Effects of Microbial Fertilizers on the Properties of Simulated Lunar Soil and Lettuce Growth

TL;DR

Microbial fertilizers improve simulated lunar soil and boost lettuce growth, offering a potential solution for extraterrestrial agriculture.

Contribution

Demonstrates that microbial fertilizers can enhance simulated lunar soil properties and lettuce growth.

Findings

Microbial fertilizers increased available nutrients and organic matter in simulated lunar soil.

Lettuce biomass increased by up to 91.61% with microbial fertilizer treatment.

Treatment improved antioxidant activity and nutrient accumulation in lettuce.

Abstract

The lunar surface soil (regolith) represents a potential substrate for crop cultivation in future extraterrestrial bases. However, the absence of indigenous microbial activity severely limits nutrient availability in lunar soil. In this study, the effects of three commercial microbial fertilizers on improving simulated lunar soil and promoting lettuce (Lactuca sativa L.) growth were experimentally evaluated. The results showed that microbial fertilizers significantly increased the contents of available nutrients (N, P, and K) and organic matter in simulated lunar soil, thereby enhancing lettuce growth and biomass accumulation. Compared with the treatment without adding microbial fertilizer application (CK), the aboveground and belowground fresh weights of lettuce increased by up to 91.61% and 89.08%, respectively, under the microbial fertilizer MLQ treatment. In addition, microbial fertilizer treatment increased nutrient accumulation and photosynthetic pigment contents in lettuce, alleviated oxidative stress by improving antioxidant system performance, and consequently enhanced lettuce quality. High-throughput sequencing analysis further revealed that the dominant bacterial genera under these conditions were Bacillus, Glutamicibacter, Acetobacter, Enterococcus, and Microbacterium, while the dominant fungal genera included Saccharomyces, Pichia, and Trigonopsis. These findings provide theoretical support for the development of functional microbial fertilizers tailored for simulating lunar soil.

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Full text

1. Introduction

Future lunar settlements missions must rely as much as possible on self-sufficiency, as transporting all materials required to sustain human crews from Earth would be prohibitively costly [1,2]. The conception and development of the bioregenerative life support systems (BLSSs) aim to provide food, oxygen, and water recycling for long-term manned missions to the Moon [3]. Within BLSSs, plant cultivation plays a central role by enabling food production, oxygen generation, and the recycling of crew waste into water and nutrients. To further reduce Earth’s input into BLSSs, the concept of in situ resource utilization (ISRU) has been proposed, which involves using local regolith and recycled organic waste as primary resources [4]. The integration of BLSSs with ISRU technologies offers a promising approach for achieving reliable food production on extraterrestrial bodies such as the Moon. Consequently, identifying suitable in-situ substrates for plant growth is essential for the development of sustainable space agriculture and future extraterrestrial bases [5].

ISRU technologies that utilize lunar soil (regolith) as a growth substrate represent a potentially sustainable crop cultivation on the Moon. Lunar soil has long been regarded as an important resource for extraterrestrial agriculture due to its abundance and its potential to serve as a physical support for plant roots and as a source of mineral nutrients [6]. Nevertheless, several challenges must be systematically addressed before lunar soil can be effectively used for plant growth. Unlike terrestrial soils, lunar soil is characterized by extremely low organic matter content and poor moisture retention, which severely limit the availability of essential plant nutrients such as nitrogen (N), phosphorus (P), and potassium (K) [7,8]. Moreover, the absence of natural weathering process and microbial activity on the lunar surface further constrains nutrient cycling and bioavailability [9].

Recent studies have demonstrated that Arabidopsis thaliana can germinate and grow in both real lunar soil and simulated lunar soil, highlighting the potential feasibility of lunar agriculture [6,10,11]. However, due to the scarcity of authentic lunar soil samples, most experimental studies rely on simulated lunar soils with mineralogical compositions similar to those of real lunar regolith. Various types simulated lunar soils have been developed for different research purposes, including JSC-1A, LHS-1 and LSS-ISAC-1 [12,13,14]. While these materials have been extensively studied for their geotechnical and thermophysical properties, relatively few investigations have focused on their suitability for plant cultivation. To address this gap, our research group developed a simulated lunar soil (CQU-1) tailored to plant growth requirements and explored strategies for improving its performance as a cultivation substrate [15].

Previous studies have primarily focused on assessing the dissolution of essential elements in simulated extraterrestrial soils following microbial inoculation [16,17]. Desert environments on Earth share several characteristics with the lunar surface, including large diurnal temperature fluctuations, nutrient scarcity, and salinization. Microorganisms inhabiting such extreme environments have evolved unique metabolic capabilities that enable them to survive and function under harsh conditions [18]. This similarity suggests that certain terrestrial microorganisms may be capable of adapting to lunar soil-like environments. Indeed, extremophilic microorganisms such as Bacillus subtilis, Microcystis and Euglena have been investigated for their survival, radiation tolerance, and adaptability under simulated extraterrestrial conditions [19]. Some of these microorganisms are capable of colonizing nutrient-poor soils and performing essential biological functions, which is of great significance for both extraterrestrial life research and space exploration.

Recent studies have shown that various soil amendments and microbial interventions can substantially enhance plant growth in simulated lunar soils, indicating promising potential for future space agriculture. For example, Jirasirichot et al. [20] reported that amending TLS-01 simulated lunar soil with coconut coir fibre significantly improved sunflower seedling development and germination rates. Romano et al. [21] demonstrated that earthworms (Eisenia fetida) could survive in simulated lunar highland soil when mixed with cow dung. Xia et al. [17] found that phosphorus-solubilizing bacteria exhibited strong survival capabilities in simulated lunar soil and significantly increased available phosphorus for plant uptake. Bacterial strains such as Bacillus sp. and Pseudomonas sp. are capable of mineralizing insoluble inorganic phosphorus, thereby enhancing soil fertility and plant growth. Previous experiments have also evaluated nitrogen-fixing legumes (e.g., Lupinus sp., Vicia sp., and Melilotus sp.) for their adaptability to nutrient-poor simulated extraterrestrial soils. Although Wamelink et al. [6] did not inoculate these crops with specific rhizobia, increased plant biomass and soil nitrogen availability were observed compared with non-inoculated soils. Supporting this observation, earlier studies have demonstrated that at least one rhizobium species can survive in simulated extraterrestrial soils [22].

