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Substrate Matters: Ionic Silver Alters Lettuce Growth, Nutrient Uptake, and Root Microbiome in a Hydroponics System

Ionic silver (Ag+) is being investigated as a residual biocide for use in NASA spacecraft potable water systems on future crewed missions. This water will be used to irrigate future spaceflight crop production systems. We have evaluated the impact of three concentrations (31 ppb, 125 ppb, and 500 ppb) of ionic silver biocide solutions on lettuce in an arcillite (calcinated clay particle substrate) and hydroponic (substrate-less) growth setup after 28 days. Lettuce plant growth was reduced in the hydroponic samples treated with 31 ppb silver and severely stunted for samples treated at 125 ppb and 500 ppb silver. No growth defects were observed in arcillite-grown lettuce. Silver was detectable in the hydroponic-grown lettuce leaves at each concentration but was not detected in the arcillite-grown lettuce leaves. Specifically, when 125 ppb silver water was applied to a hydroponics tray, Ag+ was detected at an average amount of 7 μg/g (dry weight) in lettuce leaves. The increase in Ag+ corresponded with a decrease in several essential elements in the lettuce tissue (Ca, K, P, S). In the arcillite growth setup, silver did not impact the plant root zone microbiome in terms of alpha diversity and relative abundance between treatments and control. However, with increasing silver concentration, the alpha diversity increased in lettuce root samples and in the water from the hydroponics tray samples. The genera in the hydroponic root and water samples were similar across the silver concentrations but displayed different relative abundances. This suggests that ionic silver was acting as a selective pressure for the microbes that colonize the hydroponic water. The surviving microbes likely utilized exudates from the stunted plant roots as a carbon source. Analysis of the root-associated microbiomes in response to silver showed enrichment of metagenomic pathways associated with alternate carbon source utilization, fatty-acid synthesis, and the ppGpp (guanosine 3′-diphosphate 5′-diphosphate) stringent response global regulatory system that operates under conditions of environmental stress. Nutrient solutions containing Ag+ in concentrations greater than 31 ppb in hydroponic systems lacking cation-exchange capacity can severely impact crop production due to stunting of plant growth.

lettuce↗

Effects of Residual Water System Silver on Space Crop Microbiome and Nutrient Content

Ionic silver (Ag+) is being investigated as a residual biocide for use in spacecraft potable water systems on future crewed missions. In addition to providing clean water to the crew and other life support system functions, the potable water is used to irrigate space crop production units such as the Vegetable Production System (Veggie) and the Advanced Plant Habitat (APH). We have evaluated the impact of different concentrations of Ag+ biocide solutions in comparison to a control in both substrate (arcillite-based) and substrate-less (hydroponics-based) growth set ups. Here, we provide evidence that increasing the concentration of silver in the irrigation water impacts the root zone microbiome in both setups, with plant growth and elemental nutrient content also affected in the hydroponic set up. This suggests a need for a silver removal step to achieve acceptable silver levels in irrigation water before application to space crops in a substrate-less hydroponics system. This removal step is also recommended for a substrate-based system, although it is not as critical as in a hydroponics system.

Aubrie O’Rourke↗

Preflight Definition and Verification Testing for the Plant Habitat-07 Experiment to Study Substrate Moisture Impacts on Lettuce Plant and Microbiome Development

Delivering adequate water and oxygen to root zones of crops growing in microgravity is challenging due to the complex behavior of fluids and gasses during spaceflight. Chronic excess (flood) or insufficient (drought) water levels, or intermittent watering and wilting of plants, leads to alterations in plant growth and impacts on the nutritional and microbial composition of those plants. PH-07 will apply controlled water stress to assess and quantify changes in plant growth and the microbiome of a well-tested food crop, ‘Outredgeous’ red romaine lettuce, grown in NASA’s Advanced Plant Habitat (APH) on the ISS.

Gioia Massa↗

The Microbiome of A Tomato Crop Grown Under Different Lighting Regimes on the International Space Station

The VEG-05 experiment presented here investigated the effect of red-rich and blue-rich light recipes in Veggie on the microbiome of the Veggie facility and the plant tissues of a dwarf tomato variety, Solanum lycopersicum cv. Red Robin. For food safety, the plants were screened using culture-based methods for potential human pathogens that may cause infection by consumption of the fruit. The microbiome was investigated using bacterial 16S and fungal ITS sequencing methods to enumerate and identify bacterial and fungal communities on tomato fruit, roots, leaves, rooting substrate, and Veggie facility surfaces grown under blue-rich or red-rich lighting. Comparisons of microbial communities were made between lighting treatments, as well as for flight and ground controls. This analysis determined the core microbiome and microbiological composition for tomato plants grown under a blue-rich or red-rich lighting treatment and microgravity conditions. Culture-based pathogen screening, corroborated by 16S and ITS sequencing, yielded negative results. Bacterial and fungal counts were lower for ground controls than in-flight samples. However, there were no differences in microbial counts between lighting treatments. Regardless of lighting treatment, plant components shared a core microbiome, although some differences were observed in genera between lighting treatments.

