BioNutrients: Microbial On-Demand Production of Short Shelf-Life Micronutrients in Space
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Nutrients↗
Engineering topics
Hiromi Kagawa
Publications and source records attributed to Hiromi Kagawa.
BioNutrients-1: Development of an On-Demand Nutrient Production System for Long-Duration Missions
Future long-duration missions beyond low-Earth orbit will require advances in food technologies to address the documented problem of degradation of vitamins and nutrients in supplied foods stored long-term. To begin to address the issue of nutrient degradation, we are developing and flight-testing a platform biomanufacturing technology for in situ production of target nutrients. This technology is being tested over a five-year duration on the International Space Station (ISS). As part of the BioNutrients-1 project we have developed an on-demand system for the production of two carotenoids, β-carotene and zeaxanthin, by genetically engineering distinct strains of Saccharomyces cerevisiae, more commonly known as baker’s yeast. The on-orbit nutrient production packs contain a desiccated yeast strain and edible growth substrate. Once hydrated, the contents of the production packs are intended to grow and produce a desired amount of ready-to-consume nutrients. In this current version the production packs will not be consumed and future missions will require an inactivation of microorganisms before consumption. In addition to the on-orbit hydration of the production packs a series of valuable microorganisms are currently being stored in stasis packs on the ISS including probiotics organisms, bacterial strains used in yogurt production, and organisms with potential use for future biomanufacturing. Analysis of returned ISS stasis packs and ground controls will include multi-omics studies and provide insight into long-term survival of organisms stored in a space environment. Both stasis packs and hydrated production packs will be intermittently returned to Earth for analysis. Preliminary data from long-term storage studies of stasis packs stored on the ISS for 47 days versus their ground control counterparts have shown no significant difference in viability. Currently no production packs have been processed.
Yeast↗
BioNutrients-2: Improvements to the BioNutrients-1 Nutrient Production System
The BioNutrients (BN) project aims to develop an on-demand nutrient production system capable of rapid and safe delivery of multiple nutrients in a single-use production pack for long-duration missions. The general concept of this system will be to develop an edible engineered microorganism capable of surviving during long-term storage, revival and growth of that microorganism for product production, and delivery of precise quantities of nutrients for human consumption. BN-1 is a five-year flight project currently on the International Space Station (ISS). It is testing the long-term storage of various microorganisms for the biomanufacturing of space relevant compounds and to validate the performance of the first generation (Gen-1) of production packs. The BN-2 project is a continuation of the BN-1 flight project and intends to expand the projects overall scope by introducing novel products, broadening the range of microbial food sources, and improving previously designed hardware from the BN-1 project. We are in the process of optimizing our platform biomanufacturing technology for in situ production of yogurt and kefir on the ISS. BN-2 will test the efficacy of this technology for future delivery of probiotic organisms which can be engineered to produce additional nutritional supplements (consumption by the crew is not planned in this project). Furthermore, to improve the feasibility of this technology for mission use we have transitioned from a hard-shell pack to a Gen-2, fluorinated ethylene propylene (FEP) bag. The single-use FEP bioreactor will facilitate growth of known organisms and ensure a contaminant free environment, while reducing mass and volume. These bioreactors will be employed for production of additional space relevant products in the BN-2 project. On-orbit testing of Gen-2 production packs will be necessary for future iterations, in order to optimize bag design and product production for eventual flight use by the crew.
BioNutrients↗
BioNutrients-2: Improvements to the BioNutrients-1 Nutrient Production System
The BioNutrients (BN) project aims to develop an on-demand biological production system capable of rapid and safe delivery of multiple nutrients in single-use production packs for long-duration missions. The general concept of this system is to develop edible engineered microorganisms that produce nutrients for human consumption following long-term storage, revival, and growth. BN-1 is a five-year flight project currently on the International Space Station (ISS). It is testing the long-term storage of various microorganisms for the biomanufacturing of space-relevant compounds and validating the performance of the first generation of production packs. The BN-2 project expands the BN-1 flight project scope by introducing novel products, broadening the range of microbial food sources, and improving upon production pack hardware. We are optimizing the biomanufacturing platform for in situ production of yogurt and kefir on the ISS. BN-2 also will test engineered microorganisms, including a probiotic strain, to produce carotenoids or follistatin. To improve the feasibility of this technology for mission use, we transitioned from a hard-shell pack to a second-generation fluorinated ethylene propylene (FEP) bag. The single-use FEP bioreactor facilitates growth of target organisms in a contaminant free environment, while reducing mass and volume compared to the BN-1 hardware. These bioreactors will be employed for production of additional space-relevant products in the BN-2 project. On-orbit testing of FEP production packs will enable optimization of the platform for eventual flight use by the crew.
