fmicb-11-00525 March 30, 2020 Time: 18:31 # 1 ORIGINAL RESEARCH published: 31 March 2020 doi: 10.3389/fmicb.2020.00525 Microbial Monitoring in the EDEN ISS Greenhouse, a Mobile Test Facility in Antarctica Jana Fahrion1,2, Carina Fink1, Paul Zabel3, Daniel Schubert3, Mohamed Mysara4, Rob Van Houdt4, Bernhard Eikmanns2, Kristina Beblo-Vranesevic1* and Petra Rettberg1 1 Institute of Aerospace Medicine, German Aerospace Center (DLR), Cologne, Germany, 2 Institute of Microbiology and Biotechnology, Faculty of Natural Sciences, University of Ulm, Ulm, Germany, 3 Institute for Space Systems, German Aerospace Center (DLR), Bremen, Germany, 4 Microbiology Unit, Belgian Nuclear Research Centre (SCK CEN), Mol, Belgium The EDEN ISS greenhouse, integrated in two joined containers, is a confined mobile test facility in Antarctica for the development and optimization of new plant cultivation techniques for future space programs. The EDEN ISS greenhouse was used successfully from February to November 2018 for fresh food production for the overwintering crew at the Antarctic Neumayer III station. During the 9 months of Edited by: operation, samples from the different plants, from the nutrition solution of the aeroponic Rakesh Mogul, planting system, and from diverse surfaces within the three different compartments of California State Polytechnic University, the container were taken [future exploration greenhouse (FEG), service section (SS), Pomona, United States and cold porch (CP)]. Quantity as well as diversity of microorganisms was examined by Reviewed by: 2 Tatiana A. Vishnivetskaya, cultivation. In case of the plant samples, microbial quantities were in a range from 10 to The University of Tennessee, 104 colony forming units per gram plant material. Compared to plants purchased from Knoxville, United States David Anthony Pearce, a German grocery, the produce hosted orders of magnitude more microorganisms than Northumbria University, the EDEN ISS plants. The EDEN ISS plant samples contained mainly fungi and a few United Kingdom bacteria. No classical food associated pathogenic microorganism, like Escherichia and *Correspondence: Salmonella, could be found. Probably due to the used cultivation approach, Archaea Kristina Beblo-Vranesevic [email protected] were not found in the samples. The bioburden in the nutrition solutions increased constantly over time but never reached critical values like 102–103 cfu per 100 mL Specialty section: in irrigation water as it is stated, e.g., for commercial European plant productions. This article was submitted to Extreme Microbiology, The surface samples revealed high differences in the microbial burden between the a section of the journal greenhouse part of the container and the SS and CP part. However, the numbers of Frontiers in Microbiology organisms (bacteria and fungi) found in the planted greenhouse were still not critical. Received: 19 November 2019 Accepted: 11 March 2020 The microbial loaded surfaces showed strong temporal as well as spatial fluctuations. Published: 31 March 2020 In samples of the nutrition solution and the surface, the amount of bacteria exceeded Citation: the amount of fungi by many times. For identification, 16S rRNA gene sequencing was Fahrion J, Fink C, Zabel P, performed for the isolated prokaryotic organisms. Phylogenetic analyses revealed that Schubert D, Mysara M, Van Houdt R, Eikmanns B, Beblo-Vranesevic K and the most abundant bacterial phyla were Firmicutes and Actinobacteria. These phyla Rettberg P (2020) Microbial include plant- and human-associated bacterial species. In general, it could be shown Monitoring in the EDEN ISS Greenhouse, a Mobile Test Facility that it is possible to produce edible fresh food in a remote environment and this food is in Antarctica. safe for consumption from a microbiological point of view. Front. Microbiol. 11:525. doi: 10.3389/fmicb.2020.00525 Keywords: EDEN ISS, greenhouse, bacteria, plants, surfaces, phyllosphere, space exploration Frontiers in Microbiology| www.frontiersin.org 1 March 2020| Volume 11| Article 525 fmicb-11-00525 March 30, 2020 Time: 18:31 # 2 Fahrion et al. Microbiome Within the EDEN ISS Greenhouse INTRODUCTION of vegetables improved the morale of the crew significantly (Schlacht et al., 2019). After further investigation of this Manned missions to other planets like Mars or to the Moon, psychological effect, it might be a useful tool to improve the as well as stays on the International Space Station (ISS), quality of life for crew members on space ships. Antarctica have to be well planned in advance. For a successful mission, was chosen as a testbed for several reasons. The microbial the expertise of many different scientific and technical fields burden is low in Antarctic ice (Pearce et al., 2009) and needs to be combined. One important part that has to be thus, it is easier to monitor the microbial loads inside of considered is the food supply of the crew. From the beginning the greenhouse, because microorganisms are less likely to be of space flight, astronauts had to bring everything