Microorganisms can persist in in situ lunar soil analogues and promote plant growth by improving soil physicochemical properties through the mineralization process. Additionally, microorganisms can be freeze-dried or encapsulated, significantly reducing their volume and mass during transportation to space [23,24]. These characteristics highlight the considerable potential of microbial applications for lunar soil improvement [25]. However, the use of mature commercial microbial fertilizers to enhance simulated lunar soil has received little attention, particularly with respect to their effects on soil physicochemical properties and subsequent crop performance. Moreover, due to the extreme nature of simulated lunar soil, not all microorganisms can survive or function effectively in this environment, and the identity of dominant microbial genera adapted to such conditions remains poorly understood.

Given the limited availability of real lunar soil, this study employed simulated lunar soil prepared from crushed terrestrial rocks that replicates the geological and compositional characteristics of actual lunar regolith. As humans move toward long-term lunar habitation, understanding and utilizing lunar soil for agricultural purposes will be critical for the sustainable operation of future extraterrestrial BLSSs. We hypothesized that commercial microbial fertilizers have significant potential to enhance the crop cultivation performance of simulated lunar soil. The objectives of this study were to (1) evaluate the effects of commercial bacterial fertilizers on physicochemical properties of simulated lunar soil; (2) investigate changes in the physiological and biochemical characteristics of lettuce; and (3) analyze the diversity and structural dynamics of the lettuce rhizosphere microbial community. Furthermore, dominant microbial genera capable of thriving in simulated lunar soil were identified, providing a foundation for constructing microbial consortia adapted to this environment and enhancing the agricultural potential of simulated lunar soil.

2. Results

2.1. Changes in the Physicochemical Properties of Simulated Lunar Soil

2.1.1. Bulk Density

Soil bulk density (BD) plays a crucial role in root expansion and distribution, thereby influencing nutrient and water uptake as well as overall plant growth. As shown in Figure S1, the application of different microbial fertilizers resulted in varying effects on the BD of simulated lunar soil. The XN and NFK treatments slightly increased soil BD; however, these changes were not statistically significant. Overall, no significant differences in soil BD were observed among the treatments, indicating that microbial fertilizers exerted limited influence on the physical properties of simulated lunar soil. Subsequent analyses revealed that microbial fertilizers primarily affected the chemical properties and microbial community structure of the simulated lunar soil to different extents.

2.1.2. pH, EC, and Organic Matter

The application of different microbial fertilizers significantly affected soil pH (Figure 1A), electrical conductivity (EC; Figure 1B), and soil organic matter (SOM) content (Figure 1C). Soil pH strongly influences plant growth by regulating nutrient availability, microbial activity, and the root environment. All three microbial fertilizers reduced soil pH to varying degrees. Compared with the CK treatment, the MLQ treatment caused the greatest decrease in soil pH (approximately 1.5 units), followed by the XN treatment, while the NFK treatment exhibited the smallest effect.

Microbial strains present in microbial fertilizers can secrete low-molecular-weight organic acids, such as gluconic and citric acids, which interact with alkaline ions in soil, thereby neutralizing basic components and lowering soil pH [26]. Similar trends were observed for EC and SOM. The MLQ and XN treatments significantly increased EC values and SOM contents, whereas no significant differences were detected between the NFK and CK treatments. Electrical conductivity is an important indicator of soil salinity, which affects plant water and nutrient uptake. Meanwhile, changes in SOM content can alter soil nutrient availability and influence soil structural properties.

Specifically, the MLQ and XN treatments increased EC values by 98.15% (from 0.17 ± 0.01 to 3.44 ± 0.14 mS·cm^−1^) and 86.51%, respectively. Although elevated EC levels may be associated with salinity stress depending on soil type, salt composition, and crop species [27], the results of this study indicate that higher EC values induced by microbial fertilizer application promoted lettuce growth in simulated lunar soil. This effect may be attributed to the substantial increase in nutrient salt content following microbial fertilizer application. Nevertheless, the potential long-term effects of salt accumulation warrant further investigation. Additionally, SOM content increased by 33.13% (from 1.11 ± 0.06 to 1.66 ± 0.06 g·kg^−1^) under the MLQ treatment and by 22.38% under the XN treatment.

2.1.3. Available N, P and K

The availability of nutrients in simulated lunar soil directly affects plant nutrient uptake and biomass accumulation. The contents of available nitrogen (N), phosphorus (P), and potassium (K) were significantly enhanced by all three microbial fertilizer treatments. Compared with the CK treatment, the MLQ treatment significantly increased alkali-hydrolyzable nitrogen (Figure 1D) and available phosphorus (Figure 1E) contents in simulated lunar soil by 63.75% (from 10.46 ± 1.75 to 28.85 ± 1.63 mg·kg^−1^) and 54.75% (from 9.13 ± 0.50 to 20.17 ± 0.84 mg·kg^−1^), respectively. The XN treatment exhibited moderate effects, while the NFK treatment showed the least improvement. In contrast, no significant differences were observed among the three microbial fertilizer treatments with respect to available potassium content (Figure 1F). Neither the MLQ nor the XN treatment differed significantly from the CK treatment.

2.1.4. Total N, P, and K

All three microbial fertilizers increased total nitrogen (TN) content in simulated lunar soil to varying extents. As shown in Figure 1G, TN content increased significantly following the application of XN, NFK, and MLQ treatments, reaching increases of 66.86%, 68.11%, and 69.80% (from 0.0692 ± 0 to 0.2291 ± 0 g·kg^−1^), respectively. However, no significant differences were observed among the three treatments. The MLQ treatment produced the most pronounced increase in total phosphorus (TP) content (Figure 1H), which rose by 55.21% (from 0.4436 ± 0.03 to 0.9904 ± 0.05 g·kg^−1^), followed by the NFK and XN treatments. Similarly, MLQ increased total potassium (TK) content from 10.17 ± 1.68 to 14.12 ± 0.65 g·kg^−1^ (Figure 1I). No significant differences were observed between MLQ and NFK, or between XN and CK treatments.