Natalie Ball↗
BioNutrients: Microbial on-demand production of short shelf-life micronutrients in space
Long duration deep space missions pose a significant challenge when considering adequate nutrition for human spaceflight. Many micronutrients supplied in pre-packaged foods for low Earth orbit flights have a short shelf life and will degrade over multi-year missions. One strategy to mitigate the risk of micronutrient malnutrition is to grow fresh foods to supplement a pre-packaged diet. The BioNutrients project aims to develop systems for growing edible microorganisms that are genetically engineered to produce short shelf life micronutrients. BioNutrients-1 (BN-1) is a five-year spaceflight project currently underway on the International Space Station (ISS) that tests long-term storage and production of two carotenoids, β-carotene and zeaxanthin, in genetically engineered yeast strains. BN-1 on-orbit and ground control activities indicate that the nutrient production system will be stable for at least two years in ambient storage conditions. BN-1 is also testing the effects of long-term ISS storage on viability and gene expression using “stasis packs” containing a series of microorganisms useful in food and biomanufacturing processes. Stasis pack analysis is distinguishing microorganisms with full viability after two years of storage from other species that lose viability rapidly. In parallel, BioNutrients-2 (BN-2) is being developed as a six-month flight experiment that will expand the range of foods produced on ISS to include yogurt, kefir, and probiotic yeast. BN-2 will improve upon hardware design by transitioning from a hard-shell polycarbonate bioreactor to a flexible fluorinated ethylene propylene (FEP) bag. This change reduces the mass and volume required to store production packs. The long-term goal of BioNutrients is to develop the capability for rapid and safe delivery of multiple micronutrients in single-use production packs. We expect synthetic biology approaches similar to BioNutrients will play an important role for astronaut health in future NASA missions to the Moon and Mars.
Synthetic Biology↗
Bionutrients: Microbial Production of on-Demand Nutrients on the International Space Station
Providing adequate nutrition to crew members is essential as deep-space missions cannot rely on consistent Earth-based resupply. The current NASA pre-packaged food system is designed for low-earth-orbit missions with a stated shelf-life of two years and notable vitamin degradation over time. One strategy to mitigate nutrient loss is to implement bioregenerative food sources to supplement the pre-packaged food system. The BioNutrients project is designed to provide targeted production of short shelf-life nutrients in a single-use production pack. BioNutrients-1 includes two strains of edible yeast that have been genetically engineered to produce carotenoids, β-carotene, and zeaxanthin. This five-year mission on the International Space Station (ISS), launched in 2019, tests the long-term storage and nutrient production of the microorganisms of interest. Additional microorganisms are also stored in stasis packs to determine the effects of long-duration storage on the ISS. These organisms may be useful for production of fermented foods, pharmaceuticals, or biomanufacturing processes. Improving on the BioNutrients-1 project, BioNutrients-2 has optimized the production pack hardware by reducing the overall mass and volume of the system. Furthermore, BioNutrients-2 has expanded the projects scope by investigating novel products and microbial food sources. BioNutrients-2 is a six-month mission, launched in 2022, which tests production of fresh foods such as yogurt and kefir. The BioNutrients missions seek to provide advances for in-space biomanufacturing by addressing safe and reliable production of high-value nutrients and on-demand foods for future exploration efforts.
BioNutrients↗
Biomanufacturing in Space: BioNutrients and CO2-Based Manufacturing
Biomanufacturing can provide on-demand production of mission-critical compounds and materials to support long-duration space exploration while circumventing the challenges of transporting materials from Earth. Synthetic Biology Project is developing two biomanufacturing capabilities: BioNutrients and CO2-Based Manufacturing. BioNutrients is an ongoing mission aboard the International Space Station focused on the production of perishable nutrients in an on demand for direct for consumption. The first flight experiment of this project targeted the production of carotenoids: β-carotene and zeaxanthin, in recombinant yeast strains. Since then, the project has expanded to encompass the production of the fermented consumables like yogurt and kefir for use as a nutrient delivery mechanism. The CO2-Based Manufacturing system aims to use in situ resources to allow for biomanufacturing with minimal re-supply required. The manufacturing platform is combined with an electrochemical CO2 conversion system which can produce simple carbon substrates to support microbial based biomanufacturing. A comprehensive ground-based platform for recombinant protein purification is in development with the goal of producing a thermostable carbonic anhydrase enzyme from E. coli utilizing CO2-derived acetate. Our group hopes to propel advancements in space biomanufacturing for long duration space flight by harnessing the tools of synthetic biology.
