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115 records · Page 7

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.↗

Microbiological Horticultural Internship Final Abstract

GMO dwarf plum (Prunus domestica) is being evaluated as a candidate food crop for long duration space flight missions. A project was undertaken to develop a protocol for transferring selected genetic lines of GMO plum (previously maintained in pots and propagated by cuttings at NASA's Kennedy Space Center in Florida) into in vitro tissue culture. In vitro culture may reduce the space, materials, and labor required to maintain the current lines of GMO plum and better preserve them for future study. Fresh plant material from three selected GMO plum lines (NASA-5, NASA-10, and NASA-11) and a non-modified control line (Control-5) were processed aseptically into in vitro culture on four separate occasions. The impact of multiple treatments on the successful growth of GMO plum tissue in vitro were tested: Parent explant tissue type (leaf petioles, stem nodes containing buds and internodes without buds), tissue sterilization method [soaking in 10 bleach only (5 min for petioles or 10 min for nodesinternodes), or soaking in 70 EtOH (30 sec) followed by 10 bleach (5 min for petioles and 10 min for nodesinternodes)], and media type [three Murashige and Skoog-based medias (SGM, SRM, and SRM+2,4-D) and one recipe containing woody plant media (WPM)]. 22.2 of the plates containing tissue sterilized with bleach alone developed microbial contamination after two weeks, while only 11.8 of plates containing tissue sterilized sequentially with EtOH and bleach developed contamination. Node bud tissue from all four genetic lines of plum produced leafy plantlets on SGM and SRM media after 4-6 weeks. The most numerous and well-developed plantlets were present on SGM. Upon reaching suitable size, plantlets were transferred to larger media containers for further growth. Some node bud growth occurred on SRM+2,4-D and WPM 2.5 weeks after plating, however as of yet no pieces on SRM+2,4-D have adequate development for transferring. Tissue pieces from NASA-5 plated on WPM are developing leaves and will be ready for transferring soon. Petioles and internode tissue lacking bud meristem failed to produce any plantlets on any plates, however petioles developed large masses of undifferentiated callus tissue on SRM+2,4-D media. These callused pieces were then transferred to SRM+TDZ media, which resulted in even larger callus growth but no differentiation. All four selected plum lines were successfully transitioned into in vitro culture. Nodes from NASA-5 and NASA-10 lines produced the most numerous and well-developed leafy plantlets in vitro, while those from NASA-11 and Control-5 were generally smaller, slower growing and less numerous. The best method overall was to use young stem node tissue with buds, surface sterilize the pieces sequentially with 70 EtOH and 10 bleach, and then plate them onto SGM media. Future areas of study will include introducing additional genetic lines of GMO plum into in vitro culture, attempting to induce shoot growth in petiole callus tissue, testing methods (such as cold storage) that extend the time interval between transferring explants into new media, and testing viability of plantlets transferred from in vitro culture back to traditional pot culture.

bioregenerative life support↗

Heat Melt Compaction as an Effective Treatment for Eliminating Microorganisms from Solid Waste

One of the technologies being tested at Ames Research Center as part of the logistics and repurposing project is heat melt compaction (HMC) of solid waste to reduce volume, remove water and render a biologically stable and safe product. Studies at Kennedy Space Center have focused on the efficacy of the heat melt compaction process for killing microorganisms in waste and specific compacter operation protocols, i.e., time and temperature, required to achieve a sterile, stable product. The work reported here includes a controlled study to examine the survival and potential re-growth of specific microorganisms over a 6-month period of storage after heating and compaction. Before heating and compaction, ersatz solid wastes were inoculated with Bacillus amyloliquefaciens and Rhodotorula mucilaginosa, previously isolated from recovered space shuttle mission food and packaging waste. Compacted HMC tiles were sampled for microbiological analysis at time points between 0 and 180 days of storage in a controlled environment chamber. In addition, biological indicator strips containing spores of Bacillus atrophaeus and Ceo bacillus stearothermophilus were imbedded in trash to assess the efficacy of the HMC process to achieve sterilization. Analysis of several tiles compacted at 180 C for times of 40 minutes to over 2 hours detected organisms in all tile samples with the exception of one exposed to 180 C for approximately 2 hours. Neither of the inoculated organisms was recovered, and the biological indicator strips were negative for growth in all tiles indicating at least local sterilization of tile areas. The findings suggest that minimum time/temperature combination is required for complete sterilization. Microbial analysis of tiles processed at lower temperatures from 130 C-150 C at varying times will be discussed, as well as analysis of the bacteria and fungi present on the compactor hardware as a result of exposure to the waste and the surrounding environment. The two organisms inoculated into the waste were among those isolated and identified from the HMC surfaces indicating the possibility of cross contamination.