2.2. Changes Microbial Communities Diversity and Structure in Simulated Lunar Soil

2.2.1. Microbial Community Diversity

High-throughput sequencing of 16S rRNA and ITS regions was performed to characterize microbial communities in the experimental soils, and alpha diversity indices were calculated (Table S2). Rarefaction curves for bacteria (Figure S2A) and fungi (Figure S2B), based on observed amplicon sequence variants (ASVs) and Shannon indices, gradually plateaued, indicating sufficient sequencing depth to capture microbial diversity in all samples.

The number of bacterial and fungal ASVs varied among treatments. For instance, bacterial ASV richness was relatively higher following the NFK treatment, whereas fungal ASV richness was comparatively lower. Principal component analysis (PCA) based on ASV abundance revealed clear separations among treatments for both bacterial (Figure S2C) and fungal (Figure S2D) communities, indicating that different microbial fertilizers significantly altered microbial community composition in simulated lunar soil.

2.2.2. Microbial Community Structure

Venn diagrams were used to compare shared and unique ASVs among treatments for bacteria (Figure 2A) and fungi (Figure 2C). The results showed that bacterial communities shared more ASVs than fungal communities across treatments. Analysis of core microbiota revealed that XN, NFK, and MLQ treatments contained 37, 109, and 23 core bacterial taxa, respectively, with six taxa shared among all treatments (Figure 2B). In contrast, fungal communities exhibited fewer core taxa, with six, eight, and six core taxa in XN, NFK, and MLQ treatments, respectively, and only two taxa shared across all treatments (Figure 2D).

Bar plots illustrating microbial composition at the phylum and genus levels are presented in Figure 2E–H. At the phylum level, Firmicutes dominated bacterial communities across all treatments, with relative abundance reaching up to 99.9% in the MLQ treatment. The XN and NFK treatments also exhibited substantial proportions of Proteobacteria (26.09% and 41.88%, respectively) and Actinobacteria (56.44% and 18.12%, respectively). In fungal communities, Ascomycota overwhelmingly dominated all treatments, accounting for nearly 99.9% of total abundance.

At the genus level, bacterial community composition differed markedly among treatments. Dominant genera in the XN treatment included Bacillus (5.05%), Glutamicibacter (35.61%), Enterococcus (8.58%), Lactococcus (8.92%), and Sphingobacterium (7.66%). In the NFK treatment, dominant genera were Bacillus (7.52%), Acetobacter (38.98%), Microbacterium (13.28%), Massilia (13.33%), and Lactobacillus (11.05%). The MLQ treatment was primarily dominated by Bacillus (76.49%) and Enterococcus (23.34%). For fungi, Saccharomyces was the dominant genus in XN (98.13%) and MLQ (99.99%) treatments, while Pichia (95.62%) dominated the NFK treatment.

2.2.3. Screening of Dominant Bacterial Genera

Differential abundance analysis revealed significant variations in bacterial genera among treatments (Figure S3A–D). Compared with fungal communities, bacterial communities exhibited a greater number of differentially abundant genera. The XN treatment showed the highest number of differentially abundant bacterial genera, whereas no significantly different fungal genera were detected. Linear discriminant analysis (LDA) identified a total of 23 differentially abundant bacterial genera across treatments, with 10, 10, and 3 genera enriched in XN, NFK, and MLQ treatments, respectively.

To identify dominant bacterial genera adapted to simulated lunar soil, bacterial abundance profiles were further compared (Figure 3A). Bacillus was present in all treatments, with relative abundances of 5.05%, 7.52%, and 76.49% in XN, NFK, and MLQ treatments, respectively. Additional dominant genera included Glutamicibacter, Enterococcus, Lactococcus, and Sphingobacterium in XN; Acetobacter, Microbacterium, Massilia, Lactobacillus, and Lysinibacillus in NFK; and Enterococcus in MLQ. Regarding fungi (Figure 3B), Saccharomyces dominated XN and MLQ treatments, while Pichia and Trigonopsis were enriched in the NFK treatment.

2.3. Changes in the Physiological and Biochemical Indicators of Lettuce

2.3.1. Biomass and Growth Indicators

Application of microbial fertilizers significantly promoted lettuce growth in simulated lunar soil (Figure 4A). All treatments markedly increased both aboveground and belowground fresh weight (FW) compared with the CK treatment (Figure 4B). Under the MLQ treatment, aboveground and belowground FW increased by 91.61% (from 1.46 ± 0.29 to 17.38 ± 0.99 g) and 89.08% (from 0.79 ± 0.06 to 7.28 ± 0.49 g), respectively. Similar improvements were observed in XN and NFK treatments. In contrast, no significant differences in dry weight (DW) were detected among treatments (Figure 4C). Leaf and stem growth parameters further demonstrated the positive effects of microbial fertilizers. Compared with the NFK treatment, MLQ and XN treatments more effectively increased leaf number, leaf length, and leaf width (Figure S4A–C). Relative to CK, MLQ increased leaf number, length, and width by 63.79%, 74.44%, and 73.23%, respectively. All treatments also significantly enhanced plant height and stem diameter (Figure S4D,E).

2.3.2. Nutrient Accumulation and Photosynthetic Pigments

Microbial fertilizer application significantly increased total nitrogen content in lettuce (Figure 5A). Relative to CK, total N content increased by 26.69% (from 6.25 ± 0.41 to 8.53 ± 0.08 mg·g^−1^), 19.86%, and 38.77% (from 6.25 ± 0.41 to 10.21 ± 0.16 mg·g^−1^) under XN, NFK, and MLQ treatments, respectively. No significant differences were observed among treatments for total phosphorus content (Figure 5B). Total potassium content increased by 28.59% (from 1.07 ± 0 to 1.50 ± 0 mg·g^−1^), 12.69%, and 32.15% (from 1.07 ± 0 to 1.58 ± 0.02 mg·g^−1^) in XN, NFK, and MLQ treatments, respectively (Figure 5C).

