Fig. 2c). Though winter witnessed some plants perishing at the end of their life cycles, others overwintered. Cyanobacteria flourished once more, enveloping the container surface (Fig. 2d). By the following spring, it became evident that new shoots sprouted from seeds left behind by the previous generation (Fig. 2e).

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Natural cycle observed in ecosphere 1 under natural conditions. (a) Conceptual representation of the natural cycle within Ecosphere 1. (b) Observations of Cyanobacteria proliferation during summer. (c) Appearance of regrowing clover in autumn. (d) Increase in Cyanobacteria during winter. (e) Residual clover seeds.

Growth rate of plants in a sealed environment

To closely examine the influence of a sealed environment on the growth rate of plants, observations were made within the Ecosphere 1 from January 13, 2021, to April 25, 2021, spanning approximately 15 weeks (Fig. 3a). In the sealed space, plants grew only about 10 cm over 15 weeks, whereas in an open environment, they achieved this growth in merely 5 weeks. This stunted growth is believed to be due to the accumulation of the plant hormone ethylene and a lack of essential moisture for plant growth11. It is speculated that the buildup of the plant hormone in the closed environment adversely impacted plant development. Additionally, given the characteristics of the Ecosphere, the total water content remained constant. As plants grew, the soil moisture content diminished, which was thought to further inhibit growth.

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Results of plant growth experiments in sealed environments and survival of yellow Drosophila fly. (a) Growth of clover in a sealed environment. (b) Plant growth under LED light conditions. The top image showcases basil growth in an open environment with LED lighting, while the bottom shows cabbage growth in a sealed environment with LED lighting. (c) Representation indicating the sustainability of the yellow Drosophila fly in a sealed environment. The left image is from the first day where larvae were visible, while the right image is from the last observed moment, showing them flying within the container.

Drawing inspiration from these insights, we revamped Ecosphere 1 and birthed “Ecosphere 2”, endowed with an expansive underground aquifer (Supplementary Fig. 3). Thanks to this new structure, the soil within Ecosphere 2 remained consistently moist regardless of seasonal changes or the growth status of the plants (See Supplementary materials 2 for the consideration of the quantities of water in the experiment). Within Ecosphere 2, while plants stretched taller, their leaf dimensions remained steadfast, possibly under the influence of the ethylene hormone.

Importance of the presence of a groundwater layer

This study clarified the important role that groundwater layers play in maintaining closed ecosystems. The presence of a groundwater layer in a closed system enabled a stable water supply to plants and acted as a buffer against external temperature fluctuations, greatly improving the chances of plant survival. Notably, this experiment resolved the issue of differences in plant growth, particularly height, between open and closed systems, which had been an issue in previous studies using Ecosphere1. In closed systems with a properly designed groundwater layer, plants were observed to grow in total length equal to or greater than those in open systems. On the other hand, a new interesting phenomenon was also confirmed. In closed systems, regardless of the presence or absence of a groundwater layer, the elongation of plant leaves tended to be suppressed compared to open systems. This phenomenon unique to closed systems may be caused by factors that have yet to be elucidated, such as humidity and carbon dioxide concentration within the system. These findings provide important insights into the design of sustainable closed ecosystems and future plant cultivation in space. Future research will focus on elucidating the leaf growth suppression mechanism unique to closed systems and will continue to work toward realizing a more complete closed ecosystem.

During the experimental period, in S100-1, S100-2, S200-1, and S200-2, which do not have a groundwater layer, the white clover inside reached its peak 3 and 6 days after the start of the experiment, and eventually all the individuals died (Figs. 4, 5a). On the other hand, in S400-1, S400-2, S600-1, and S600-2, which have a groundwater layer, several individuals were still surviving after 15 days. In S400-2, all the germinated seeds survived to the end (Figs. 4, 5a). However, in S600-1 and S600-2, which had the maximum amount of water from the groundwater layer, the soil was too wet, causing root rot, making it difficult for some individuals to survive.

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Experiment showing the importance of groundwater. Measurements were taken for 15 days. The state after 3, 6, 9, 12, and 15 days from the start of the experiment.