Hummerick, Mary P.↗

Heat Melt Compaction as an Effective Treatment for Eliminating Microorganisms from Solid Waste

One of the technologies being tested at Ames Research Center as part of the logistics and repurposing project is heat melt compaction (HMC) of solid waste to reduce volume, remove water and render a biologically stable and safe product. Studies at Kennedy Space Center have focused on the efficacy of the heat melt compaction process for killing microorganisms in waste and specific compacter operation protocols, i.e., time and temperature required to achieve a sterile, stable product. The work. reported here includes a controlled study to examine the survival and potential re-growth of specific microorganisms over a 6-month period of storage after heating and compaction. Before heating and compaction, ersatz solid wastes were inoculated with Bacillus amyloliquefaciens and Rhodotorula mucilaginosa, previously isolated from recovered space shuttle mission food and packaging waste. Compacted HMC tiles were sampled for microbiological analysis at time points between 0 and 180 days of storage in a controlled environment chamber. In addition, biological indicator strips containing spores of Bacillus atrophaeus and Geobacillus stearothermophilus were imbedded in trash to assess the efficacy of the HMC process to achieve sterilization. Analysis of several tiles compacted at 180deg C for times of 40 minutes to over 2 hours detected organisms in all tile samples with the exception of one exposed to 180deg C for approximately 2 hours. Neither of the inoculated organisms was recovered, and the biological indicator strips were negative for growth in all tiles indicating at least local sterilization of tile areas. The findings suggest that minimum time/temperature combination is required for complete sterilization. Microbial analysis of tiles processed at lower temperatures from 130deg C-150deg C at varying times will be discussed, as well as analysis of the bacteria and fungi present on the compactor hardware as a result of exposure to the waste and the surrounding environment. The two organisms inoculated into the waste were among those isolated and identified from the HMC surfaces indicating the possibility of cross contamination.

Hummerick, Mary P.↗

The Future of Astronaut Nutrition: Daily Production of Kefir Nutrient Packs in a Lunar Analog Mission

Astronaut nutrition will need to be supplemented with fresh nutrients and probiotics for long duration space travel. Kefir is a fermented milk beverage that provides probiotic bacteria, protein, calcium, and vitamin K. It has been historically cultured for thousands of years and is a modern-day health food. During the BioNutrients-2 experiment, one generation of kefir was cultured by astronauts on the ISS in fluorinated ethylene propylene (FEP) bags. This follow-on experiment aimed to test the hypothesis that kefir can be produced at ambient temperatures and safely passaged to make a series of nutrient packs in an analog environment. The Hawaiian Space Exploration Analog and Simulation (HI-SEAS) is a semi-controlled habitat located on the Mauna Loa volcano on the island of Hawaii at 2,500 m elevation. The recent EMMIHS (EuroMoonMars, International MoonBase Alliance, HI-SEAS) missions comprised of interdisciplinary, international crews for mock lunar missions. During a six-day mission in March 2024, crew members tested continuous passaging of kefir cultures (C-FIR commercial strain). After approximately 24 hours of growth, a portion of culture was used to seed the next generation of culture bags for four passages. A color board and pH indicator allowed the crew to easily determine when the culture had reached optimal pH. Laboratory analysis at NASA Ames Research Center showed that the pH met Kefir standards (<5). The yeast and lactic acid bacteria were in normal range and no contamination was detected in the final passage. By successfully accomplishing this experiment the team has demonstrated the ability to safely produce daily cultures of kefir in a microgravity applicable growth system.

Astronaut nutrition↗

CESO 22-4: Parabolic and Suborbital Glovebox in Support of Space Crop Production

Space crop production research approaches and technologies can be validated for microgravity using parabolic or suborbital flight opportunities, and this will save considerable risk, time, and money for implementing new strategies in spaceflight. While the durations of microgravity in these tests are insufficient to grow crops, there are numerous examples of the value of microgravity testing for subsystems such as plant water and nutrient delivery, where multiphase fluid flow can be elucidated in short durations, and horticultural operations, such as harvesting and produce sanitation where containment and contamination can be assessed. Containment in any operational test is essential, as crop operations involve fluids and biological samples, which are potential hazards. This project consists of designing, developing, and constructing a parabolic/suborbital glovebox for experiment containment. The design involves modifications and upgrades to an existing glovebox developed at the University of Louisville. The University of Louisville glovebox was used by KSC researchers for space crop parabolic flight tests in 2021, and lessons learned from that testing have driven design modifications and improvements in the KSC-generated glovebox. Requirements were identified, parts were ordered, an operational science glovebox was fabricated, and a detailed materials specification list was generated. Analyses that are required for flight, remain to be performed to meet airworthiness requirements, and that work will have to be conducted in the future before use in flight.

Food Production↗

PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours. Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.

Venkateswaran, Kasthuri J.↗