Veggie↗

Persistence of a Potential Pathogen on a Plant Microbiome-Does Seed Sanitization Matter?

Microbial interactions on, in, and around the seeds can have profound effects on plant growth, development, and productivity. These interactions can be casual or intimate in nature, but ultimately they all contribute in varying degrees to an ever-evolving microbiome. Plant microbiomes have been investigated over decades and new data continue to reveal how microbiomes play an important role in the plant’s success. Many eubacteria and fungi have been found to have a symbiotic relationship with plants and other microorganisms. The source of the plant microbiome on adult plants is provided by the seed via vertical transmission to leaf, root, flowers or fruit, as well as the surrounding environment.

Anirudha R. Dixit↗

Seed surface sanitization and persistence of E.coli through different tissues of ‘Red Robin’ Tomato (Solanum lycopersicum cv. Red Robin)

Seed surface sanitization via chemical processes removes/reduces microbes from the external surfaces of the seed and thereby could have an impact on the plants’ health or productivity. To determine the impact of seed surface sanitization on the plants’ microbiome, sanitized and unsanitized seeds from ‘Red Robin’ Tomato (Solanumlycopersicum cv. Red Robin) were exposed to Escherichia coli (E. coli) and grown in a controlled environment growth chamber simulating environmental conditions aboard the International Space Station (ISS). Plants were harvested at four intervals, days 11, 33,42 and 76 post-germination. Changes in the microbial communities of leaf, stem, root, and fruit because of E. coli exposure and the persistence of E. coli itself were investigated using aerobic plate count (APC), qPCR and 16S rRNA sequencing. It was determined that E. coli persisted for longer periods of time in plants from sanitized versus unsanitized seeds and was identified in root tissue more frequently than in leaf or stem tissue. E. coli was not detected in fruits raised from either sanitized or unsanitized seeds. The 16S rRNA sequencing showed dynamic changes in the abundance of members of the phylum Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes in all tissue types studied. We observed minimal or no changes in the alpha diversity of leaf stem and fruit tissue with time, or between sanitized and unsanitized seeds. Roots showed significant differences in alpha diversity with time and seed sanitization status. Beta-diversity showed that time had more of an influence on all samples versus the E. coli treatment. Members of phyla Proteobacteria and Bacteroidetes were found to be differentially abundant across leaf, stem and root tissue. Our results indicated that the seed surface sanitization, although a requirement for sending seeds to space, might influence the developing microbiome. This research was funded by NASA’s Space Life and Physical Sciences Research and Applications.

Anirudha R Dixit↗

Space Crop Production

As astronauts venture farther from Earth, and for longer periods, food will become increasingly critical. Crop production can supplement a packaged diet to provide additional nutrients and variety for astronauts. Testing with the Veggie and Advanced Plant Habitat chambers on the International Space Station is allowing us to understand the impacts of gravity and spaceflight on crop growth and nutritional content, and the importance of plants to astronauts living and working away from our home planet. The space environment presents unique challenges to crop production, and technology and knowledge gaps have been identified. Key gaps include how to: - Identify and demonstrate effective options to provide both water and oxygen to the root zone in microgravity - Understand the deep space radiation impacts on seeds and plants - Investigate the relationship between microbiomes and food safety - Store and handle seeds to ensure they are viable, free of contaminants and long-lived - Identify / develop potential crops suitable for the space environment - Understand automation and human factors - Determine scalability for different concepts and architectures Filling these gaps will help enable future human exploration and move us toward Earth-independence.

Space Crop Production↗

Space Crop Production

As astronauts venture farther from Earth, and for longer periods, food will become increasingly critical. Crop production can supplement a packaged diet to provide additional nutrients and variety for astronauts. Testing with the Veggie and Advanced Plant Habitat chambers on the International Space Station is allowing us to understand the impacts of gravity and spaceflight on crop growth and nutritional content, and the importance of plants to astronauts living and working away from our home planet. The space environment presents unique challenges to crop production, and technology and knowledge gaps have been identified. Key gaps include how to: • Identify and demonstrate effective options to provide both water and oxygen to the root zone in microgravity • Understand the deep space radiation impacts on seeds and plants • Investigate the relationship between microbiomes and food safety • Store and handle seeds to ensure they are viable, free of contaminants and long-lived • Identify / develop potential crops suitable for the space environment • Understand automation and human factors • Determine scalability for different concepts and architectures Filling these gaps will help enable future human exploration and move us toward Earth-independence.

Veggie↗

Does Seed Sanitization Affect the Plant Rhizosphere Microbiome and Its Ability to Compete with the Human Associated Pathogen, E. coli on Salad Crops?