Photosynthetic pigment contents were also significantly enhanced by microbial fertilizer treatments. Compared with CK, chlorophyll a, chlorophyll b, and total chlorophyll contents increased in all treatments (Figure S5A–C), with the greatest increases observed under the MLQ treatment.

2.3.3. Antioxidant Defence Systems

There are two main types of antioxidant defence systems by which plants remove reactive oxygen species, namely, enzymatic antioxidant defence system and nonenzymatic antioxidant defence system. In this study, the changes in the activities of antioxidant enzyme activities and malondialdehyde content in lettuce under different treatments were used to reflect the antioxidant defence system of lettuce and the damage of cell membrane in lettuce, in simulated lunar soil, which could reflect the stress degree of lettuce during its growth. Different microbial fertilizer treatments obviously changed the activities of antioxidant enzymes in lettuce. As shown in Figure 6, after the microbial fertilizers were added into the simulated lunar soil, the activities of the SOD (Figure 6A), CAT (Figure 6B), and GR (Figure 6E) decreased significantly, while the activities of the above enzymes POD (Figure 6C) and APX (Figure 6D) significantly increased. Compared with the untreated control, the XN and MLQ treatments could more effectively alleviate the stress of lettuce. In addition, the different microbial fertilizer treatments significantly changed the malondialdehyde content (Figure 6F), and oxidative damage of membrane lipids in lettuce. The MDA content of XN, NFK and MLQ were decreased by 19.45%, 5.62%, and 14.53%, respectively.

Furthermore, the changes in the contents of substances within the nonenzymatic antioxidant system were analysed. Different microbial fertilizers can alleviate the stress on lettuce to varying degrees, increase its stress resistance, and promote the accumulation of biomass. Among the three microbial fertilizer treatments, MLQ demonstrated the greatest efficacy. Compared with the untreated control, it can most effectively relieve the inhibitory impact of simulated lunar soil on lettuce growth, and can decrease the contents of Pro (Figure 7A) and GSH (Figure 7B) by 37.29% and 57.57%, respectively, and increase the content of GSSG (Figure 7C) by 16.93%. When lettuce adapts to the simulated lunar soil environment, it continues to accumulate secondary metabolites in the body to adapt to the effect of the external environment. That is, in the changes in total phenol content (Figure 7D), the XN, NFK, and MLQ treatments decreased it by 28.63% (from 2.86 ± 0 to 2.04 ± 0.02 mg·g^−1^), 14.62%, and 27.74%, respectively; in the changes in flavonoid content (Figure 7E), and the XN, NFK, and MLQ treatments decreased it by 14.52%, 5.35%, and 16.72% (from 43.19 ± 0.36 to 35.97 ± 0.96 mg·g^−1^), respectively; in terms of the change in alkaloid content (Figure 7F), the XN, NFK, and MLQ treatments reduced it by 61.84%, 38.91%, and 66.72% (from 2.15 ± 0.08 to 0.72 ± 0.03 mg·g^−1^), respectively. Secondary metabolite content showed no significant difference between XN and MLQ treatments.

According to Spearman’s correlation analysis (Figure S6), MDA, Pro, GSH, Flavonoid, Total phenolics and Alkaloids were significantly and positively correlated with CAT, SOD and GR (p < 0.001), while they were significantly and negatively correlated with POD (p < 0.001). Additionally, GSSG was significantly and positively correlated with APX (p < 0.001). It can be seen that MDA, Pro, GSH and secondary metabolites (Flavonoid, Total phenolics and Alkaloids) in lettuce are closely related to its antioxidant activity, possess certain antioxidant capacity and are important antioxidant active components, playing a significant role in the process of lettuce adapting to the simulated lunar soil growth environment. Based on the above results, the treated plants exhibited significantly increased biomass accumulation (Figure 4B,C) and improved photosynthetic parameters (Figure S5), indicating an overall improvement in the physiological status of lettuce. In this context, the decrease in antioxidant enzyme activities is more likely to reflect a reduced requirement for antioxidant defense rather than aggravated oxidative damage, suggesting that cellular redox homeostasis tends to stabilize.

2.3.4. Vitamin C, Soluble Protein and Soluble Sugars

The nutritional value and functions of lettuce are primarily reflected in its content of vitamin C, soluble protein, and soluble sugars. Therefore, we also analysed the changes in the contents of vitamin C (Figure 8A), soluble protein (Figure 8B) and soluble sugar (Figure 8C) in lettuce after treatment with the three microbial fertilizers. XN, NFK and MLQ increased the content of vitamin C in lettuce significantly. The contents of vitamin C in lettuce increased by 4.49% (from 6.93 ± 0.12 to 7.26 ± 0.10 mg·g^−1^), 4.13% and 3.53%, respectively, but the difference was not significant. The content of soluble protein in lettuce increased by 32.16% (from 1.37 ± 0.08 to 2.02 ± 0.09 mg·g^−1^), 13.63%, and 31.47% after treatment with XN, NFK, and MLQ, respectively, but no marked variation was observed between XN and MLQ. Additionally, as shown in the changes in the content of soluble sugars in lettuce, the three microbial fertilizers significantly improved alkaloids and alkaloid contents in lettuce increased by 36.24%, 31.82%, and 40.55% (from 7.80 ± 0.17 to 13.13 ± 0.59 mg·g^−1^), respectively.

