Multi-omics characterization of biofilms in the C.R.O.P.® urine recycling system for space-relevant closed-loop life support

Kurzfassung

The C.R.O.P.® (Combined Regenerative Organic food Production) system is a vertical trickle filter designed to recover nutrients from human urine to generate a nitrate-rich plant fertilizer solution to provide closed-loop food production during long-duration Moon habi tation. Expanded clay particles in the filter are used as carrier material to host a functional biofilm that enables the nitrification process from urine to nitrate. This thesis combined 16S rRNA gene sequencing with LC-MS/MS metaproteomics, and ion chromatography to characterize the complex community of microorganisms across four different C.R.O.P.® filter units, and to assess functional nitrogen transformation throughout a 14-month study period. The microbial communities of the studied filter units significantly differed from each other. The substrate switch from synthetic to real human urine and adjusted synthetic urine con centration strongly promoted a shift in community composition, taxonomic richness, and phylogenetic diversity. This finding was verifiable despite a complex boundary partially linked to the separate sequencing in two batches. The presence of mussel shells for sys tem buffering showed to drive a moderate effect on the community and significantly im proved nitrification performance. Additionally, a shift in community structure was observed linked to the spatial position within a C.R.O.P.® trickle filter along the vertical axis. Metaproteomic analysis complements the metagenomic finding with confirmed active ex pressions of enzymes catalyzing both steps of the nitrification process. However, the pro tein-level results showed no substrate-associated changes or temporal development in accordance with the genomic analysis or with the development of measured nitrogen chemistry. In contrast, the nitrifier abundance detected in 16S rRNA gene sequencing showed a strong correlation with both, ammonia consumption and nitrate production, in agreement with the nitrification process. While partially agreeing with each other, signals in microbial abundance and protein expression display two distinctly different approaches on the nitrogen cycle activity within the system. The C.R.O.P.® filter system demonstrated efficient and stable nitrogen recovery under substantially more acidic conditions than ideally reported for nitrification during urine pro cessing, highlighting its relevance for nutrient recovery in extreme and resource-limited conditions, such as Lunar habitation or terrestrial circular agriculture.