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Plant growth and survival in relation to groundwater layers. (a) Abundance ratio (= number of birthed and survived seeds/total number of seeds) of the five enclosed seeds in each sample observed during the experimental period. (b) Comparison of total length of white clover plants surviving inside ce-600, ce-400, and nce measured 15 days after the start of the experiment. Error bars indicate mean \(\pm\) standard error.

The samples in which white clover survived until the end were S400-1, S400-2, S600-1, S600-2, S1, S2, and S3. Here, S400-1 and S400-2, which have the same amount of groundwater, were collectively referred to as closed environment 400 (ce-400), S600-1 and S600-2 were collectively referred to as closed environment 600 (ce-600), and the open systems S1, S2, and S3 were collectively referred to as non-closed environment (nce) to compare the measurement results.

These results show that in a closed ecosystem with a groundwater layer, white clover can grow to a length like that of an open system. In the ce-400 system, growth was better than in the open system, despite the same sunshine conditions (Figs. 5b, 6b). However, the leaf length of the closed systems ce-400 and ce-600 tended to be relatively smaller than that of the open system (Supplementary Table 4).

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Groundwater and white clover. (a) The state of the groundwater in each sample. Water vapor was observed in S100-1, S100-2, S200-1, and S200-2, but no groundwater was observed. Groundwater was seen to have accumulated in S400-1, S400-2, S600-1, and S600-2. (b) The white clover collected and measured 15 days after the start of the experiment.

These experimental results show that the presence of a groundwater layer is essential for the long-term survival of white clover in a closed system, and that an appropriate amount of moisture (ce-400) produces the best growth results, while excessive moisture (ce-600) causes root rot. In particular, a properly designed closed system (ce-400) can achieve length growth equal to or greater than that of an open system (nce). However, while the closed system shows good length growth, the leaf length tends to be smaller than that of the open system. These findings provide important insights into the design of sustainable closed ecosystems, emphasizing the importance of a groundwater layer and the need for appropriate moisture management.

Plant cultivation in a sealed environment using LED lighting

During outdoor maintenance of Ecosphere1, many plants succumbed to the intense summer heat. In extraterrestrial environments, such as the moon, there is no thick atmosphere like on Earth. Consequently, the heat from sunlight is directly transferred, compounded by intense radiation. In response, we experimented with plant cultivation using the SP312 LED light (NARRNA), which has less heat impact than natural sunlight. We chose basil (Ocimum basilicum) and cabbage (Brassica oleracea var.capitata) for the study, given their resistance to diseases and pests, and their low sunlight requirements and cold tolerance.

Initially, to verify the suitability of the LED lights for plant growth, we conducted germination and growth experiments with basil in open conditions, and with cabbage in enclosed environments (Fig. 3b, top panel). By day 15, the cabbage germinated normally, and the basil growth was progressing well (Fig. 3b, bottom panel). An observation of cabbage germination in an open environment, without soil, confirmed its normal germination by day 15 (Supplementary Fig. 4).

Building on these results, we attempted plant cultivation in a closed space. We planted clover and cabbage simultaneously, monitoring their growth under the LED lights. Both plants were able to sustain life. However, a characteristic challenge in enclosed spaces was observed: the tendency for leaves to be smaller. Nevertheless, this tendency was also observed in natural sunlight conditions. Thus, we decided that using LED lights wouldn’t pose issues for the plant growth experiments.

Observations on sustaining life in sealed environments

In the preliminary stages of our experiment with Ecosphere1, a dead fly was discovered 1 month after the experiment’s inception. However, no other fly remains were detected thereafter. Based on this initial test, we introduced an advanced model, “Biosealed”, incorporating clover, cabbage, and artificially added Cyanobacteria. Biosealed, made of glass, doesn’t feature an underground aquifer; instead, it uses soil enriched with vermiculite to enhance moisture retention.