3. Discussion

Establishing permanently inhabited lunar bases is a major direction of future lunar exploration. To sustain long-duration human presence on the Moon, bioregenerative life support systems (BLSSs) will be indispensable [28]. Large-scale, long-term plant cultivation within BLSSs requires substantial amounts of growth substrate [29]. However, the long Earth–Moon distance makes transporting cultivation media from Earth logistically difficult and prohibitively expensive. In this context, lunar regolith—an abundant in situ resource composed of fine mineral particles (approximately 50 μm) and essentially devoid of organic matter and indigenous organisms—has considerable potential as a plant growth substrate [30]. Utilizing lunar soil for crop cultivation would therefore contribute to lunar self-sufficiency and reduce operational costs, thereby improving BLSS feasibility and long-term stability.

Lunar regolith contains a range of mineral elements relevant to plant nutrition, such as silicon, iron, phosphorus, and calcium [29]. Nevertheless, several major constraints must be addressed before it can function effectively as a cultivation medium. In particular, lunar soil has extremely low organic matter and poor water retention. Moreover, nutrients may be present largely in mineral-bound forms that are not readily bioavailable to plants, and the absence of weathering and microbial activity limits nutrient cycling [4,16]. Consistent with these limitations, previous studies have shown that plants can grow in lunar soil, but growth is often inhibited and stress phenotypes are evident. For example, Arabidopsis can germinate and grow in lunar regolith, yet growth rates are reduced and stress-related phenotypes are pronounced [11]. These findings indicate that lunar soil is not inherently phytotoxic, but requires targeted improvement to meet the functional demands of BLSS plant production. Our research findings suggest that the application of microbial fertilizers in simulated lunar soil markedly increases N, P, and K content, promotes lettuce growth, and accelerates lettuce biomass accumulation. In short, the prospects of promoting lettuce growth with various amendments and microbial measures are promising in simulated lunar soil. In addition, there is one more point that needs to be noted. In long-term cultivation, an increase in EC typically indicates the accumulation of soil salts, which may lead to inhibited crop growth, soil hardening, and an increased risk of salinization. Excessive EC values can impair water absorption and exacerbate nutrient imbalances, reducing soil fertility and permeability, thus negatively affecting agricultural productivity [31,32]. Therefore, the influence of changes in EC values on the growth of crops in simulated lunar soil should be continuously monitored in the future.

Although lunar soil contains elements required for plant growth, these elements cannot be directly taken up and utilized by plants in many cases. Due to the absence of biological activity in extraterrestrial regolith, microbially mediated soil processes are largely absent, restricting the bioavailability of mineral-bound nutrients [4]. Microbial fertilizer application can partially restore these biological processes by introducing metabolically active microorganisms, thereby altering soil biochemical activity and facilitating nutrient mobilization. Consequently, soil conditions supporting plant growth may be improved through enhanced nutrient availability and rhizosphere function [17].

Plant growth-promoting rhizobacteria (PGPR) can facilitate plant growth and development via multiple mechanisms, including nitrogen fixation, phosphate solubilization, phytohormone production, induction of systemic resistance, suppression of pathogens, and enhancement of tolerance to abiotic stress, as well as rhizosphere colonization [23,24]. Because PGPR communities can exhibit multifunctionality, robustness, and adaptability, the use of consortia rather than single strains has attracted increasing attention. Common PGPR genera include Rhizobium, Bacillus, Enterobacter, and Pseudomonas [33]. Notably, Pseudomonas species are widely recognized as phosphate-solubilizing bacteria and can improve phosphorus availability and promote crop growth [16]. Xia et al. [17] reported that Bacillus sp. and Pseudomonas sp. can survive under simulated lunar soil conditions. Similarly, in this study, several microbial genera capable of thriving in simulated lunar soil were identified, including Bacillus, Glutamicibacter, Acetobacter, Enterococcus, Microbacterium, Massilia, Lactobacillus, Lactococcus, Sphingobacterium, Enterobacter, as well as fungal genera such as Saccharomyces, Pichia, and Trigonopsis. These taxa provide a foundation for assembling functional microbial consortia tailored to simulated lunar soil, thereby supporting future research on microbial-based soil improvement. It is also worth noting that a high relative abundance of Saccharomyces is often associated with carbon-rich environments; while a transient increase is not necessarily detrimental, sustained dominance could indicate microbial community imbalance and potentially influence nutrient cycling and root performance under specific conditions [34].

Inoculation with plant growth-promoting microorganisms under simulated lunar soil conditions has been reported to increase alpha diversity (e.g., Shannon index) and alter beta diversity, with positive relationships observed between microbial diversity and concentrations of bioavailable nutrients in soil and plant tissues [16]. These observations are broadly consistent with our results, as significant differences in Shannon and Simpson indices were detected among treatments, together with clear separation of microbial communities. Beyond nutrient mobilization, PGPR may also support plant performance through hormonal regulation. Some PGPR produce indole-3-acetic acid (IAA) or modulate ethylene biosynthesis, thereby influencing root and shoot development [35]. Auxin-mediated responses can promote root elongation and enhance root system architecture, facilitating nutrient acquisition and stress tolerance [36]. In addition, PGPR can suppress pathogens through antimicrobial compound production and competitive exclusion, which is particularly relevant in closed BLSS environments [37]. Some rhizobacteria also produce siderophores that can act as biocontrol agents by limiting pathogen access to iron [38].

Despite these advantages, whether such microorganisms can consistently promote plant growth under true spaceflight conditions remains uncertain [39]. The selection of suitable PGPR will depend on regolith properties, plant species, and cultivation practices, and lunar regolith mineralogy may vary substantially across different lunar regions [40,41]. In addition, the potential biosafety risks of introducing certain microorganisms into life support systems must be carefully evaluated, particularly regarding opportunistic pathogenicity [39]. However, studies of fresh produce aboard the International Space Station (ISS) have suggested that microbial loads on plants do not necessarily correlate with compromised food safety, and no human pathogens were detected using cultivation-based methods and sequencing analyses [42]. Beneficial microorganisms, especially endophytes, can improve host nutrient status and enhance resistance to biotic and abiotic stresses [43]. Overall, plant–microbe symbioses may support plant survival and productivity under extreme environmental conditions relevant to space missions [44].

