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. 2025 Jul 2;11:31. doi: 10.1038/s41526-025-00491-y

Soil conditioning for enhancing plant growth using biochar and hydrochar under microgravity

Charles Wang Wai NG 1, Yu Chen Wang 1,✉
PMCID: PMC12222869  PMID: 40603864

Abstract

Cultivating plants in outer space is crucial for bioregenerative life support systems in human space exploration. This study aims to investigate the effects of soil conditioning with biochar and hydrochar on the growth and production of Malabar Spinach in microgravity conditions. Peanut shell biochar and wood hydrochar were applied at a 3% dosage by mass. Two gravity conditions were considered, including 1 g and microgravity simulated by a Random Positioning Machine (RPM). After an 18-day plant growth period, microgravity reduced the fresh biomass accumulation of Malabar Spinach by up to 71%. This reduction was attributed to inhibited leaf and root growth, which decreased light interception and nutrient uptake. In microgravity, biochar was more effective than hydrochar in enhancing plant production, mitigating the growth inhibition caused by microgravity. In the presence of biochar, microgravity significantly enhanced the biosynthesis of chlorophyll a and carotenoids by up to 36%. Furthermore, biochar and hydrochar treatments in microgravity conditions significantly increased the nutrient contents, such as K and P, in Malabar Spinach leaves. These findings indicate that biochar and hydrochar are promising soil conditioners for enhancing plant development in low-gravity conditions.

Subject terms: Plant sciences, Environmental sciences, Agriculture

Introduction

As humanity expands beyond Earth, the ability to cultivate plants becomes increasingly important for extended missions to outer space destinations1. Plants play a crucial role in life support systems by producing oxygen through photosynthesis and serving as a renewable food source during long-term space exploration, including human habitation and potential immigration to habitable exoplanets2. Given these functions, plants are considered a key component of bio-regenerative life support systems. However, plants growing in space face significant environmental challenges. For instance, nutrient deficiency, limited water and altered gravity in space induce harsh environments for plant cultivation3. Developing methods to condition the soil to help plants better adapt to these space conditions is crucial for supporting future space missions.

Techniques for applying soil conditioners to enhance soil quality and promote plant production have been developed. Biochar is produced through the pyrolysis of biomass, such as peanut shells, in the absence of oxygen. Previous studies have reported that biochar can improve soil fertility, enhance water retention, and promote beneficial microbial activity4. Its porous structure increases soil aeration and nutrient availability, making it an effective amendment for improving crop yields and soil quality5. Hydrochar is produced by hydrothermal carbonization. It involves treating biomass with heat and pressure in the presence of water6. Hydrochar offers similar benefits to biochar, including improved soil structure and nutrient retention. Hydrochar typically has a lower pH than biochar7, which can enhance its effectiveness in certain soil types or conditions. Some studies have reported the potential contaminant leaching from biochar/hydrochar derived from sewage sludge and livestock manure8,9. In addition, the heavy metal immobilization capacity of biochar/hydrochar has also been reported due to its abundant functional groups and porous structure4,10. It seems that the variability of biochar/hydrochar properties depending on the feedstock used. Nevertheless, the effects of these soil conditioners on plant development in low gravity in space are still not well understood.

Previous studies have investigated the effects of low gravity on plants. Microgravity conditions were simulated using a 3D clinostat, resulting in increased chlorophyll loss, along with elevated levels of jasmonates (JAs) and abscisic acid (ABA) in oat leaves under microgravity11. These findings contrast with the results that chlorophyll content in rice seedlings increased under simulated microgravity12. These discrepancies suggest that the effects of microgravity on plant metabolism and physiological characteristics may vary depending on the specific plant species. Previous studies also discovered significant changes in the cell proliferation rate and regulation of cell cycle progression in Arabidopsis thaliana seedlings13. This aligns with the findings reporting increased cell proliferation but reduced cell growth in Arabidopsis thaliana under low gravity conditions simulated by RPM14. Additionally, several studies have explored the effects of low gravity on vegetables and crops. Lettuce was grown aboard the Tiangong Spacelab, where it was observed to exhibit increased height and a deeper green colour under microgravity conditions15. In contrast, an investigation of rice growth in a space environment reported that spaceflight resulted in decreased germination rates and plant height16. Overall, there remains a need for further research to enhance our understanding of the coupled effects of microgravity and soil conditioners (i.e., biochar, hydrochar) on the production of food plants, such as vegetables.

