Slippery lubricant-infused silica nanoparticulate film processing for anti-biofouling applications.

Microbial biofilm build-up in water distribution systems can pose a risk to human health and pipe material integrity. The impact is more devastating in space stations and to astronauts due to the isolation from necessary replacement parts and medical resources. As a result, there is a need for coatings to be implemented onto the inner region of the pipe to minimize the adherence and growth of biofilms. Lubricant-infused surfaces has been one such interesting material for anti-biofouling applications in which their slippery property promotes repellence to many liquids and thus prevents bacterial adherence. Textured and porous films are suitable substrate candidates to infuse and contain the lubricant. However, there is little investigation in utilizing a nanoparticulate thin film as the substrate material for lubricant infusion. A nanoparticulate film has high porosity within the structure which can promote greater lubricant infusion and retention. The implementation as a thin film structure aids to reduce material consumption and cost. In our study, we utilized a well-studied nanoporous thin film fabricated via layer-by-layer assembly of polycations and colloid silica and then calcination for greater stability. The film was further functionalized to promote fluorinated groups and improve affinity with a fluorinated lubricant. The pristine nanoporous film was characterized to determine its morphology, thickness, wettability, and porosity. The lubricant-infused film was then tested for its lubricant layer stability upon various washing conditions and its performance against bacterial biofilm adherence as a result of its slippery property. Overall, the modified silica nanoparticulate thin film demonstrated potential as a base substrate for lubricant-infused surface fabrication that repelled against ambient aqueous solvents and as an anti-biofouling coating that demonstrated low biofilm coverage and colony forming unit values. Further optimization to improve lubricant retention or incorporation of a secondary function can aid in developing better coatings for biofilm mitigation.

Biofilm formation is correlated with low nutrient and simulated microgravity conditions in a Burkholderia isolate from the ISS water processor assembly.

The International Space Station (ISS) Water Processor Assembly (WPA) experiences intermittent dormancy in the WPA wastewater tank during water recycling events which promotes biofilm formation within the system. In this work we aimed to gain a deeper understanding of the impact of nutrient limitation on bacterial growth and biofilm formation under microgravity in support of biofilm mitigation efforts in exploration water recovery systems. A representative species of bacteria that is commonly cultured from the ISS WPA was cultured in an WPA influent water ersatz formulation tailored for microbiological studies. An isolate of Burkholderia contaminans was cultured under a simulated microgravity (SµG) treatment in a vertically rotating high-aspect rotating vessel (HARV) to create the low shear modeled microgravity (LSMMG) environment on a rotating wall vessel (RWV), with a rotating control (R) in the horizontal plane at the predetermined optimal rotation per minute (rpm) speed of 20. Over the course of the growth curve, the bacterial culture in ersatz media was harvested for bacterial counts, and transcriptomic and nutrient content analyses. The cultures under SµG treatment showed a transcriptomic signature indicative of nutrient stress and biofilm formation as compared to the R control treatment. Further analysis of the WPA ersatz over the course of the growth curve suggests that the essential nutrients of the media were consumed faster in the early stages of growth for the SµG treatment and thus approached a nutrient limited growth condition earlier than in the R control culture. The observed limited nutrient response may serve as one element to explain a moderate enhancement of adherent biofilm formation in the SµG treatment after 24 h. While nutrients levels can be modulated, one implication of this investigation is that biofilm mitigation in the ISS environment could benefit from methods such as mixing or the maintenance of minimum flow within a dormant water system in order to force convection and offset the response of microbes to the secondary effects of microgravity.

Spatial Characterization of Microbial Communities on Multi-Species Leafy Greens Grown Simultaneously in the Vegetable Production Systems on the International Space Station.

The establishment of steady-state continuous crop production during long-term deep space missions is critical for providing consistent nutritional and psychological benefits for the crew, potentially improving their health and performance. Three technology demonstrations were completed achieving simultaneous multi-species plant growth and the concurrent use of two Veggie units on the International Space Station (ISS). Microbiological characterization using molecular and culture-based methods was performed on leaves and roots from two harvests of three leafy greens, red romaine lettuce (Lactuca sativa cv. 'Outredgeous'); mizuna mustard, (Brassica rapa var japonica); and green leaf lettuce, (Lactuca sativa cv. Waldmann's) and associated rooting pillow components and Veggie chamber surfaces. Culture based enumeration and pathogen screening indicated the leafy greens were safe for consumption. Surface samples of the Veggie facility and plant pillows revealed low counts of bacteria and fungi and are commonly isolated on ISS. Community analysis was completed with 16S rRNA amplicon sequencing. Comparisons between pillow components, and plant tissue types from VEG-03D, E, and F revealed higher diversity in roots and rooting substrate than the leaves and wick. This work provides valuable information for food production-related research on the ISS and the impact of the plant microbiome on this unique closed environment.

Persistence of Escherichia coli in the microbiomes of red Romaine lettuce (Lactuca sativa cv. 'Outredgeous') and mizuna mustard (Brassica rapa var. japonica) - does seed sanitization matter?