Integrating soil-based plant cultivation systems with microbial technologies could improve BLSS performance and astronaut self-sufficiency during deep-space exploration. Microorganisms are attractive candidates for supporting human space exploration due to their functional versatility, controllability, and compact transport requirements, especially when formulated via freeze-drying or encapsulation [45]. Microbial technologies can contribute to higher closure efficiency of BLSSs and support ISRU-related processes such as material production, resource recovery, and energy-related applications [46,47,48]. Compared with purely physical or chemical approaches, microbial systems can be self-sustaining, require relatively low energy input, and demand minimal monitoring under appropriate design conditions [46,47,48]. In particular, the plant microbiome is increasingly recognized as essential for successful space crop production, influencing plant growth, hormone regulation, disease suppression, immune responses, and stress mitigation [49]. For instance, Azotobacter cyanogenes reportedly grows in simulated lunar soil and promotes nitrogen-related fertility and mineral nutrient availability [50]. Zaets et al. [25] also demonstrated that inoculation of microbial communities (e.g., Pseudomonas sp., Stenotrophomonas maltophilia, Paenibacillus sp., Klebsiella oxytoca, and Pantoea agglomerans) into lunar regolith supported microbial survival and biomineralization, thereby increasing French marigold biomass. These findings suggest that agroprobiotics warrant serious consideration for enabling stable and productive extraterrestrial agriculture during extended missions. In BLSS, dominant microbial genera such as Bacillus, Enterococcus, Microbacterium, Glutamicibacter, Acetobacter and the dominant genera (Saccharomyces, Pichia, Trigonopsis) play important functional roles but also require biosafety consideration. Some genera (e.g., Enterococcus, Bacillus, Microbacterium) are recognized opportunistic pathogens and may exhibit toxin production, biofilm formation, or antimicrobial resistance, posing potential health risks under long-term confinement [51,52,53,54]. Although yeasts such as Saccharomyces and Pichia are generally regarded as safe, invasive infections and outbreaks have been reported in susceptible populations, highlighting the need for careful exposure control in closed systems [55,56,57]. Therefore, continuous microbial monitoring, strain-level identification and biosafety-oriented management are essential to ensure system stability and crew health in BLSS.

Most current studies aimed at improving simulated lunar soil primarily focus on modifying physicochemical properties and increasing nutrient availability to promote plant growth. Wamelink et al. [6] reported that crops grown in simulated lunar soil may exhibit severe growth inhibition or mortality after germination, likely due to high pH and poor water retention. Contreras et al. [58] showed that wheat and peas grown in simulated lunar soil exhibited decreased chlorophyll contents and elevated reactive oxygen species, indicating oxidative stress. The addition of monogastric animal manure has been reported to improve simulated lunar soil properties by increasing water-holding capacity and nutrient levels; however, excessive manure addition can inhibit lettuce growth and adversely affect root development [59,60]. In these studies, increasing manure ratios elevated mineral nutrient uptake (particularly phosphate, potassium, and sodium), yet lettuce fresh biomass, nitrate content, antioxidant activity, and total phenolics declined with higher amendment rates [61]. Similarly, Hosamani et al. [62] reported that simulated lunar soil exhibited high pH (9.61) but low EC and low N, P, and K contents, and that cocopeat could alleviate growth inhibition and increase crop biomass. Biochar has also been shown to promote lettuce seedling growth, with an optimal amendment ratio reported for improving simulated lunar soil [63].

In the present study, Spearman correlation analysis indicated that MDA, proline, GSH, and secondary metabolites (flavonoids, total phenolics, and alkaloids) were closely associated with antioxidant-related responses in lettuce grown in simulated lunar soil. These compounds can function as important antioxidant-related indicators and may reflect plant adaptation to stressful growth conditions. Previous studies have suggested that transient increases in MDA may also act as signaling cues that activate antioxidant pathways and defense gene expression, potentially enhancing stress resistance [64]. Proline metabolism contributes to antioxidant defense and cellular homeostasis and can reduce the risk of lipid peroxidation under stress [65]. GSH content and the GSH/GSSG ratio are sensitive indicators of cellular redox status and oxidative damage [66]. Secondary metabolites such as alkaloids, flavonoids, and phenolics play important regulatory roles in plant oxidative stress responses; when reactive oxygen species accumulate, pathways such as the phenylpropanoid pathway are activated, often leading to secondary metabolite accumulation [67]. Phenolic compounds are widely recognized as secondary metabolites that accumulate in response to various abiotic stresses, functioning as antioxidant defense molecules. Under low-stress or optimal growth conditions, the biosynthesis of these compounds is down regulated due to reduced requirement for stress mitigation, which explains the observed decrease in their levels without implying a reduction in crop quality [68]. In this study, microbial fertilizer application increased aboveground and belowground lettuce fresh weight by up to 91.61% and 89.08%, respectively, and increased plant height, stem diameter, and leaf number, while reducing oxidative damage indicators, suggesting alleviation of growth stress following soil improvement.

In space environments, plant physiology is influenced by multiple abiotic and biotic factors. Abiotic stressors include altered gravity, ionizing radiation, magnetic field changes, atmospheric pressure, and light regimes, whereas biotic factors involve interactions with both beneficial and pathogenic microorganisms [69]. Microgravity can affect plant organ and cellular functions and has been reported to alter cell wall composition, including lignin, cellulose, hemicellulose, and pectin [70]. Ionizing radiation may induce genetic and epigenetic effects, although responses can vary substantially across species and experimental conditions [71]. Magnetic field intensity can also influence plant physiological processes, and geomagnetic fields can penetrate plant tissues more readily than electric fields [71]. Understanding plant adaptation to low-pressure and other atypical environments is important for managed agriculture in orbital and extraterrestrial settings [71]. Microorganisms that constitute the plant microbiome can play critical roles in plant growth and stress resistance, and plant–microbe symbioses may enhance plant survival and health under harsh space-relevant conditions [43,72,73,74].