Therefore, this study aims to investigate the effects of biochar and hydrochar treatments on plant development under microgravity conditions. An RPM was utilised to simulate microgravity conditions. The tested plant species was Malabar Spinach, which was a commonly grown vegetable. The experiment duration was 18 days. Plant morphology, physiological properties, and productivity were measured to evaluate plant development. This study represents the first effort to enhance our mechanistic understanding of how biochar and hydrochar influence vegetable plant growth in microgravity.

Results

Plant morphological properties

Figure 1 illustrates the increase in leaf area per pot during the plant growth period under different gravity and soil conditions. At the midpoint of the growth period, which was 8 days, the gravity-induced changes in leaf area enlargement were not significant. Under microgravity conditions, leaf area enlargement was observed to decrease by 5% and 6% with biochar and hydrochar treatments (p > 0.05), respectively. As the growth duration extended to 16 days, microgravity inhibited leaf area enlargement by up to 30% (p > 0.05), regardless of the soil condition. This indicates that as the duration of plant growth increases, the inhibition of leaf development caused by microgravity becomes more pronounced. In this figure, both biochar and hydrochar significantly enhanced leaf area enlargement. Specifically, on the 8th day, biochar and hydrochar increased leaf area enlargement by up to 148% and 117% (p < 0.05), respectively. By the 16th day, the application of biochar and hydrochar resulted in improvements of up to 109% (p < 0.05) and 57% (p > 0.05) in leaf area enlargement, regardless of gravity conditions. This suggests that peanut shell biochar is more effective than wood hydrochar in promoting leaf development in Malabar Spinach.

Fig. 1. Variations in the increase of leaf area under coupled effects of microgravity and soil conditioners.

Fig. 1

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Data are presented as mean value ± standard deviation (n = 3). Different letters above the bars indicate significant differences (p < 0.05) between the groups under different treatments.

Figure 2 shows the root surface area of Malabar Spinach at different soil depths under the combined effects of microgravity and soil conditioners. Among all conditions, the root surface area was relatively large at a depth of 10–20 mm. However, as the depth increased beyond 30 mm, the root surface area decreased. Similar to the negative impact of microgravity on leaf area growth, the root surface area was also reduced under microgravity conditions, particularly in the control soil and biochar-treated groups at depths of 10–30 mm. For instance, at a soil depth of 10–20 mm, the root surface area was reduced by 26%, 29%, and 12% under control, biochar-treated, and hydrochar-treated conditions, respectively. When the soil depth exceeded 30 mm, the microgravity-induced changes in root surface area became less pronounced.

Fig. 2. Variations in root surface area with depth under coupled effects of microgravity and soil conditioners.

Fig. 2

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Data are presented as mean value ± standard deviation (n = 3).

Leaf pigment content

Figure 3 shows the contents of plant pigments, including chlorophyll a, chlorophyll b, and carotenoids in the leaves of Malabar Spinach, under the combined effects of microgravity and soil conditioners. These three pigments play essential roles in plant photosynthesis by capturing light energy and protecting plants from excessive radiation17. Microgravity conditions led to increases in chlorophyll a content of 4%, 36%, and 6% under control, biochar-treated, and hydrochar-treated conditions, respectively. The most significant enhancement in chlorophyll a content due to microgravity was observed in the biochar treatment (p < 0.05). Chlorophyll b, an accessory pigment to chlorophyll a, also exhibited increases of 2%, 44%, and 6% under microgravity conditions for the control, biochar-treated, and hydrochar-treated groups, respectively, compared to normal 1 g conditions on Earth (p > 0.05). Similarly, microgravity improved carotenoid biosynthesis in the leaves of Malabar Spinach by 1%, 33%, and 8% under the control, biochar-treated, and hydrochar-treated conditions, respectively. Among the various soil conditions, only the presence of biochar resulted in a significant increase in carotenoid content under microgravity (p < 0.05). The alterations in plant pigments in the leaves of Malabar Spinach induced by biochar and hydrochar were not significant under either microgravity or 1 g-gravity conditions (p > 0.05).

Fig. 3. Variations in plant pigment contents, including chlorophyll a, chlorophyll b, and carotenoids, under coupled effects of microgravity and soil conditioners.