with them. introduced from the outside. Moreover, it is a cold and hostile For example, lots of water and food have to be packed environment and therefore has properties that come closest to and brought to the ISS (Perchonok et al., 2012). Because the conditions in space and on other planets. The command flights to other planets or the Moon take much longer than center for the EDEN ISS container is located in Germany, to the ISS and also require longer stays in space, it is of the supervisor there has to remotely monitor the functionality high importance to find technical solutions to secure food of the greenhouse, and the operator within the greenhouse production and water recycling in-flight as well as in future has to get along with what he already has on site (Zeidler extraterrestrial habitats. So-called bio-regenerative life support et al., 2019). Therefore, the framework conditions are similar to systems not only provide fresh food but can also be used for a space mission. oxygen production, recycling of carbon dioxide, water recycling, Plants in general as well as edible vegetables host vast and waste management (Zabel et al., 2015). One approach to amounts of microorganisms on their surfaces (Lindow and develop such a bio-regenerative life support system was the Brandl, 2003; Lopez-Galvez et al., 2014). While the root EDEN ISS project (Evaluation and Design of Environmentally microbiota, also called rhizosphere microbiota, has been a closed Nutrition-sources; funded by the European Union‘s subject to research for many decades now, the leaf microbiota Research and Innovation Action program Horizon 2020; grant is still inadequately investigated. The origin of microorganisms agreement ID: 636501). In 2015, the main goal of the project on leaves is not completely understood until today. The leaf was defined as “the adaptation, integration, fine-tuning, and and fruit surface of plants, the so-called phyllosphere, is demonstration of higher plant cultivation technologies and very diverse and organisms can be introduced via air, soil, operation procedures for safe food production on-board ISS other plants, animals, and water (Vorholt, 2012). The fact and for future human space exploration missions” (Zabel et al., that many plants re-establish their microbial communities 2015). The EDEN ISS greenhouse is a mobile test facility each year leads to the assumption that there is a core at the Neumayer III station in Antarctica. It was designed, microbiota in the soil and on the seeds that is responsible tested, and operated by a team of 14 international companies, for this effect (Knief et al., 2010). Because the leaves are institutes, and universities between March 2015 and April 2019, the entry site for many plant pathogens (Schulze-Lefert and including a test phase in Antarctica from December 2017 Robatzek, 2006), it is of high importance that the microbial to December 2018. At first, the EDEN ISS greenhouse was burden on the leaf surfaces is under strong control in assembled, integrated, and tested in Bremen, Germany, in order greenhouses in general and especially in greenhouses used in to investigate its functionality in a laboratory setting. Afterward, future space missions. it was disassembled, packed into two containers, and loaded onto Even though the EDEN-ISS plants were not germinated the German icebreaker research vessel Polarstern and transported gnotobiotically, some of the factors that usually contribute via Cape Town in South Africa to Antarctica. 400 m from to the microbial diversity of the phyllosphere are minimized the German Neumayer III station, the two containers were in the EDEN ISS system. For instance, the introduction of assembled and connected again to build one unit consisting soil microorganisms is improbable since the plants were of the greenhouse, the service section (SS), and a cold porch cultivated in an aeroponic system. The strong evolutionary (CP; Schlacht et al., 2019). The greenhouse is confined half- pressure from the harsh environment can give rise to autonomous and contains its own nutrient delivery system specially adapted cells with high heat and desiccation (NDS), special LED lights with adapted wavelengths for the tolerances (Mora et al., 2016). Therefore, not only the specific plants, and an air management system (Zabel et al., natural accumulation of microorganisms in nutrient- 2015; Imhof et al., 2018). The EDEN ISS greenhouse was closed rich greenhouses can pose a risk, but also these special with the only exception of the entering and leaving of the same circumstances. Due to this, it is of high importance to operator once per day for reasons, like sowing, harvesting, filling monitor the general microbial burden in order to evaluate the nutrient tanks, and cleaning work. There was no direct the risks that may arise. exchange between the greenhouse itself and the environment, The goal of this study was to provide a survey of since the operator was entering the container via the CP and the global situation of the microbial community inside the the SS.