To this Biosealed model, we introduced eggs of the fruit fly (Drosophila melanogaster), assessing life sustenance potential under conditions similar to the previous experiment. Thanks to a consistently maintained temperature in a stable environment, larvae were identified within a week. Under these conditions, the fruit fly eggs hatched and matured into adults. Notably, the last observation of a living fruit fly was on day 37 post-introduction. Considering that the average lifespan of a fruit fly in natural conditions (At an average temperature of 27 ℃) is known to be 40 days, we can infer that life sustenance in the enclosed environment was a success (Fig. 3c). We speculate that the extended life span could be attributed to the more stable conditions than those found in nature.

From the above findings, it is evident that life can be sustained in enclosed environments for a definite period.

Plant cultivation in exoplanetary simulated soils

In this study, we examined the variations in plant cultivation environments within sealed spaces using multiple extraterrestrial simulated soils. Notably, in the microbe-free Ryugu simulated soil, germination of plants was not observed (Fig. 7a). Given that the aqueous pH value of the relevant simulated soil stood at 8.45, we ruled out pH levels as the inhibiting factor for germination. Observations of darkened clover seeds and the presence of fine particles in the Ryugu simulated soil led us to postulate that these particles might have adhered to the clover seeds, potentially inhibiting their respiration.

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Germination experiments of plants in exoplanetary simulated soils and importance of microbial presence. (a) Germination rate comparison between plants in exoplanetary simulated soils with similar components, with and without introduced microbes. (b) Plant cultivation experiments under conditions resembling lunar soil, using Fly ash and Clinker ash. (c) Plant growth in a sealed environment without sterilizing the microbes. (d) Plant growth in a sealed environment post microbial sterilization.

Contrastingly, in the microbe-introduced Ryugu simulated soil, 6 out of 10 seeds successfully germinated (Fig. 7a). This suggests the potential of microbes to ameliorate the extreme conditions of the Ryugu simulated soil, laying the groundwork for further analysis. In the lunar simulated soil, irrespective of the presence of microbes, all seeds germinated. These results have been consolidated in Fig. 7a.

In both Fly ash and Clinker ash, germination and growth of clover were confirmed regardless of microbial presence (Fig. 7b). However, Fly ash, owing to its fine particles, exhibited heightened water repellency, posing a constraint to plant growth. Building upon prior research which reported difficulties in microbe fixation to soil12, efforts to increase the porosity of Fly ash and Clinker ash were undertaken. Initially, Fly ash mixed with water was found to be fragile and had issues with excessive water absorption, rendering it unsuitable (Supplementary Fig. 5e). In contrast, solids made from cement and bentonite were porous but lacked adequate moisture retention, showing no signs of plant germination. Using a bonding agent, Booncrete exhibited moderate water absorption (Supplementary Fig. 5 h, j, k), yet its excessive hardness was deemed unsuitable for plant cultivation, indicating room for improvement.

To evaluate the extent to which microbes contribute to forming a plant cultivation environment in sealed spaces, a comparative experiment between sterilized and non-sterilized soils was conducted. Consequently, non-sterilized soils showed favorable growth of clover (Fig. 7c), whereas growth in sterilized soils was minimal (Fig. 7d). These findings underscore the significance of microbial presence in improving conditions within extreme and sealed environments.

Plant growth and microbial interactions in sealed environments

To elucidate the interactions between plant growth and microbes, we conducted a metagenomic analysis. Initially, the relative abundance of microbes was depicted in Fig. 6a using entropy (alternatively known as ASV-based Shannon diversity), a measure commonly utilized to indicate biological diversity and distribution. Ecosphere samples (ES1, ES2) displayed lower entropy compared to other samples, suggesting a pronounced presence of specific bacteria within the ecosphere. However, the time elapsed post-microbial introduction into the Indicator soil’s simulated soil was shorter than for ES1 and ES2, suggesting we might be observing a transitional state of microbial communities. Indicator soil is primarily composed of heterotrophic microbes. In the case of endogenous heterotrophic succession, it’s proposed that an intermediate supply of all limiting resources and a peak diversity in the organic carbon pool may occur13.