Cultivation of crops in controlled environmental agricultural systems may limit microbial colonization and reduce diversity of the microbial communities. Practices like seed and growth medium sanitization may further impact microbial communities in the mature plant and the plant’s capacity to limit the growth of pathogens through competition. As humans expand their travels to space, understanding plant growth, health, and development in closed environments will be critical to the success of producing a safe, supplemental food source for astronauts. To determine the persistence of a potential human pathogen in plant growth and development, sanitized and unsanitized seeds from, mizuna (Brassica rapa var japonica) and red romaine lettuce (Lactuca sativa cultivar ‘Outredgeous’), were inoculated with Escherichia coli, ATCC 21445, germinated under simulated International Space Station (ISS) environmental conditions and harvested every 7 days until maturity. The persistence of E. coli in the rhizosphere was determined by plating on selective media, real time PCR (Polymerase Chain Reaction) and community sequencing of the rhizosphere communities. E. coli was detected in the crops’ roots and leaves for several weeks post germination. At day 28, plants from sanitized seeds had significantly higher counts of E. coli on the roots than those from unsanitized seeds. E. coli was also detected on a few uninoculated plants indicating airborne cross contamination among plants in the same growth chamber and suggesting an influence of the natural microbiome on human pathogen survival and persistence in leafy greens. Sequencing analysis revealed variations in composition and diversity between the communities. Understanding the microbial community of the rhizospheric microbiome is only the first step in determining the relationships between plants. Additional studies to include genotypic and phenotypic variations in the plants should be considered to determine if the natural microbes in the rhizosphere may contribute to the health and therefore, safety of the edible plants.

Khodadad, Christina L. M.↗

Plant Microbiomes May Provide Vital Information to Plant Success

Plant associated microbiomes, the rhizosphere and phyllosphere, are composed of communities of bacteria and fungi that may be mutualistic or pathogenic. These communities have the potential to influence plant health and development and can affect plant growth. Crop plants are being investigated as a fresh and safe supplement to astronauts’ diet and it is critical to understand and characterize these microbial communities. Multi-species crops, Mizuna mustard (Brassica rapa var japonica), ‘Outredgeous’ red romaine lettuce (Lactuca sativa), and Waldman’s Green lettuce (Lactuca sativa) were grown in two Veggie units on the International Space Station (ISS) for three grow outs in various combinations of plant types. Upon harvest, plant and pillow samples were frozen and returned to Earth for analysis. Bacterial and fungal community analyses for plant leaf and root, as well as pillow components, wick and media, were completed using next generation sequencing with the goal of surveying the composition of the entire community and identifying any potential pathogens. Bacteria were identified using the 16S rRNA gene whereas, fungi were identified with the internal transcribed spacer (ITS). The community composition for these three crops was compared between crop types and between plant tissue types. It is vital to mission success for the short term and long term to add nutritious, safe to eat vegetables providing a supplement to the crew members’ dietary requirements as well as to develop planning for deep space missions as we reach for the moon and on to Mars. Veggie technology validation tests were supported by NASA’s Space Biology Program.

Khodadad, Christina L.↗

Veggie: Space Vegetables for the International Space Station and Beyond

The Veggie vegetable production system was launched to the International Space Station (ISS) in 2014. Veggie was designed by ORBITEC to be a compact, low mass, low power vegetable production system for astronaut crews. Veggie consists of a light cap containing red, blue, and green LEDs, an extensible transparent bellows, and a baseplate with a root mat reservoir. Seeds are planted in plant pillows, small growing bags that interface with the reservoir. The Veggie technology validation test, VEG-01, was initiated with the first test crop of 'Outredgeous' red romaine lettuce. Prior to flight, lettuce seeds were sanitized and planted in a substrate of arcillite (baked ceramic) mixed with controlled release fertilizer. Upon initiation, astronauts open the packaged plant pillows, install them in the Veggie hardware, and prime the system with water. Operations include plant thinning, watering, and photography. Plants were grown on the ISS for 33 days, harvested, and returned frozen to Earth for analysis. Ground controls were conducted at Kennedy Space Center in controlled environment chambers reproducing ISS conditions of temperature, relative humidity, and CO2. Returned plant samples were analyzed for microbial food safety and chemistry including elements, antioxidants, anthocyanins and phenolics. In addition the entire plant microbiome was sequenced, and returned plant pillows were analyzed via x-ray tomography. Food safety analyses allowed us to gain approvals for future consumption of lettuce by the flight surgeons and the payload safety office. A second crop of lettuce was grown in 2015, and the crew consumed half the produce, with the remainder frozen for later analysis. This growth test was followed by testing of a new crop in Veggie, zinnias. Zinnias were grown to test a longer duration flowering crop in preparation for tests of tomatoes and other fruiting crops in the future. Zinnias were harvested in February. Samples from the second harvest of lettuce and the zinnia harvest are frozen on the ISS and will return with the next cargo return flight. Some challenges occurred in all tests, especially in the area of watering, with plants receiving insufficient or excess water leading to stressed growth. Zinnia plants were also challenged with fungal growth. Initial tests with Veggie have given us great insight into future crop production scenarios as we work to develop regular supplemental salad crop production on ISS and larger food production systems for our journey to Mars. Funding for this research comes from NASA's Space Biology Program.

Food Safety↗