Matthew Brian Paddock↗
Strategies for Determining Safety of Fermented Foods Produced in Space
The BioNutrients experiments examine the feasibility of using microbially produced foods – yogurt, kefir, fermented food products– to supplement the current prepackaged food system to provide freshly synthesized nutrients and genetically engineered protein therapeutics to support crew health for sustainable and long duration crewed exploration. Safety of the products must be established as direct consumption of such products is a risk due to potential contamination with pathogenic organisms. Identifying pathogen contamination is particularly challenging as the fermenting cultures can mask the presence of contaminating organisms. Here we consider current methods of detecting coliforms, aerobic colonies, non-lactic acid bacteria, molds (yeasts), and specific pathogens including Staphylococcus aureus and salmonella. We also consider alternate and additional testing including sequencing and quantitative PCR. We investigate the use of the E-Nose developed by NASA Ames Research Center. This portable device senses volatile organic compounds in real time and can be trained to recognize “good” uncontaminated products from contaminated ones as a means of predicting food safety. Finally, we consider methods of in-flight pasteurization to increase safety and decrease overall microbial load of these products. This presentation will summarize testing status and outline the strategy that will be employed in the BioNutrients-3 ISS fermented foods experiment.
Natalie Ball↗
Bionutrients: Microbial Production of on-Demand Nutrients on the International Space Station
Providing adequate nutrition to crew members is essential as deep-space missions cannot rely on consistent Earth-based resupply. The current NASA pre-packaged food system is designed for low-earth-orbit missions with a stated shelf-life of two years and notable vitamin degradation over time. One strategy to mitigate nutrient loss is to implement bioregenerative food sources to supplement the pre-packaged food system. The BioNutrients project is designed to provide targeted production of short shelf-life nutrients in a single-use production pack. BioNutrients-1 includes two strains of edible yeast that have been genetically engineered to produce carotenoids, β-carotene, and zeaxanthin. This five-year mission on the International Space Station (ISS), launched in 2019, tests the long-term storage and nutrient production of the microorganisms of interest. Additional microorganisms are also stored in stasis packs to determine the effects of long-duration storage on the ISS. These organisms may be useful for production of fermented foods, pharmaceuticals, or biomanufacturing processes. Improving on the BioNutrients-1 project, BioNutrients-2 has optimized the production pack hardware by reducing the overall mass and volume of the system. Furthermore, BioNutrients-2 has expanded the projects scope by investigating novel products and microbial food sources. BioNutrients-2 is a six-month mission, launched in 2022, which tests production of fresh foods such as yogurt and kefir. The BioNutrients missions seek to provide advances for in-space biomanufacturing by addressing safe and reliable production of high-value nutrients and on-demand foods for future exploration efforts.
BioNutrients↗
Strategies for Determining Safety of Fermented Foods Produced in Space
The BioNutrients experiments examine the feasibility of using microbially produced foods – yogurt, kefir, fermented food products– to supplement the current prepackaged food system to provide freshly synthesized nutrients and genetically engineered protein therapeutics to support crew health for sustainable and long duration crewed exploration. Safety of the products must be established as direct consumption of such products is a risk due to potential contamination with pathogenic organisms. Identifying pathogen contamination is particularly challenging as the fermenting cultures can mask the presence of contaminating organisms. Here we consider current methods of detecting coliforms, aerobic colonies, non-lactic acid bacteria, molds (yeasts), and specific pathogens including Staphylococcus aureus and salmonella. We also consider alternate and additional testing including sequencing and quantitative PCR. We investigate the use of the E-Nose developed by NASA Ames Research Center. This portable device senses volatile organic compounds in real time and can be trained to recognize “good” uncontaminated products from contaminated ones as a means of predicting food safety. Finally, we consider methods of in-flight pasteurization to increase safety and decrease overall microbial load of these products. This presentation will summarize testing status and outline the strategy that will be employed in the BioNutrients-3 ISS fermented foods experiment.
