Microbial monitoring, microbial diversity and evaluation of cleaning events in the EDEN-ISS greenhouse, a test facility for plant-cultivation in space

Kurzfassung

The storage requirements for pre-packaged supplies like food, water and medicine limit the distance and duration of current crewed space flight missions. Bio-regenerative Life Support Systems (BLSS) aim to remedy these problems by utilizing the regenerative capabilities of plants and microbes for the recycling of resources. The EDEN ISS greenhouse is a platform that was used to test technologies for the cultivation of plants for food production in a BLSS context. It was in operation in Antarctica from 2018 to 2022 and produced various fresh vegetables and herbs. The monitoring of the microbiome of the greenhouse as well as the application of sufficient cleaning protocols are both important to identify and remove potentially harmful microbes. For this purpose, surface samples were taken from various locations in the greenhouse before and after the greenhouse was cleaned between growing seasons. Samples were also taken from the subfloor over the duration of an entire growing season. The bioburden of the sampled surfaces was evaluated by extracting the cells from the surface swabs and plating them in ascending dilutions on agar plates selective for either bacteria or fungi and counting the number of colonies after incubation at room temperature (~22 °C). Sporeformers were identified by heat shocking the samples and processing them as described previously. Morphologically distinct bacterial colonies were isolated and both them and the bacterial and archaeal sequences present in the DNA directly extracted from surfaces swabs were identified with 16S rRNA sequencing. The results showed that the effectiveness of the cleaning protocol could not be accurately evaluated, as plant cultivation began again before the post-cleaning samples were taken, making it unclear if the lack of reduction in bioburden was caused by insufficient cleaning or the re-introduction of plants. Nonetheless both the cultivation of the heat shocked samples as well as the 16S rRNA sequencing revealed an increase in potential sporeformers too, indicating that the current cleaning protocol was not sufficient in the removal of bacterial spores. The bioburden of the subfloor underwent large fluctuations, which might have been caused by a combination of harvests, excessive condensation and cleaning around the sampling location. The composition of its microbiome also changed over time, with distinct phases being identifiable. This might have been caused by an influx of bacteria from recently harvested plants. Phases of high bioburden went together with low alpha diversity of the samples as a small number of genera began dominating the sampling location. The presence of humans and plants in the greenhouse seemed to be the driving factors of its microbiome composition, with waterassociated genera, most likely derived from the nutrient solution, also playing a role. Genera that contain potentially harmful species were observed, highlighting the importance of microbial monitoring and suitable cleaning protocols for BLSSs.