Fig. 3

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Data are presented as mean value ± standard deviation (n = 3). Different letters above the bars indicate significant differences (p < 0.05) between the groups under different treatments.

Plant biomass and water use efficiency under microgravity

Figure 4 shows the increase in fresh biomass per pot during the plant growth period under the coupled effects of microgravity and soil conditioners. Compared with the control soil condition, the biomass accumulation of Malabar Spinach could be enhanced by 344% with biochar treatment (p < 0.05) and by 65% with hydrochar treatment (p > 0.05) in microgravity level. This suggests that biochar is more effective than hydrochar in enhancing the biomass accumulation of Malabar Spinach, which aligns with the observations of plant leaf and root development shown in Figs. 1 and 2. In comparison to 1 g normal gravity, microgravity induced a 69% reduction in fresh biomass accumulation for the control soil condition (p > 0.05) and up to a 71% reduction for the biochar and hydrochar treated groups (p < 0.05). This indicates a more statistically significant reduction in biomass accumulation due to microgravity in the presence of biochar/hydrochar, suggesting an interaction between microgravity and soil conditioner affecting plant biomass accumulation.

Fig. 4. Variations in the increase of fresh biomass per pot under coupled effects of microgravity and soil conditioners.

Fig. 4

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Data are presented as mean value ± standard deviation (n = 3). Different letters above the bars indicate significant differences (p < 0.05) between the groups under different treatments.

Figure 5a shows the transpiration rate of Malabar Spinach under coupled effects of microgravity and soil conditioners. In microgravity, the transpiration rates decreased by 31%, 24%, and 1% for the control, biochar, and hydrochar-treated groups, respectively. However, these changes were not statistically significant (p > 0.05). Compared to the control soil condition, biochar and hydrochar significantly reduced the transpiration rates by 47% and 49% in microgravity, respectively (p < 0.05). Figure 5b shows the water use efficiency (WUE) of Malabar Spinach under coupled effects of microgravity and soil conditioners. WUE is defined as the ratio of biomass produced to the amount of water consumed during plant growth, reflecting how effectively a plant utilises water for growth and productivity18. Compared to control soil, biochar increased WUE by 278% (p < 0.05), while hydrochar increased it by up to 67% (p > 0.05) in microgravity condition. The significant increase in WUE with biochar treatment was attributed to its promotion of plant productivity and a slight decline in transpiration (as shown in Figs. 4 and 5a). In microgravity, Malabar Spinach experienced a 65–68% reduction in WUE compared to 1 g gravity conditions. This significant decrease was particularly evident in the biochar and hydrochar-treated groups, resulting from a notable decline in biomass accumulation and a slight increase in transpiration due to microgravity (as shown in Figs. 4 and 5a). Consequently, this led to a significant inhibition of water use efficiency.

Fig. 5. Variations in a transpiration and b water use efficiency under coupled effects of microgravity and soil conditioners.

Fig. 5

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Data are presented as mean value ± standard deviation (n = 3). Different letters above the bars indicate significant differences (p < 0.05) between the groups under different treatments.

Plant nutrient uptake and potentially toxic metal accumulation

The concentrations of nutrient elements, including K, P, Ca, Mg, N, and C, in Malabar Spinach leaves are presented in Table 1. Under biochar treatment, K content in the leaves increased by up to 174%, while hydrochar treatment resulted in a 55% increase (p < 0.05). Microgravity further enhanced plant K uptake by 22% and 25% in the presence of biochar and hydrochar, respectively. Additionally, the application of soil conditioners, particularly hydrochar, significantly increased plant P content, resulting in a 38–52% higher leaf P concentration (p < 0.05). Microgravity also improved leaf P levels, especially under hydrochar treatment, by 31%. Regarding Ca, its concentration in Malabar Spinach leaves increased by up to 73% and 26% under biochar and hydrochar treatments, respectively. Significant increases in plant Ca content were observed only in the biochar-treated condition (p < 0.05). Unlike K and P, the Ca concentration in plant leaves was not significantly affected by changes in gravity levels. For the other three elements (i.e., Mg, N and C), their concentrations in Malabar Spinach leaves were not significantly influenced by either soil conditioner (biochar or hydrochar) or microgravity conditions.

Table 1.