We employed BALSAMICO (BAyesian Latent Semantic Analysis of MIcrobial COmmunities)14 to identify bacterial communities from microbiome data and to explore the association between environmental factors and these communities. BALSAMICO integrates a non-negative matrix factorization approach, factoring in environmental influences to model community structures. As illustrated in Fig. 8b, the analysis reveals that while bacterial community 1 predominantly characterizes ES1, ES2 is mainly defined by bacterial community 2, and the simulated soil is dominated by bacterial community 3. Intriguingly, the significant difference in bacterial communities between ES1 and ES2 suggests that humidity might have a profound impact on the bacterial community composition. Water activity is a known determinant of microbial communities11,15, which might play a more significant role than other environmental factors in distinguishing ES1 and ES2.

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Metagenomic analysis results to examine the impact of microbes on plant growth. (a) Entropy for each environment. (b) Microbial community composition for each environment, as estimated by BALSAMICO. (c) Predominant microbes in each community, as inferred by BALSAMICO. (d) Effects of environmental factors on the estimated microbial community composition by BALSAMICO (expressed as log odds ratio).

Delving deeper into the bacterial communities, Community 1, as evident from data analysis in Fig. 6d and 8c and Supplementary Table 2, is primarily dominated by aerobic chemoautotrophic bacteria such as Brevibacillus from the phylum Firmicutes. Notably, the presence of oxygenic photosynthetic nitrogen-fixing bacteria, like Leptolyngbya from the phylum Cyanobacteria, is suggested. In Community 1, not only plants but also these bacteria might play a role in supplying both oxygen and organic compounds within the Ecosphere. In contrast, Communities 2 and 3 have fewer oxygenic photosynthetic bacteria. Community 2 mainly consists of aerobic chemoautotrophic bacteria such as Rhodothermaceae from the phylum Bacteroidetes and Trueperaceae from the phylum Thermi, as well as anaerobic chemoautotrophic bacteria like Anaerolineae from the phylum Chloroflexi. Notably, Acidimicrobiales from the phylum Actinobacteria suggest a potential role in supporting organic compound provision through autotrophic iron ion oxidation. Additionally, Comamonadaceae from the phylum Proteobacteria is a notably abundant species. Finally, Community 3 is primarily composed of aerobic chemoautotrophic bacteria like Arthrobacter, Nocardioidaceae, and Agromyces from the phylum Actinobacteria. Especially, Arthrobacter has been reported to survive under stressful conditions16,17.

Conclusion

In our research, we crafted innovative enclosures dubbed “Ecosphere” and “Biosealed” to delve into the creation and upkeep of compact, naturally sustainable ecosystems within confined spaces. Our experiments furnished pivotal insights relevant to life support systems in space exploration:

  • Natural circulation in sealed habitats We discerned the symbiotic synergy between plants and Cyanobacteria in the Ecosphere, which facilitate oxygen generation during daylight. This interdependence is integral to life preservation in sealed settings. Additionally, leveraging LED lighting augments plant growth, even sans sunlight—a revelation with profound implications for space missions with limited solar access.

  • Decoding growth dynamics: We observed that a diminished plant growth rate correlated with ethylene gas buildup and moisture deficits. To address these challenges, an Ecosphere blueprint featuring a subterranean water reservoir has been proposed to ensure consistent moisture availability. Furthermore, variations in leaf dimensions, modulated by plant hormones, emerged as morphological feature indicative of metabolic function. Since water is used to form new plant tissues, there is a possibility of water shortage occurring. Therefore, in Ecosphere2, we created an underground water layer to resolve the water shortage issue.

  • Navigating planetary soil terrains: Under severe soil conditions, the incorporation of microorganisms seems promising in enhancing seed sprouting. This underscores the merit of microbial symbiosis as a viable strategy for life support in inhospitable extraterrestrial conditions. The soil’s intrinsic properties, such as granular size, hydrophobicity, and cohesiveness, significantly impact plant development. Recognizing these factors is vital in refining life support system architectures.