It should be noted that the present experiments were conducted under terrestrial environmental conditions and did not incorporate key lunar surface factors such as reduced gravity, vacuum, and elevated radiation. These factors are likely to influence both microbial survival and plant–microbe interactions. Replicating such conditions comprehensively on Earth remains technically challenging. Nevertheless, our findings demonstrate that microbial fertilizers can improve simulated lunar soil properties, alleviate the inhibitory effects of simulated lunar soil on lettuce growth, and enhance biomass and nutrient accumulation. Therefore, functional microbial fertilizers represent a promising approach for lunar regolith improvement and deserve further attention. Future work will focus on assembling tailored microbial consortia for simulated lunar soil to maximize soil improvement and crop productivity. However, given biosafety concerns, comprehensive pathogenicity assessments must be performed for all candidate microorganisms prior to their introduction into BLSSs at extraterrestrial bases to ensure biosafety control and to prevent risks to crew health or other organisms. Overall, microbial technologies hold substantial promise for enabling sustainable space agriculture, improving BLSS closure efficiency during long-term missions, and minimizing dependence on Earth-supplied resources.

4. Materials and Methods

4.1. Simulated Lunar Soil and Lettuce Seeds

The simulated lunar soil (CQU-1) was provided by the Center of Space Exploration, Ministry of Education, Chongqing University, Chongqing, China. CQU-1 is comparable to Apollo 14 regolith in terms of particle size distribution, mineralogical composition, and elemental composition; detailed information is provided in Mei et al. [15]. The simulated soil had the following physicochemical properties: bulk density 1.30 ± 0.06 g·cm^−3^; pH 8.07 ± 0.05; electrical conductivity (EC) 0.08 ± 0 mS·cm^−1^; organic matter (OM) 1.19 ± 0.03 g·kg^−1^; total nitrogen 0.07 ± 0 g·kg^−1^; total phosphorus 0.31 ± 0.01 g·kg^−1^; and total potassium 52.06 ± 0.87 g·kg^−1^. The simulated lunar soil was sterilized by gamma irradiation (25 kGy) to ensure that its mineral composition, particle structure, and surface properties remained unchanged [75].

Lettuce (Lactuca sativa L.) seeds were purchased from Shandong Shouhe Seed Industry Co., Ltd. (Weifang City, Shandong Province, China). Prior to sowing, seeds were surface-sterilized by soaking in 5% hydrogen peroxide solution and then rinsed several times with sterile water. Seeds were soaked for 6 h and subsequently incubated in the dark at 20 ± 1 °C for 48 h [76].

4.2. Commercial Microbial Fertilizers

Three commercially available microbial fertilizers were selected and purchased from Jinning XiaNong Biotechnology Co., Ltd. (Jining City, Shandong Province, China), NongFuKang Biotechnology Co., Ltd. (Zhengzhou City, Henan Province, China), and MiLiQi Alida Bioengineering Co., Ltd. (Zibo City, Shandong Province, China). Based on the manufacturer names, the fertilizers were designated XN, NFK, and MLQ, respectively, for subsequent treatments. The key characteristics of these microbial fertilizers are summarized in Table S1.

Because the simulated lunar soil used in this study was gamma-sterilized and therefore considered sterile, microbial fertilizer application was intended both to improve soil properties and to increase microbial abundance. Based on Huang et al. [77], the microbial fertilizer application rate was set to 2% (w/w).

4.3. Experiment Settings

4.3.1. Soil Incubation Under Sterile Conditions

Four treatments were established: untreated control (CK), XN, NFK, and MLQ, each with three replicates (12 experimental units in total). For each replicate, 100 g of simulated lunar soil was placed into a sterile culture bottle. Then, 3 g of microbial fertilizer was added to the corresponding bottles. After thorough mixing, sterile deionized water was added as required for subsequent analyses. The bottles were incubated in a biochemical incubator at 37 °C for 14 days in the dark.

All equipment was sterilized prior to use, and aseptic procedures were strictly followed throughout. After incubation, each soil sample was divided into two subsamples. One subsample was collected using sterile sampling tools and immediately stored on dry ice for microbial community analysis. The other subsample was air-dried at room temperature and used for physicochemical property determination.

4.3.2. Lettuce Growth Experiment

Four treatments were established: CK, XN, NFK, and MLQ, each with three replicates (12 experimental units in total). For each replicate, 500 g of simulated lunar soil was placed into a plastic pot (9.5 × 9.5 × 9.7 cm^3^). Microbial fertilizer was thoroughly mixed with the soil, and sterile deionized water was added as needed. Germinated lettuce seeds were then sown, and the pots were transferred to a greenhouse. Six seeds were sown per pot at a depth of approximately 1.0–1.5 cm. When seedlings reached the stage of three true leaves with a visible central bud, thinning was performed to retain two seedlings per pot. Greenhouse conditions were maintained at 20 ± 1.0 °C with relative humidity of 60 ± 5.0%. A 12 h light/12 h dark photoperiod was provided using artificial lighting at an intensity of 80 μmol·m^−2^·s^−1^. The greenhouse in this study used ambient air without added carbon dioxide to minimize the impact of higher carbon dioxide concentrations on plant photosynthesis and biomass accumulation [78]. Soil moisture was maintained at 60% field capacity by adding deionized water every two days using the gravimetric (weighing) method.

4.4. Determination of Physicochemical Properties of Simulated Lunar Soil

After soil incubation, samples were air-dried indoors and analyzed for physicochemical properties. Bulk density (BD) was determined using a modified mass-per-volume method. Due to limited sample volume, ring cutters were used [79,80,81]. Soil pH was measured with a glass electrode pH meter (PHB-3, SANXIN, Shanghai, China) in a soil suspension. Soil EC was measured using a microprocessor-based conductivity meter [81]. Soil organic matter was determined using the K_2_Cr_2_O_7_–H_2_SO_4_ oxidation method followed by FeSO_4_ titration [82]. Alkali-hydrolyzable nitrogen was measured by 1.0 M NaOH extraction as an indicator of available N [83]. Available phosphorus was determined using 0.5 M NaHCO_3_ following Olsen et al. [84]. Available potassium was extracted with 1 M NH_4_OAc (pH 7) and quantified by atomic absorption spectrophotometry.