The concentrations of nutrient elements in plant leaves

Plant growth condition Nutrient concentration in plant leaves
Gravity Soil treatment K (g/kg) P (g/kg) Ca (g/kg) Mg (g/kg) N (%) C (%)
μg Control 11.7 ± 1.7d 2.5 ± 0.3bc 7.7 ± 0.6c 5.6 ± 0.6b 1.4 ± 0.3a 45.4 ± 1.0a
Biochar treated 32.0 ± 3.4a 2.9 ± 0.4b 13.3 ± 1.5ab 7.6 ± 0.9ab 1.3 ± 0.5a 45.9 ± 2.5a
Hydrochar treated 18.1 ± 1.8c 3.8 ± 0.4a 9.7 ± 2.9bc 7.6 ± 1.4ab 1.7 ± 0.5a 44.3 ± 2.7a
1 g Control 11.8 ± 1.4 d 2.1 ± 0.4c 8.6 ± 1.1c 6.2 ± 0.2ab 1.7 ± 0.3a 44.3 ± 1.3a
Biochar treated 26.3 ± 2.5b 2.5 ± 0.0bc 13.9 ± 1.4a 8.3 ± 1.2a 1.2 ± 0.4a 46.9 ± 2.2a
Hydrochar treated 14.5 ± 1.6 cd 2.9 ± 0.1b 10.1 ± 1.7bc 6.1 ± 0.9b 1.5 ± 0.2a 45.6 ± 0.6a
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Data are presented as mean value ± standard deviation (n = 3). Different letters (e.g., a, b, c) indicate significant differences (p < 0.05) between the groups under different treatments.

Figure 6 shows the accumulation of soil pollutants (i.e., heavy metal Cu and Cd) in the leaves of Malabar Spinach under coupled effects of microgravity and soil conditioners. The heavy metal content in vegetation is a crucial factor for assessing plant quality. Under microgravity conditions, plant Cu concentration increased by up to 24%, regardless of soil conditions. A significant increase in plant Cu uptake due to microgravity was observed in the non-biochar/hydrochar-treated groups (p < 0.05). The application of biochar and hydrochar reduced plant Cu concentration by 7% (p > 0.05) and 17% (p < 0.05), respectively, under microgravity. Compared with Cu, Cd poses greater ecotoxicity and requires particular attention. Microgravity significantly enhanced plant Cd accumulation by up to 114% (p < 0.05), regardless of soil conditions. Under microgravity level, biochar reduced plant Cd accumulation by 9% (p > 0.05), while hydrochar decreased it by 36% (p < 0.05).

Fig. 6. Variations in the concentrations of plant Cu and Cd under coupled effects of microgravity and soil conditioners.

Fig. 6

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Data are presented as mean value ± standard deviation (n = 3). Different letters above the bars indicate significant differences (p < 0.05) between the groups under different treatments.

Multivariate analysis of variance

Table 2 shows the results of multivariate analysis of variance (MANOVA) for the plant characteristics related to the morphology, physiology and production of Malabar Spinach. Among these characteristics, gravity significantly reduced plant biomass and water use efficiency (WUE) (p < 0.05), regardless of soil conditions (as shown in Figs. 4 and 5). This reduction was attributed to altered metabolism and decreased levels of carbon fixation and photosynthetic capacity in plants under microgravity19. Conversely, the accumulation of heavy metals, including Cu and Cd, in plants significantly increased under microgravity (p < 0.05) (as shown in Fig. 6). This increase was due to microgravity-related changes in uptake kinetics and enhanced membrane fluidity under low gravity19,20. Biochar significantly increased leaf area, plant biomass, and WUE (p < 0.05), while hydrochar did not have a significant impact on these parameters (p > 0.05) (as shown in Figs. 1, 4, and 5). This effect was attributed to the abundant mineral nutrients provided by biochar (Table S2). In comparison, hydrochar demonstrated higher efficiency in soil Cd immobilisation, thereby reducing plant Cd accumulation (as shown in Fig. 6). An interaction of gravity level and soil condition was observed on the biomass and WUE of Malabar Spinach (p < 0.05) (as shown in Figs. 4 and 5). Microgravity could not significantly affect the increase in biomass and WUE when soil conditioner was absent (p > 0.05). In the presence of biochar or hydrochar, microgravity significantly reduced the biomass accumulation and WUE (p < 0.05). Nevertheless, peanut shell biochar could significantly enhance biomass accumulation and WUE in microgravity conditions.