  • Microbial dynamics in extraterrestrial soils: Understanding microbial populations thriving in simulated alien soils is essential, as it could foreshadow potential pathogen proliferation during extraterrestrial colonization. Given the pivotal link between gut microbiome and factors like healthspan, this exploration is of utmost importance.

While our study provided substantial insights, there are limitations to consider. Firstly, the “Ecosphere” and “Biosealed” systems, despite emulating extraterrestrial conditions, were still influenced by Earth’s specific gravitational and atmospheric conditions. This may not entirely encapsulate the challenges posed by actual space environments. The dependence on LED lighting, while demonstrating potential, raises questions about energy requirements and the feasibility of maintaining such systems during extended space voyages. Moreover, while we have charted the behavior of specific microbial communities in our simulated environments, the full spectrum of microbial interactions and potential risks in genuine extraterrestrial contexts remains uncertain. Collectively, these findings provide a robust framework to enhance the design and functionality of life support systems in the evolving realm of space exploration.

Materials and methods

Preparation of the ecosphere container

The Ecosphere utilized in this research was crafted from a sealed glass container. Plastic containers were not chosen due to their vulnerability to heat, making them unsuitable for prolonged experiments. The lid of the container was fortified with silicone and further sealed at the mouth with Parafilm for added protection.

Construction of Ecosphere1

Soil samples were taken from various locations in the Kumayama mountains in Okayama, Japan, and mixed with soil where clover grew. Water collected from our immediate environment was used. The collected soil, water, and plant seeds were placed in the glass container and sealed. No sterilization process was implemented. This procedure, including the collection and use of plant materials, was conducted in strict compliance with both domestic and international guidelines and legislation relevant to experimental research and plant material collection.

Construction of Ecosphere2

Large grain Akadama soil (Supplementary Fig. 6b) was laid at the bottom of the container. Cultivated soil (Supplementary Fig. 6a), leaf mold, chemical fertilizer with a nitrogen (N), phosphorus (P), and potassium (K) ratio of 6:6:6, and soil from clover-growing regions were mixed and placed on top. Water from the surrounding environment was poured over the Akadama soil, followed by the mixed soil. Clover seeds were then spread over this layer and the container was sealed. The interior was composed of air, soil, and groundwater layers with respective ratios of 13:5:4.

Construction of biosealed

The same cultivated soil, leaf mold, and fertilizer with a ratio of 6:6:6 N:P:K, as used in Ecosphere2, were combined. To enhance moisture retention, zeolite (Supplementary Fig. 6c) and vermiculite (Supplementary Fig. 6d) were added. Eggs of the yellow Drosophila fly, cabbage, and clover seeds were introduced, and the glass container was sealed.

Creation of exoplanetary simulated soils

Lunar simulated soil

We employed a lunar simulated soil with the same composition as lunar regolith (provided by Hideaki Miyamoto, Supplementary Fig. 6a). Refer to Supplementary Fig. 6 for the chemical composition.

Ryugu simulated soil

Ryugu Simulated Soil provided by Hideaki Miyamoto (Supplementary Fig. 6b) are possessing a similar composition to Ryugu soil. Refer to reference 13 for more information18,19,20,21.

Lunar regolith simulated soil

Fly ash and Clinker ash, by-products from coal-fired power plants resembling lunar regolith properties, were utilized (provided by Chugoku Electric Power Co.). For the chemical composition, see Supplementary Fig. 6 and Supplementary Table 3.

Creation of porous fly ash using bentonite as a binder

Ten grams of Fly ash (produced by Chugoku Electric Power Co.—Misumi Power Plant) was mixed with 1 g of bentonite (Kunigel V1 from Kunimine Industries). This mixture was kneaded with 5 mL of water and molded. The firing process involved a gradual increase from room temperature to 900 °C over 1 h, maintaining 900 °C for 2 h, and then naturally cooling inside the furnace, as detailed in the time program (Koizumi et al.22, Supplementary Fig. 6).

