To enable long-distance space travel, the development of a highly efficient and robust system to recover nutrients from waste streams is imperative. The inability of the current physicochemical-based environmental control and life support system (ECLSS) on the ISS to produce food in situ and to recover water and oxygen at high enough efficiencies results in the need for frequent resupply missions from Earth. Therefore, alternative strategies like biologically-based technologies called bioregenerative life support systems (BLSSs) are in development. These systems aim to combine biological and physicochemical processes, which enable in situ water, oxygen, and food production (through the highly efficient recovery of minerals from waste streams). Hence, minimalizing the need for external consumables. One of the BLSS initiatives is the European Space Agency’s (ESA) Micro-Ecological Life Support System Alternative (MELiSSA). It has been designed as a five-compartment bioengineered system able to produce fresh food and oxygen and to recycle water. As such, it could sustain the needs of a human crew for long-term space exploration missions. A prerequisite for the self-sufficient nature of MELiSSA is the highly efficient recovery of valuable minerals from waste streams. The produced nutrients can be used as a fertilizer for food production. In this review, we discuss the need to shift from the ECLSS to a BLSS, provide a summary of past and current BLSS programs and their unique approaches to nitrogen recovery and processing of urine waste streams. In addition, compartment III of the MELiSSA loop, which is responsible for nitrogen recovery, is reviewed in-depth. Finally, past, current, and future related ground and space demonstration and the space-related challenges for this technology are considered.

We developed a procedure for extracting maximal amounts of high-quality RNA from low-biomass producing (autotrophic) bacteria for experiments where sample volume is limited. Large amounts of high-quality RNA for downstream analyses cannot be obtained using larger quantities of culture volume. The performance of standard commercial silica-column based kit protocols and these procedures amended by ultrasonication or enzymatic lysis were assessed. The ammonium-oxidizing Nitrosomonas europaea and nitrite-oxidizing Nitrobacter winogradskyi were used as model organisms for optimization of the RNA isolation protocol. Enzymatic lysis through lysozyme digestion generated high-quality, high-yield RNA samples. Subsequent RNA-seq analysis resulted in qualitative data for both strains. The RNA extraction procedure is suitable for experiments with volume and/or biomass limitations, e.g., as encountered during space flight experiments. Furthermore, it will also result in higher RNA yields for whole transcriptome experiments where sample volume and/or biomass was increased to compensate the low-biomass characteristic of autotrophs.

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.

Bioregenerative life support systems (BLSS) are currently in development to tackle low recovery efficiencies, high energy demands, as well as food, water, and oxygen production challenges through the regeneration of nutrients from waste streams. The MELiSSA pilot plant has been developed as a testbed for regenerative life support system bioreactor operation and characterization. As nitrogen is a vital resource in such systems, we studied the functional composition of a new packed-bed nitrifying bioreactor inoculated with a co-culture of Nitrosomonas europaea (ATCC 25978) and Nitrobacter winogradskyi (ATCC 25391). After 840 days of autotrophic continuous cultivation, the packed-bed was sampled at five vertical positions, each with three horizontal positions, and the biomass at each position was characterized via qPCR, 16S amplicon sequencing, and liquid chromatography tandem mass spectrometry. The total number of cells within the different sections fluctuated around 8.95 ± 5.10 × 10⁷ cells/mL of beads. Based on 16S amplicons and protein content, N. europaea and N. winogradskyi constituted overall 44.07 ± 11.75% and 57.53 ± 12.04% of the nitrifying bioreactor, respectively, indicating the presence of a heterotrophic population that, even after such a long operation time, did not affect the nitrification function of the bioreactor. In addition, DNA-based abundance estimates showed that N. europaea was slightly more abundant than N. winogradskyi, whereas protein-based abundance estimates indicated a much higher abundance of N. europaea. This highlights that single-method approaches need to be carefully interpreted in terms of overall cell abundance and metabolic activity.

RNA-sequencing for whole transcriptome analysis requires high-quality RNA in adequate amounts, which can be difficult to generate with low-biomass-producing bacteria where sample volume is limited. We present an RNA extraction protocol for low-biomass-producing autotrophic bacteria Nitrosomonas europaea and Nitrobacter winogradskyi cultures. We describe steps for sample collection, lysozyme-based enzymatic lysis, and a commercial silica-column-based RNA extraction. We then detail evaluation of RNA yield and quality for downstream applications such as RNA-Seq. For complete details on the use and execution of this protocol, please refer to Verbeelen et al.¹.