Table 2.

MANOVA of gravity and soil treatment on plant characteristics (p-value)

Factor Plant characteristics
Leaf area Biomass WUE Chlorophyll a Chlorophyll b Carotenoids Plant Cd Plant Cu
Gravity 0.443 0.000 0.000 0.116 0.171 0.121 0.000 0.005
Soil treatment 0.003 0.000 0.000 0.502 0.736 0.464 0.011 0.068
Gravity×soil treatment 0.816 0.000 0.003 0.297 0.336 0.313 0.689 0.144
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WUE stands for water use efficiency.

Discussion

The reduction in leaf area enlargement due to low gravity might be attributed to decreased plant cell growth14. Additionally, microgravity during spaceflight could hinder Ca2+ utilization, impacting processes such as the formation of plant cell wall architecture and lignin biosynthesis21. Furthermore, the negative impact of microgravity on signal transduction might cause the reduction of leaf development22. Both biochar and hydrochar demonstrate greater efficiency in stimulating leaf growth during the relatively early stages of plant development. This can be explained by the fact that although wood hydrochar contains higher organic matter, peanut shell biochar has greater levels of soil-available nutrients such as K, P, N, Mg, and Ca (as shown in Table S2). These mineral nutrient contents contribute to improved leaf growth and larger leaf area. The inhibition of root growth in microgravity was attributed not only to decreased plant cell growth but also to hampered signalling via Ca2+ in physiological processes14,21. Low gravity could decrease the water infiltration rate, thus reducing water loss through leaching3. Consequently, relatively high soil moisture could sometimes hinder root system development23, leading to a reduced root surface area under microgravity conditions. Furthermore, compared to the control, peanut shell biochar and wood hydrochar increased the root surface area, indicating enhanced root growth and development. This improvement was attributed to the enhanced soil mineral nutrients and organic content, respectively (as shown in Table S2).

Microgravity conditions could induce changes in biological systems related to chlorophyll biosynthesis and upregulate plastid-encoded transcript expression, leading to increased levels of pigments in plant leaves24,25. Research has demonstrated that microgravity enhances plant sensitivity to various environmental cues26. Additionally, nutrient supply (e.g., N, K, P) from biochar could influence the biosynthesis of chlorophyll and carotenoids27. Therefore, when microgravity occurred in conjunction with biochar treatment, plant sensitivity was enhanced, leading to an amplified stimulation of plant pigment biosynthesis. The reduced leaf area and root surface area observed under microgravity conditions (as shown in Figs. 1 and 2) limited the light interception and nutrient uptake of Malabar Spinach, resulting in lower biomass accumulation. Additionally, microgravity could alter plant metabolism and inhibit carbon fixation19. Plant photosynthesis was also restricted during spaceflight in microgravity1, contributing to the decline in biomass accumulation. These results highlight concerns about reduced vegetable production when growing plants in space. Biochar remains a recommended soil conditioner in microgravity conditions to enhance plant production.

The reduction in transpiration rate under microgravity contrasts with some previous findings2, reporting increased leaf temperatures in microgravity leading to suppressed leaf transpiration due to lower free air convection. Conversely, some other studies observed that enhanced transpiration rates in microgravity were associated with faster plant water uptake, likely due to the upregulation of genes related to water uptake28. Additionally, plants might maintain auxin levels that keep stomata open under microgravity28. The water movement in the soil-plant-atmosphere system is governed by Darcy’s law. Therefore, differences in hydraulic heads (i.e., controlled by total suction) between the plant and soil could affect the plant water uptake4. The abundant ions released from the surface of biochar and hydrochar into the soil water (as indicated by the EC value in Table S1) could lead to increased soil osmotic suction and total suction. Consequently, this resulted in reduced plant water uptake and transpiration. The reduced WUE under microgravity conditions highlights the importance of effective irrigation and soil water retention for plant growth and productivity in space. Biochar presents a promising soil amendment to enhance plant WUE under microgravity conditions in space.

