1. Ground-based research for soybean cultivation in BLSSs: harmonization of plant structure and physiology in hydroponics Paradiso R., De Micco V., Arena C., Aronne G., Pepe O., De Pascale S. University of Naples Federico II - Italy ISLSWG Workshop Bioregenerative Life Support Turin, 18-19 May 2015

2. Osteoporosis risk Khalil et al., 2002 J. Nutr. 132: 2605-2608 Proteolysis of myofibrillar proteins Tada and Yokogoshi, 2002 J. Nutr. Sci. Vit. 48(2): 115-119 Peroxidation products in brain Chowdhury and Soulsby, 2002 Ann. Cl. Lab. Sci. 32(2):188-192 Why soybean? Nutritional value of seeds Hoff et al., 1982 NASA Contract Report 166324

3. I. to develop an objective theoretical procedure to identify candidate cvs De Micco et al., 2012 - Proceedings 63 rd Intl. Astronautical Congress (IAF) De Micco et al., 2012 - Adv. in Space Res. 49: 1415-21 II. to evaluate plant behavior in hydroponics in controlled environment Paradiso et al., 2012 - Adv. in Space Res. 50: 1501-11 Paradiso et al., 2014 – Plant biology, 16(suppl. 1): 69-78 III. to compare nutritional composition of seeds in hydroponics/open field Palermo et al., 2012 - J. Agric. Food Chem., 60: 250-255 Objectives of research on Soybean within MELiSSA project ( Micro Ecological Life Support System Alternative) FC Phase 1

4. Interaction of soybean plants with favourable microorganisms FC Phase 2 IV. to evaluate the effects of beneficial microorganisms on plant performance in closed-loop hydroponics in controlled environment Myco Madness microbial mix (Humboldt nutrients) to evaluate the effects of root symbiosis with nitrogen fixing bacteria on plant performance in closed-loop hydroponics in controlled environment

5. Root nodulation and seeds yield and quality of plants inoculated with B. japonicum in cooperation with School of Environmental Sciences (University of Guelph) Aim  to evaluate the effect of inoculation with Bradyrhizobium japonicum (soybean bacterial symbiont) in 2 hydroponic systems (NFT and cultivation on rockwool) with urea as alternative N-source to nitrate Results  Root inoculation did not influence plant performance, regardless of the N-source  Cultivation on rockwool positively influenced root nodulation and plant growth and yield, without affecting the composition of seeds, compared to NFT  Urea improved root symbiosis but it reduced plant growth and yield. Hydroponic cultivation of soybean for BLSSs: the effect of nitrogen source and bacterial root symbiosis Paradiso et al., Proceedings 63 rd Intl. Astronautical Congress (IAF) Paradiso et al., 2014 - Adv. in Space Res. 53: 574-84

6. Growth chamber & environmental control Materials & Methods 200  300 cm Osram PlantaStar ® 600W Fog system

7. PPFD 500  mol m -2 s -1 light/dark 12/12 h Temperature 26/20 °C RH 60  80% Environmental parameters Modified Hoagland & Arnon ½ strength 0.25 Materials & Methods EC target 1.2 dS m -1 C - 1.4 dS m -1 pH target 5.8 Fertigation strategy (Wheeler et al., 2008)

8. Inoculation protocol SOYBEAN STERILIZED SEEDS cultivar ‘Pr91m10’ (Pioneer Intl.) Myco Madness mix preparation 1/4-strength Ringer’s solution Seeds inoculation (12 h) Washing in sterilized distilled water Sowing 22 °C, darkness, 8 days Cell count (5x10 5 cells/ml) Thoma cell counting chamber, Microscope Nikon Eclipse E200 Seedling roots inoculation Nutrient solution inoculation 3 times in 3 weeks Materials & Methods (Bashan, 1986. Soil Biol. and Bioch. 18(3): 297-301)

9. Materials & Methods Gas exchanges & Chlorophyll fluorescence Infra red gas analyzer Walz HCM-1000 Junior – Pam Walz Leaf greenness & Chlorophyll content Hach 4000 Spectrophotometer Spad Minolta 502 Leaf anatomical features Rotative mnicrotome Light & Epi-fluorescence microscopy on leaf thin section Plant growth and seed yield and quality Proximate composition of seeds (AOAC, 1995) S. Pieters & E. Dulière, Institut Paul Lambin, Louvain, Belgium Rhizosphere microbiome characterization Microorganisms DNA Sequencing C. Sheridan & D. Geelen, Dep. Plant Production, Gent University Measurements

10. Photosynthesis and transpiration rate Plants at reproductive phase, Temperature 26 °C, RH 70%, PPFD 400  mol m -2 s -1 Results Measurements of gas exchanges revealed higher CO 2 assimilation and transpiration rate in Inoculated plants compared to Control, starting from the beginning of flowering.

11. Photosynthesis: Photochemical efficiency (Plants at reproductive phase, Temperature 26°C, RH 70%, PPFD 400  mol m -2 s -1 ) Results Similarly, Chl fluorescence measurements demonstrated better photochemical performance in treated plants in the reproductive phase, with higher values of quantum yield of Photosystem II (  PSII) a b

12. Leaf anatomy: stomata size and frequency C I Results Changes in plant behaviour were also due to modifications in anatomical traits: Inoculated plants showed higher frequency of smaller stomata than control, which accounts for a better control of gas exchanges

13. 100 micron Leaf anatomy: lamina structure C I Results Moreover, Inoculated plants showed a mesophyll structure (thicker layer of palisade parenchyma and larger intercellular space) favouring the control of gas-exchanges

14. Plant growth and Seed yield Vegetative growth Flowering & Fruit setting Pod filling Seed drying Results Inoculation determined higher values of plant height and leaf area. Consistently, seed yield was higher in Inoculated plants compared to Control, with no change in nutritional quality.

15. Christopher Sheridan & Danny Geelen Dep. Plant Production, Gent University Rhizosphere microbiome analysis C I Root endosphere Root exosphere C I Results At the end of the experiment, dominant microbial composition differed between control and treated plants in both the regions of rhizosphere: In the exosphere, Control communities were dominated by Chitinophagaceae bacteria and Ascomycetes fungi, while they were much more heterogeneous in Inoculated plants, with a mix of Actinobacteria, Bacilli and Betaproteobacteria. In the endosphere, Control was mainly composed by Ascomycetes, while treated samples were dominated by Ochrobactrum, which is a plant growth-promoting taxon.

16. -Root inoculation of soybean with Myco Madness mix enhanced plant growth and seed productivity, inducing functional anatomical modifications and enhancing physiological performance compared to control -Improved mineral nutrition and hormone activity by plant-growth promoting bacteria ( Ochrobactrum spp.) in the endosphere of treated plants may explain these results CONCLUSIONS Inoculation with beneficial microorganisms can be considered as a tool to improve performance of soybean in hydroponics, under controlled environment!