BACKGROUND: Seed 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 sanitization on the plants' microbiome and pathogen persistence, sanitized and unsanitized seeds from two leafy green crops, red Romaine lettuce (Lactuca sativa cv. 'Outredgeous') and mizuna mustard (Brassica rapa var. japonica) were exposed to Escherichia coli and grown in controlled environment growth chambers simulating environmental conditions aboard the International Space Station. Plants were harvested at four intervals from 7 days post-germination to maturity. The bacterial communities of leaf and root were investigated using the 16S rRNA sequencing while quantitative polymerase chain reaction (qPCR) and heterotrophic plate counts were used to reveal the persistence of E. coli. RESULT: E. coli was detectable for longer periods of time in plants from sanitized versus unsanitized seeds and was identified in root tissue more frequently than in leaf tissue. 16S rRNA sequencing showed dynamic changes in the abundance of members of the phylum Proteobacteria, Firmicutes, and Bacteroidetes in leaf and root samples of both leafy crops. We observed minimal changes in the microbial diversity of lettuce or mizuna leaf tissue with time or between sanitized and unsanitized seeds. Beta-diversity showed that time had more of an influence on all samples versus the E. coli treatment. CONCLUSION: Our results indicated that the seed surface sanitization, a current requirement for sending seeds to space, could influence the microbiome. Insight into the changes in the crop microbiomes could lead to healthier plants and safer food supplementation.

A Microbial Monitoring System Demonstrated on the International Space Station Provides a Successful Platform for Detection of Targeted Microorganisms.

Closed environments such as the International Space Station (ISS) and spacecraft for other planned interplanetary destinations require sustainable environmental control systems for manned spaceflight and habitation. These systems require monitoring for microbial contaminants and potential pathogens that could foul equipment or affect the health of the crew. Technological advances may help to facilitate this environmental monitoring, but many of the current advances do not function as expected in reduced gravity conditions. The microbial monitoring system (RAZOR® EX) is a compact, semi-quantitative rugged PCR instrument that was successfully tested on the ISS using station potable water. After a series of technical demonstrations between ISS and ground laboratories, it was determined that the instruments functioned comparably and provided a sample to answer flow in approximately 1 hour without enrichment or sample manipulation. Post-flight, additional advancements were accomplished at Kennedy Space Center, Merritt Island, FL, USA, to expand the instrument's detections of targeted microorganisms of concern such as water, food-borne, and surface microbes including Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Escherichia coli, and Aeromonas hydrophilia. Early detection of contaminants and bio-fouling microbes will increase crew safety and the ability to make appropriate operational decisions to minimize exposure to these contaminants.

Fusarium oxysporum as an Opportunistic Fungal Pathogen on Zinnia hybrida Plants Grown on board the International Space Station.

A plant production system called Veggie was launched to the International Space Station (ISS) in 2014. In late 2015, during the growth of Zinnia hybrida cv. 'Profusion' in the Veggie hardware, plants developed chlorosis, leaf curling, fungal growth that damaged leaves and stems, and eventually necrosis. The development of symptoms was correlated to reduced air flow leading to a significant buildup of water enveloping the leaves and stems in microgravity. Symptomatic tissues were returned to Earth on 18 May 2016 and were immediately processed to determine the primary causal agent of the disease. The presumptive pathogen was identified as Fusarium oxysporum by morphological features of microconidia and conidiophores on symptomatic tissues; that is, by epifluorescent microscopy (EFM), scanning electron microscopy (SEM), metabolic microarrays, and ITS sequencing. Both EFM and SEM imaging of infected tissues showed that germinating conidia were capable of stomatal penetration and thus acted as the primary method for infecting host tissues. A series of ground-based pathogenicity assays were conducted with healthy Z. hybrida plants that were exposed to reduced-airflow and high-water stress (i.e., encased in sealed bags) or were kept in an unstressed configuration. Koch's postulates were successfully completed with Z. hybrida plants in the lab, but symptoms only matched ISS-flown symptomatic tissues when the plants were stressed with high-water exposure. Unstressed plants grown under similar lab conditions failed to develop the symptoms observed with plants on board the ISS. The overall results of the pathogenicity tests imply that F. oxysporum acted as an opportunistic pathogen on severely high-water stressed plants. The source of the opportunistic pathogen is not known, but virulent strains of F. oxysporum were not recovered from unused materials in the Veggie plant pillow growth units assayed after the flight.

Microbiological and Nutritional Analysis of Lettuce Crops Grown on the International Space Station.

The ability to grow safe, fresh food to supplement packaged foods of astronauts in space has been an important goal for NASA. Food crops grown in space experience different environmental conditions than plants grown on Earth (e.g., reduced gravity, elevated radiation levels). To study the effects of space conditions, red romaine lettuce, Lactuca sativa cv 'Outredgeous,' plants were grown in Veggie plant growth chambers on the International Space Station (ISS) and compared with ground-grown plants. Multiple plantings were grown on ISS and harvested using either a single, final harvest, or sequential harvests in which several mature leaves were removed from the plants at weekly intervals. Ground controls were grown simultaneously with a 24-72 h delay using ISS environmental data. Food safety of the plants was determined by heterotrophic plate counts for bacteria and fungi, as well as isolate identification using samples taken from the leaves and roots. Molecular characterization was conducted using Next Generation Sequencing (NGS) to provide taxonomic composition and phylogenetic structure of the community. Leaves were also analyzed for elemental composition, as well as levels of phenolics, anthocyanins, and Oxygen Radical Absorbance Capacity (ORAC). Comparison of flight and ground tissues showed some differences in total counts for bacteria and yeast/molds (2.14 - 4.86 log10 CFU/g), while screening for select human pathogens yielded negative results. Bacterial and fungal isolate identification and community characterization indicated variation in the diversity of genera between leaf and root tissue with diversity being higher in root tissue, and included differences in the dominant genera. The only difference between ground and flight experiments was seen in the third experiment, VEG-03A, with significant differences in the genera from leaf tissue. Flight and ground tissue showed differences in Fe, K, Na, P, S, and Zn content and total phenolic levels, but no differences in anthocyanin and ORAC levels. This study indicated that leafy vegetable crops can produce safe, edible, fresh food to supplement to the astronauts' diet, and provide baseline data for continual operation of the Veggie plant growth units on ISS.