The notable enhancement of mineral nutrients (K, P, and Ca) in the leaves due to biochar was attributed to the abundance of mineral nutrient ions supplied by biochar when mixed with soil (as shown in Table S2), leading to improved nutrient uptake. Microgravity can positively affect plant uptake of nutrient elements such as K, P, and Ca, but does not significantly influence the content of N and C in the leaves. The increase in nutrient element concentration of plant leaves induced by microgravity is highly dependent on the specific type of nutrient. The reduced concentrations of Cu and Cd in plant leaves with biochar/hydrochar application were due to the immobilisation of heavy metals in the soil (as shown in Table S3), which decreased their bioavailability and subsequent plant uptake. The improved heavy metal uptake by Malabar Spinach under microgravity was attributed to altered uptake kinetics19. Additionally, the enhanced water uptake (shown in Fig. 5a) under microgravity resulted in the greater absorption of heavy metal ions dissolved in the soil water, leading to elevated Cd content in plant leaves20,28. This indicates that compared with Earth gravity, partial gravity and microgravity in space facilitate the accumulation of heavy metals in plant tissues. Therefore, it is essential to select higher-quality, less contaminated soil for planting and agriculture in space. Future studies are encouraged to explore the mechanisms by which recycled materials, such as biochar and hydrochar, for conditioning lunar or Martian regolith simulants in a low-gravity environment in space. This research could provide valuable insights into agricultural development and habitat construction on extraterrestrial bodies.

Methods

Soil conditioning

The soil tested in this study was laterite, collected from Bijie City, Guizhou Province, China (27°24’N, 105°20’E). The soil in Guizhou Province is suitable for plant growth due to its relatively high water retention capacity. A 2 mm mesh was used to sieve the soil from the field prior to testing. After sieving, the pH of the laterite soil was measured using a pH metre according to ASTM D4972-01 (2007)29, yielding a result of 4.45. Electrical conductivity (EC) was determined30, resulting in a measurement of 113 μS/cm. The total organic carbon content of the tested soil sample was analysed using a total organic carbon analyser (Shimadzu, TOC-VCPH), which indicated a value of 0.88%.

The biochar analysed in this study was derived from peanut shells commercially provided by Sanli New Energy Co., Ltd. in Shangqiu, Henan province4. It was produced at a pyrolysis temperature of 500 °C with a resident time of 1 h. X-ray photoelectron spectroscopy (XPS) analysis was performed using an Axis Ultra DLD instrument from Kratos Analytical Limited. The results determined its chemical composition, consisting of 36.9% C, 33.6% O, 8.0% Si, 5.2% Ca, 4.1% Al, 3.2% N, 3.1% Cl, 2.1% Fe, 1.9% K, 1.1% Mg and 0.8% Na. The hydrochar utilised in this research was produced from wood sawdust of an apple tree through Hydrothermal Carbonization (HTC) at a temperature of 200 °C, with a residence time of 1 h and a feedstock-to-deionized water ratio of 1:5 (w/w). Following the pyrolysis process, the reactor was allowed to cool to room temperature. The hydrochar was then collected after undergoing filtration, washing, and oven-drying at 100 °C6. The XPS analysis of the wood hydrochar revealed a chemical composition of 66.6% C, 31.8% O, 0.61% N, 0.45% Ca, 0.29% Si, and 0.15% Al. Peanut shell biochar and wood hydrochar were each uniformly mixed with soil at a dosage of 3% by mass. Deionized water was then added to achieve a soil water content of 40%. The treated soil was subsequently incubated until it was ready for testing.

Set up of plant experiment

The trapezoidal pots featured an upper diameter of 50 mm and a bottom diameter of 20 mm. The height of the pots was 55 mm. Healthy seedlings of Malabar Spinach, approximately 40 days old and commercially purchased, were selected for the tests based on similar size and biomass. Each seedling was planted in an individual pot. The selected seedlings were planted in soil that contained either peanut shell biochar or wood hydrochar at a dosage of 3%. Control seedlings were planted in soil without any biochar or hydrochar for comparison. Six pots were prepared for each soil condition. Throughout the 18-day growth period, each pot was irrigated with a pipette every two days to maintain a water content of approximately 40%.

