Whole transcriptome analysis highlights nutrient limitation of nitrogen cycle bacteria in simulated microgravity.

Regenerative life support systems (RLSS) will play a vital role in achieving self-sufficiency during long-distance space travel. Urine conversion into a liquid nitrate-based fertilizer is a key process in most RLSS. This study describes the effects of simulated microgravity (SMG) on Comamonas testosteroni, Nitrosomonas europaea, Nitrobacter winogradskyi and a tripartite culture of the three, in the context of nitrogen recovery for the Micro-Ecological Life Support System Alternative (MELiSSA). Rotary cell culture systems (RCCS) and random positioning machines (RPM) were used as SMG analogues. The transcriptional responses of the cultures were elucidated. For CO₂-producing C. testosteroni and the tripartite culture, a PermaLife^TM PL-70 cell culture bag mounted on an in-house 3D-printed holder was applied to eliminate air bubble formation during SMG cultivation. Gene expression changes indicated that the fluid dynamics in SMG caused nutrient and O₂ limitation. Genes involved in urea hydrolysis and nitrification were minimally affected, while denitrification-related gene expression was increased. The findings highlight potential challenges for nitrogen recovery in space.