The MELiSSA (Micro-Ecological Life Support Alternative) loop is an artificial ecosystem intended as a regenerative life support system for long-term space missions. It is important to know whether the bacterial actors in such a system are susceptible to intra- and extracellular threats. One such an intracellular threat is represented by genomic instability caused by latent prophages and transposons, which may become induced or activated by a particular environmental factor, in particular (cosmic) radiation. Extracellular threats include, among others, physical stressors such as temperature and light, as well as biological ones such as bacteriophages. Bacteriophage incidence is likely to be high in the biodiverse waste streams that feed the system and could spread readily throughout the system.
Our first set of experiments was to determine the possible roles of (latent) bacterio(pro)phages on the main bacterial actors in the second and fourth compartment of the MELiSSA loop, i.e. Rhodospirillum rubrum S1H and Arthrospira sp. PCC8005. In silico investigation of their genomes indicated that the presence of functional prophages was unlikely. Subsequently, the absence of inducible prophages was confirmed by applying DNA damage-induced stress, which would induce any prophages present in the genome. This stress was created by applying a range of concentrations of mitomycin C, using a concentration optimized for Lactococcus lactis as a starting point and adjusted for use with Arthrospira sp. PCC8005 and R. rubrum S1H by screening for mitomycin C toxicity on 96 well plates. Then a concentration range between no noticeable inhibition of growth by mitomycin C and complete inhibition of growth was screened in shake flasks, and the absence of induced viral particles was confirmed by flow cytometry and viral DNA extraction.
The second set of experiments was aimed at investigating the likelihood of R. rubrum S1H and Arthrospira sp. PCC8005 becoming cross-contaminated with bacteriophages from a previous compartment, therefore a wide variety of dissimilar sources of domestic wastewater from Ireland, the Netherlands and Belgium, grease traps, a sample from a pilot installation of compartment I and a compost sample were tested for the presence of bacteriophages with lytic activity against those strains. Thus far, no bacteriophages with lytic activity against R. rubrum S1H and Arthrospira sp. PCC8005 have been found, indicating a minimal risk of cross-contamination.