Food↗
Benchmarking Computational Tools for Calling SNPs and Indels in Complex Microbial Populations
The NASA BioNutrients missions seek to understand the suitability of microorganisms for bioproduction during space flight. One topic of interest is the stability of microbial genomes during long-term ambient storage and subsequent rehydration and growth. To address these questions, samples from 8 species were flown to ISS for 5 years of desiccated storage at ambient temperature (Stasis Packs) and 2 species were packaged along with powdered media inside a bioreactor system to allow hydration and growth in microgravity (Production Packs). For both systems, Whole Genome Sequencing (WGS) of the DNA extracted from the returned samples and paired ground controls will be conducted to identify changes in genome stability due to time, storage conditions and growth in space. Across the technical replicates, ground controls, 10 timepoints, and multiple experimental conditions, ~300 samples have been selected for initial analysis with WGS sequencing to 100x coverage. A flexible and resource efficient mutation calling pipeline is needed to process this large dataset and allow for comparisons between species. Many bioinformatics tools for calling Indels and Single Nucleotide Variants (SNVs) are designed for use with pure isolates, where true variations from the reference genome are expected to dominate the reads aligning to the location of mutation. In contrast, DNA from the Stasis Pack (SP) samples was collected directly after recovery from desiccated storage and the Production Pack (PP) samples were collected after fermentation. In this context, reads with mutations are expected to be less frequent than reads that align with the reference genome, as each sample will include multiple lines of cells. Thus, BioNutrients samples are expected to be similar to samples from cancer cell or “pooled” sequencing approaches. In preparation for the analysis of the BioNutrients samples, we have tested three mutation calling tools (GATK for Microbes, BreSeq and DiscoSNP) designed for complex samples. A challenge of validating mutation identification pipelines is a lack of “Ground Truth” datasets, especially for complex samples. To compare these three tools, we sought to identify mutations in pre-existing WGS data collected from populations of Chlamydomonas reinhardtii that were exposed to UV mutagenesis and growth in LEO as part of the Space Algae-1 mission. Here we present a summary of these tools against the analysis originally conducted using the CRISP tool. Critical metrics are compared such as runtime, the number of SNPs, the number and size of Indels, and patterns of transversion and transitions identified by each tool are reported. By sharing these benchmarking results collected in support of the BioNutrients mission, we aim to guide others seeking to identify SNVs in similarly complex microbial samples.
Biology↗
Developing a Genetic Variant Calling Pipeline for Quantifying the Complex Mutagenic Load Accumulated in BioNutrients-1 Production Pack Samples
Microorganisms hold great promise for on demand production of labile nutrients and pharmaceuticals as well recycling and in situ resource utilization. The utilization of microorganisms for such tasks on space missions is hindered by the limited data on how microbes respond to spaceflight. For example, the genetic stability of microorganisms, and the genomic engineered traits added to deliver desired functions, over long-term storage in the spacecraft environment is poorly understood. The BioNutrients-1 (BN-1) mission conducted a 5-year study of desiccated storage in Low Earth Orbit (LEO) to evaluate the suitability of eight synthetic biology chassis organisms for long-duration space missions. We are employing high-depth, whole genome sequencing (WGS) to determine the mutagenic load that accumulated during long-term storage. Mutation analysis pipelines are well established for homogenous culture grown from a single colony, but the mutational landscape of the BN-1 samples present a unique analysis challenge, as every cell in the BN-1 samples had a unique genetic journey of DNA damage and repair. Consequently, sequence variants are expected at low allele frequency within samples. To address this genetic complexity, we apply two distinct computational approaches to identify mutations in pre-existing WGS data collected from populations of Chlamydomonas reinhardtii that were exposed to UV mutagenesis and growth in LEO. For reference genome free mutation detection, we utilized DiscoSNP++, which is a de Bruijn graph approach. For reference genome-based mutation detection we utilize GATK for Microbes, which is a Bayesian probabilistic approach. We will benchmark these approaches against the mutations originally identified using CRISP, a method optimized for pooled samples. Ultimately, quantifying the mutation load imposed by storage or growth on the ISS will help identify chassis organisms with both high levels of genome stability and viability, which are desirable traits for implementation of bioproduction in long-duration missions.