Methods of experimentation to demonstrate the importance of the existence of groundwater layers

To investigate the importance of the groundwater, we conducted the experiment. Eight Ecospheres were created using sun-dried soil and a container capable of storing 200 ml of water (Fig. 6a).

There were four types of ecospheres:

  1. 1.

    S100-1, and S100-2: Samples containing 100 ml of water in the soil

  2. 2.

    S200-1, and S200-2: Samples containing 200 ml of water in the soil

  3. 3.

    S400-1, and S400-2: Samples containing 200 ml of water in the soil and 200 ml in the groundwater layer (400 ml in total)

  4. 4.

    S600-1, and S600-2: Samples containing 200 ml of water in the soil and 400 ml in the groundwater layer (600 ml in total)

In addition, three open systems were also created for comparison.

Five clover seeds were enclosed in each of the 11 samples, and the samples were observed for 15 days. The lids of the Ecospheres were fixed with caulking material. The lids were shifted so that the amount of sunlight supplied to the open system was equal to that of the closed system. The containers used in the experiment were 250 ml glass bottles.

Metabolome measurement and analysis

DNA extraction from soil samples

Soil samples were lyophilized with VD-250R Freeze Dryer (TAITEC) and the lyophilized samples were smashed with Multibeads Shocker (Yasui Kikai) at 1500 rpm for 2 min. Lysis Solution F (Nippon Gene) was added to the smashed samples. After incubation for 10 min at 65 C, the samples were centrifuged at 12,000×g for 2 min. The supernatants were mixed with Purification Solution (Nippon Gene) and chloroform, and centrifuged at 12,000×g for 15 min. DNAs of the supernatants were purified with MPure-12 system and MPure Bacterial DNA Extraction Kit (MP Bio).

DNA extraction from water samples

Environmental DNA and cellular fragments were concentrated from water samples using Sterivex filters (Merck Millipore, 0.22 μm). Post-filtration, Lysis Solution F (Nippon Gene) was added, and the samples were shaken at 1500 rpm for 2 min using the Shake Master Neo (bms). After a 10-min incubation at 65 °C, the samples were centrifuged at 12,000×g for a minute, and the supernatant was collected. DNA was subsequently extracted using the Lab-Aid 824 s DNA Extraction kit (ZEESAN).

Library creation and sequencing

The Synergy LX (Bio Tek) and QuantiFluor dsDNA System (Promega) were employed to measure the DNA solution concentration. A library was prepared using the 2-step tailed PCR method. The Synergy H1 (Bio Tek) and QuantiFluor dsDNA System were then utilized to measure the library concentration, while the Fragment Analyzer and dsDNA 915 Reagent Kit (Agilent Technologies) confirmed the library’s quality. Sequencing was executed using the MiSeq system and MiSeq Reagent Kit v3 (Illumina) with a 2 × 300 bp setup.

16S rRNA data analysis

Using the FASTX-Toolkit (ver. 0.0.14), reads that matched the used primer sequences were isolated. Primer sequences in the extracted reads were removed, and the sequences were processed using various bioinformatics tools and plugins. Lineages were inferred by comparing the obtained representative sequences with the Greengene (ver. 13_8) 97% OTU database. The phylogenetic tree was constructed using the Alignment and phylogeny plugins. Supplementary Table 4 provides a correspondence between ASV IDs, read counts, and estimated phylogenetic lineages for each sample.

Microbial community identification

During the preprocessing phase of the analysis, OTUs derived from eukaryotic organisms, specifically “chloroplasts” and “mitochondria,” were removed from the dataset. Subsequently, we identified the bacterial community and applied BALSAMICO (BAyesian Latent Semantic Analysis of MIcrobial COmmunities) to investigate the association between the bacterial community and experimental conditions14. BALSAMICO incorporates a non-negative matrix factorization approach that considers environmental factors, thereby modeling community structure. The number of bacterial community clusters, L = 3, was determined using ten-fold cross-validation in BALSAMICO. Moreover, we designated “environmental condition” as the explanatory variable in BALSAMICO, selected “Indicator-soil” as the baseline, and incorporated “ES1,” “ES2,” and “others.”