Microbial engineering of soil structure: Insights from synthetic Mars simulant soils

Soil degradation is one of the most urgent global challenges, threatening food security, ecosystem functioning, and sustainable land management. Soil aggregate stability is crucial for maintaining soil health because it influences water retention, root growth, and nutrient cycling. Microorganisms play a vital role in soil aggregation through their biomass, necromass, metabolic activity, and production of extracellular polymeric substances (EPS) and enzymes. These microbial processes bind soil particles, enhance soil organic matter (SOM), and improve soil structure. This thesis investigates how microbial inoculation using individual bacterial strains, bacterial consortia, and complex microbial communities affects soil aggregate stability and soil chemical properties. To explore these mechanisms under controlled and nutrient-limited conditions, Mars simulant soils MGS-1 and MMS-1 were used as standardized substrates that mimic highly degraded terrestrial soils. Together, the studies provide a mechanistic understanding of how microbial metabolism and community dynamics contribute to soil aggregate formation. Chapter 2 examined the influence of individual bacterial strains and a bacterial consortium on soil aggregation in Mars Regolith Simulant MMS-1. Using controlled inoculations and micro-computed tomography (Micro-CT), the results showed that microbial biomass and necromass substantially increased SOM and soil porosity, both essential for aggregate stability. Even under nutrient-poor conditions, bacterial consortia improved soil structure and demonstrated the potential of microbial inoculants for soil restoration. Chapter 3 focused on the technical challenge of extracting high-quality DNA from Mars simulant soils. High silicate, iron, and metal oxide contents interfered with DNA extraction and sequencing. Several extraction methods were compared, and the MP DNA extraction kit was identified as the most effective. These methodological improvements enabled accurate microbial community characterization in mineral-rich and nutrient-poor soils and provided a foundation for further microbial ecological analysis. Chapter 4 investigated microbial community assembly in sterile MGS-1 soil buried within donor soils differing in microbial diversity. Microbial colonization improved soil aggregate stability, although the effects varied among donor soils. While microbial diversity within MGS-1 did not directly correlate with aggregate stability, higher donor soil diversity promoted the establishment of bacteria that enhanced soil carbon content and indirectly supported aggregation. These findings emphasize the role of microbial-derived carbon and community interactions in improving soil structure. Chapter 5 explored how an EPS-producing bacterial community isolated from the rhizosphere of Chrysanthemum indicum L. affected soil aggregation in MGS-1, MMS-1, and sand. Inoculation significantly enhanced aggregation across all substrates. Metagenomic analysis linked functions related to biofilm formation, energy metabolism, and amino sugar metabolism to increased stability. Random Forest modeling identified microbial biomass, necromass, and EPS as key drivers of aggregation. Cryo-SEM and SEM-EDX imaging provided visual evidence of EPS-mediated particle binding. Overall, this thesis demonstrates that microbial inoculation using single strains, consortia, or communities enhances soil aggregate stability even under nutrient-limited conditions. Microbial biomass, necromass, and EPS production consistently improved SOM and soil structure, offering insights into how microbes can be harnessed to restore and stabilize degraded soils