Set up of microgravity system

The simulated microgravity condition was created using a Random Positioning Machine (RPM, yuri GmbH, Germany). This machine operates through three main components (as shown in Fig. S1): the RPM instrument, a computer with RPM control software, and a controller box. The RPM instrument features a sample container connected to two independent axes, allowing it to rotate at random speeds in all directions. This random motion causes the sample to experience gravitational forces from every direction, resulting in an average gravitational effect of approximately 10−3 g over time. When the changes in direction of the object on the RPM are faster than the response of the object to gravity, it produces effects similar to those of true microgravity in space14. Half of the prepared pots containing Malabar Spinach seedlings, under three soil conditions (non-conditioner as control, 3% biochar, and 3% hydrochar), were fixed to the RPM instrument to simulate microgravity condition. Meanwhile, the other half of the pots were placed on a test table under 1 g of Earth gravity for comparison. To prevent soil particles from spilling during the rotation of the RPM instrument, the pots with Malabar Spinach seedlings were wrapped in plastic membranes with small holes. Each condition included three replicates.

Measurements of plant characteristics

During the 18-day growth period, leaf area was measured every two days to monitor the development of the Malabar Spinach leaves under different gravity and soil conditions. Leaf area was calculated using ImageJ based on high-resolution photographs. Prior to harvesting, a few fresh leaves were collected to assess plant pigment content, including chlorophyll a, chlorophyll b, and carotenoids31. At harvesting, the plants were gently washed with Milli-Q water, and the roots of the Malabar Spinach were scanned using an EPSON scanner (model: STD4800) to obtain the root surface area along the soil depth32. The increase in fresh biomass was calculated by subtracting the initial seedling biomass from the biomass at harvest. Fresh and dry biomasses were weighted using an electric balance with a precision of 0.0001 g, both before and after an oven-drying process at 60 °C for 24 h4. Transpiration of Malabar Spinach during the 18-day growth period was estimated33. Water use efficiency (WUE) was calculated as the ratio of dry biomass to water loss through transpiration18. Mineral nutrients (K, P, Ca, Mg, etc.) in plant tissues were extracted using 65% nitric acid (HNO3)34. Heavy metals (Cu, Cd) in Malabar Spinach tissues (0.1–0.5 g) were extracted with concentrated nitric acid (5 mL) and allowed to cold digest in a fume cupboard overnight. The temperature was then increased to 140 °C for digestion until only 1 mL remained35. Subsequently, the extracted elements from plant samples were analysed using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) after dilution.

Measurements of soil property

After harvesting, soil samples were collected after the removal of any root residues. The samples were air-dried, ground, and sieved through a 2 mm mesh. Subsequently, pH and electrical conductivity (EC) were measured according to ASTM D4972-01 (2007)29 and the methods outlined by Chowdhury et al.30 (results are presented in Table S1). Plant available N and dissolved organic carbon (DOC) in the soil were extracted by the methods reported by Carter and Gregorich36, Jones and Willett (2006)37. Then they were determined using a TOC/TN analyser (Shimadzu, TOC-VCPH). The plant available concentrations of K, P, Mg, Ca, Cu and Zn in the vegetated soil after harvest were extracted using the Mehlich 3 Extraction Method38. The concentrations of plant available heavy metals (Cu and Cd) were extracted following the protocol established by Park et al.35. Subsequently, they were analysed by ICP-OES. The available concentrations of soil nutrients and heavy metals in the root zone soil after planting are summarised in Tables S2 and S3, respectively.

Statistical analysis

Statistical analyses were performed using SPSS 20 (2011). One-way analysis of variance (ANOVA) was employed to assess statistical differences among the various treatments, with post-hoc Tukey’s honestly significant difference (HSD) test applied. Multivariate analysis of variance (MANOVA) was conducted to evaluate the significance of the combined effects of gravity and soil conditions on plant development. Results were deemed statistically significant when the p-value was less than 0.05, corresponding to a 95% confidence interval. Different letters (e.g., a, b, and c) were used to indicate statistically significant differences (p < 0.05) among groups.

Supplementary information

Supplemental materials (164KB, pdf)

Acknowledgements

The authors acknowledge the research grant no. 51778166, U20A20320 awarded by the National Natural Science Foundation of China and the Research Grants Council (RGC) of the HKSAR (Grant no. C5033-23G).

Author contributions

Charles W.W. Ng: Conceptualization, Methodology, Supervision, Writing-Review & Editing. Y.C. WANG: Investigation, Resources, Writing-Original Draft, Visualization.

Data availability

All data generated or analysed in this study are included in this published article and its supplementary information files.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

The online version contains supplementary material available at 10.1038/s41526-025-00491-y.

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