SNP↗
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↗
BioNutrients Bioreactors: Design and Materials Considerations for Synthetic Biology
The BioNutrients project aims to develop an on-demand biological production system capable of producing dietary nutrients and other products of interest. A key element of this system is the bioreactor itself, which needs to provide a suitable environment for growth of the selected microorganism(s). To support respiration of the organisms, the bioreactor should have gas-permeable elements to allow metabolically required gases (e.g., oxygen) to enter and byproduct gases (e.g., carbon dioxide) to escape. For systems utilizing yeast for precision fermentation, the system also needs to prevent or minimize the release of ethanol, which has impacts on the Environmental Control and Life Support System (ECLSS). Additionally, the bioreactor must include interfaces for hydrating the microorganism(s), sampling, and crew interface. For experiments on the International Space Station (ISS), the bioreactor must also comply with ISS-levied offgassing, cold stowage, and other safety requirements. This presentation aims to explore these design considerations, using the examples of the bioreactors used for the BioNutrients ISS payload series.
vitamins↗
BioNutrients-3: Precision Fermentation, Pasteurization, and Pathogen Detection - Towards Safe Fermentation and Production of Nutrients in Space
The BioNutrients (BN) project is developing a microbial manufacturing approach to supplement the NASA food system to address known nutrient degradation associated with long-term storage. BN uses synthetic biology to deliver high-value nutrients and therapeutics through genetic engineering of microbes and production of fermented food products like yogurt and kefir, in a fully dehydrated system. On-demand production of nutrients for human consumption requires rigorous safety protocols to ensure contaminants are not introduced during the fermentation process. The third iteration of the BN flight project, BN-3, further develops the BN project by investigating strategies for pathogen detection and pasteurization in microgravity. BN-3 will test the limits of pathogen detection using whole genome sequencing, standard microbial assays and the NASA Ames E-Nose, a volatile carbon nano tube-based sensor array, to detect unwanted microbes. BN-3 also expands on the number of nutrients produced in a single bioreactor to enhance efficiency of the system by combining production of B vitamins in conjunction with the carotenoids, beta-carotene and zeaxanthin. Serial production of food products through yogurt passaging, new updates to the fluorinated ethylene propylene bags to allow crew access via a straw, as well as use of a food safe pH indicator dye to indicate readiness of the food product, will further enhance the useability of this system. This presentation will provide status of the BN-3 flight project with the aim of advancing in-space biomanufacturing for on-demand microbially based food production for future space exploration.
Biomanufacturing↗
Bionutrients-1: Utilizing Genomics and Transcriptomics to Assess the Reliability of Microorganisms for In Situ Nutrient Production on Long Duration Missions
The resupply of current long-duration crewed missions to the ISS relies on ground-launched supplies. As NASA looks toward Mars, ground-based resupply will no longer be an option. Critical nutrients, including vitamin C, vitamin K, folate, and thiamin, degrade during long-term storage, and regular consumption of these nutrients is essential for astronaut health. Another challenge of current food systems is the difficulty of consuming sufficient calories when subsisting on the limited flavors of freeze-dried food, which can lead to weight loss. The inclusion of microorganism-based food systems could alleviate both concerns. For example, the fermentation of rehydrated milk into yogurt with microorganisms genetically incorporating genes to produce critical vitamins would allow for both in situ production of nutrients and a fresh food product with additional flavor profiles. In comparison to plant food production, microorganisms require less flight infrastructure. The BioNutrients-1 mission is demonstrating viability of microbial fermentation food production in microgravity and testing the reliability of this approach for long-duration missions lacking resupply. While the BioNutrients-1 mission includes the collection of multiple phenotypic measurements, this status update will focus on the processing of samples for genomics and transcriptomics analyses as well as the planned analysis pipelines. First, the BioNutrients-1 mission seeks to identify microorganisms capable of surviving long-duration storage at ambient temperatures while maintaining genetic fidelity. To achieve this, nine commonly employed microbial species were stored at ambient temperatures in Stasis Packs on the ISS for five years. The viability and mutation rates will be measured at multiple time points for both flown and ground control samples. From an omics perspective, the changes in the bulk rates of point mutations and genetic rearrangements across the Stasis Pack species during the five years of storage will be determined, providing valuable insights into the potential of these microorganisms for long-duration space missions. Second, the BioNutrients-1 mission is characterizing the impact of microgravity on fermentation. Two strains of the yeast Saccharomyces cerevisiae, each encoding antioxidants (β-carotene or zeaxanthin) were flown to ISS for storage and fermentation within simplified bioreactors (Production Packs). The impact of microgravity on the expression of the antioxidant production genes and general metabolic genes will be determined using RNA sequencing. Ultimately, the transcriptome data will be compared to phenotypic measurements, such as the antioxidant yield, end-state biomass, and the production of EtOH, to determine the impacts of microgravity and long-term storage on microbial fermentation. The findings from this research will be instrumental in understanding the challenges and opportunities of microorganism-based food systems in space missions.
BioNutrients↗