Total nitrogen (N) was determined using the Kjeldahl digestion and distillation method. Total phosphorus (P) was quantified using the molybdenum–antimony colorimetric method after alkaline digestion, and total potassium (K) was extracted by fusion with molten NaOH and analyzed by atomic absorption spectrophotometry. These methods were described previously by Sparks et al. [85].

4.5. Determination of Physiological and Biochemical Indices of Lettuce

Plants were harvested after 55 days of growth for physiological and biochemical analyses.

Fresh and dry biomass: Roots were carefully removed from soil to minimize root loss. Shoots and roots were separated using scissors. Surface moisture was removed with filter paper, and fresh weight (FW) was recorded for shoots and roots. Samples were then dried at 105 °C for 1 h, followed by drying at 80 °C to constant weight to obtain dry weight (DW).

Morphological traits: Leaf number was counted per plant. Leaf length and leaf width were measured on the fifth fully expanded leaf (counted from the inner leaves outward), with leaf length recorded at the longest point. Plant height was measured from the plant base to the top of the most developed upper leaf sheath using a flexible ruler. Stem diameter was measured at the widest point of the aboveground stem using a vernier caliper.

Plant nutrient contents: Dried plant tissues were ground to powder and digested with concentrated H_2_SO_4_–H_2_O_2_. Total N was determined by the Kjeldahl method, total P by the molybdenum–antimony method, and total K by flame atomic absorption spectrometry [81].

Photosynthetic pigments: Fresh leaves (0.1 g) were cut into small pieces, rinsed with distilled water, and homogenized in the dark. The extract was transferred to a 10 mL test tube and brought to volume with extraction solution. Tubes were incubated in the dark for 3 h until the tissue residue became nearly colorless, indicating complete extraction. Pigment contents were determined using a commercial kit (Suzhou Comin Biotechnology Co., Ltd., Suzhou City, Jiangsu Province, China) according to the manufacturer’s instructions. Detailed procedures are provided in the Supplementary Materials and Methods.

Antioxidant system and quality indices: Enzymatic antioxidant parameters including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), and malondialdehyde (MDA) were measured. Non-enzymatic indicators including proline (Pro), reduced glutathione (GSH), oxidized glutathione (GSSG), total phenols, total flavonoids, and alkaloids were also quantified. Quality-related indices (vitamin C, soluble protein, and soluble sugars) were determined using commercial assay kits (Suzhou Comin Biotechnology Co., Ltd., China) following the manufacturer’s protocols. Detailed methods are provided in the Supplementary Materials and Methods.

4.6. Microbial Community Sequencing and Bioinformatics Analysis

Total DNA was extracted from soil samples according to the manufacturer’s protocol. DNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo, Waltham City, MA, USA). Purified PCR products were used for library preparation with the NEXTFLEX Rapid DNA-Seq Kit and sequenced on the Illumina NextSeq 2000 platform (Majorbio, Shanghai, China; https://cloud.majorbio.com (accessed on 1 October 2025)). For bacteria, primers 338F/806R were used to amplify the V4–V5 region of 16S rRNA; for fungi, primers ITS1F/ITS2 were used to amplify the ITS1 region. Raw reads were quality-filtered using fastp and merged using FLASH based on overlap and primer sequences, followed by orientation correction. Amplicon sequence variants (ASVs) and their abundance profiles were obtained using the QIIME2 pipeline with default parameters, including denoising of quality-controlled sequences.

4.7. Statistical Analysis

Microbial community analyses were performed on the Majorbio Cloud platform (https://cloud.majorbio.com (accessed on 1 October 2025)). Alpha diversity indices were calculated using Mothur, and differences in alpha diversity among groups were assessed using the Wilcoxon signed-rank test. Principal component analysis (PCA) based on Bray–Curtis distances was used to evaluate similarities among microbial communities. PERMANOVA was applied to test whether between-group differences in community structure were statistically significant. Venn diagrams illustrating shared and unique ASVs were generated using jvenn. Taxonomic profiles at the phylum and genus levels were visualized using stacked bar plots in R (v3.3.1), with taxa below 1% relative abundance grouped as “Others”. Differences in relative abundance at genus and species levels were evaluated using the Wilcoxon rank-sum test or Student’s t-test in R (v3.3.1), with significance set at p < 0.05. LEfSe analysis was used to identify taxa with significantly different abundances among groups from phylum to genus level, using an LDA threshold > 2 and p < 0.05.

All experiments were performed in triplicate, and data are presented as mean ± standard deviation. Statistical analyses were performed using SPSS 22.0, and figures/statistical calculations were prepared using Origin 2025b (OriginLab, Northampton City, MA, USA). Prior to one-way ANOVA, normality and homogeneity of variance were assessed. When significant treatment effects were detected (p < 0.05), Duncan’s multiple range test (DMRT) was used for post hoc comparisons.

5. Conclusions

This study demonstrated that the application of commercial microbial fertilizers to simulated lunar soil not only improved soil properties but also promoted lettuce growth, enhanced biomass accumulation, alleviated oxidative damage, and strengthened stress tolerance. These results highlight the potential of microbial fertilizers as an effective strategy for improving simulated lunar soil for crop cultivation. In addition, dominant microbial genera capable of thriving in simulated lunar soil were identified. Dominant bacterial genera included Bacillus, Glutamicibacter, Acetobacter, Enterococcus, Microbacterium, Massilia, Lactobacillus, Lactococcus, Sphingobacterium, and Enterobacter, while dominant fungal genera included Saccharomyces, Pichia, and Trigonopsis. Identification of these taxa provides an important foundation for the future formulation of functional microbial consortia tailored for simulated lunar soil improvement.