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At least 19 records

Neurospora intermedia from a traditional fermented food enables waste-to-food conversion

AbstractFungal fermentation of food and agricultural by-products holds promise for improving food sustainability and security. However, the molecular basis of fungal waste-to-food upcycling remains poorly understood. Here we use a multi-omics approach to characterize oncom, a fermented food traditionally produced from soymilk by-products in Java, Indonesia. Metagenomic sequencing of samples from small-scale producers in Western Java indicated that the fungus Neurospora intermedia dominates oncom. Further transcriptomic, metabolomic and phylogenomic analysis revealed that oncom-derived N. intermedia utilizes pectin and cellulose degradation during fermentation and belongs to a genetically distinct subpopulation associated with human-generated by-products. Finally, we found that N. intermedia grew on diverse by-products such as fruit and vegetable pomace and plant-based milk waste, did not encode mycotoxins, and could create foods that were positively perceived by consumers outside Indonesia. These results showcase the traditional significance and future potential of fungal fermentation for creating delicious and nutritious foods from readily available by-products.

59 BASIC BIOLOGICAL SCIENCES

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

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

Fermented Foods Microbial Genomes Database

This database contains ~4,300 microbial genomes assembled from diverse fermented foods. These genomes were obtained from a larger set of 13,850 microbial genomes by clustering them at 99% average nucleotide identity (ANI) to create a "species"-representative database.

59 BASIC BIOLOGICAL SCIENCES

Harnessing Synthetic Biology for Biomanufacturing in Space: ​from Fermented Foods to Industrial Enzymes

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.

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

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

Synthetic Biology to Support Human Exploration of Deep Space

The International Space Station (ISS) has enabled a continuous human presence in space since November 2000. The ISS is in low Earth orbit, facilitating regular resupply missions to deliver air, water, food, spare parts, and science experiments. NASA’s Moon to Mars campaign seeks to return humans to the Moon and prepare for crewed missions to Mars. Increased distance from Earth poses logistical challenges to provide all resources needed by humans for deep space missions. NASA Ames Research Center is conducting a series of synthetic biology projects to test the use of microbes for on-demand biosynthesis of human micronutrients. The BioNutrients spaceflight experiments test an implementation concept to produce fermented food products in which microbe growth enhances micronutrient content. On-demand production of carotenoids was engineered into two yeast (Saccharomyces cerevisiae) strains that have been tested in nearly 5 years of storage. One of the BioNutrients strains will be incorporated into the Lunar Explorer Instrument for Space Biology Applications (LEIA) project. LEIA is developing an instrument suite to be delivered to the lunar south pole region by the Commercial Lunar Payload Services (CLPS) program. The LEIA instrument suite will be used to measure multiple yeast strains for growth, metabolic activity, and synthetic biology-enabled production of carotenoids, while taking real time measurements of biologically relevant radiation exposure on the lunar surface. LEIA data will be used to assess the impact of lunar surface radiation and reduced gravity on the production of engineered traits.

Synthetic Biology

Reference-free structural variant detection in microbiomes via long-read co-assembly graphs

Motivation: The study of bacterial genome dynamics is vital for understanding the mechanisms underlying microbial adaptation, growth, and their impact on host phenotype. Structural variants (SVs), genomic alterations of 50 base pairs or more, play a pivotal role in driving evolutionary processes and maintaining genomic heterogeneity within bacterial populations. While SV detection in isolate genomes is relatively straightforward, metagenomes present broader challenges due to the absence of clear reference genomes and the presence of mixed strains. In response, our proposed method rhea, forgoes reference genomes and metagenome-assembled genomes (MAGs) by encompassing all metagenomic samples in a series (time or other metric) into a single co-assembly graph. The log fold change in graph coverage between successive samples is then calculated to call SVs that are thriving or declining. Results: We show rhea to outperform existing methods for SV and horizontal gene transfer (HGT) detection in two simulated mock metagenomes, particularly as the simulated reads diverge from reference genomes and an increase in strain diversity is incorporated. We additionally demonstrate use cases for rhea on series metagenomic data of environmental and fermented food microbiomes to detect specific sequence alterations between successive time and temperature samples, suggesting host advantage. Our approach leverages previous work in assembly graph structural and coverage patterns to provide versatility in studying SVs across diverse and poorly characterized microbial communities for more comprehensive insights into microbial gene flux.

59 BASIC BIOLOGICAL SCIENCES

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

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

Innovative Polyhydroxyalkanoates (PHA) Production with Microbial Electrochemical Technology (MET)

The project “Innovative Polyhydroxyalkanoates (PHA) Production with Microbial Electrochemical Technology (MET)” addressed food waste disposal challenges by successfully converting food waste to bioplastics (known as PHAs). The novel process created by our team of researchers from universities, national labs, and industry substantially enhanced overall carbon conversion efficiency of food waste processing (> 50%), while reducing disposal costs (> 25%). The project showed economic viability potential at community scale through pilot-scale demonstration at a relevant scale (50 L reactor volume) with more than 100 hours of PHA production using realistic conditions. The project goal was to valorize food waste by shunting traditional anaerobic digestion processing and creating a value-added PHA processing route that improves the economics and sustainability of local, community-scale, wet organic waste treatment. First, the food waste undergoes microbial-based, dark fermentation to break down the food to small carbon chains known as volatile fatty acids (VFAs). Instead of microorganisms converting the VFAs into methane using normal anaerobic digestion processing, our innovative process inhibits methane production. This preserves the produced VFAs for extraction and use by a novel Haloferax mediterranei (HM) archaea, which effectively converts the VFAs to bioplastics. The project added microbial electrochemical cells (MEC) to the dark fermentation process to enhance the VFAs produced and optimize the type of bioplastics formed.

36 MATERIALS SCIENCE

Hybrid biological-chemical strategy for converting polyethylene into a recyclable plastic monomer using engineered Corynebacterium glutamicum

Converting polyethylene (PE) into valuable materials, particularly ones that are better for the environment than the incumbent plastics, not only helps mitigate environmental issues caused by plastic waste but also alleviates the long-standing problem of microbial fermentation competing with food supplies. However, the inherent robustness of PE due to its strong carbon-carbon bonds and high molecular weight necessitates harsh decomposition conditions, resulting in diverse decomposition outcomes that present significant challenges for downstream applications, especially for bioconversion. In this study, we demonstrate a hybrid biological-chemical conversion process for PE, converting its decomposition products, namely short-chain diacids, into a monomer, β-keto-δ-lactone (BKDL), for highly recyclable polydiketoenimine plastics using engineered Corynebacterium glutamicum. Since BKDL synthesis requires a substantial supply of malonyl-CoA, we employed an alternative biosynthesis pathway that leverages C. glutamicum's natural proficiency in amino acid production. We optimized this pathway in vivo by minimizing carbon loss to CO2 and byproducts, improving the transporter system, and maximizing co-factor regeneration. Furthermore, we co-optimized the PE deconstruction process to produce predominantly C4 to C6 diacids and integrated three catabolic pathways into the engineered strain to enhance diacid utilization, maximizing the carbon conversion from PE. Finally, an engineered polyketide synthase was introduced into C. glutamicum to enable BKDL synthesis. This work demonstrates the potential of a chemo-biological hybrid strategy for recycling plastic waste, highlighting its promise in addressing environmental challenges and promoting sustainable materials.

Zhan, Chunjun

Technoeconomic and Life Cycle Analysis of an Integrated Fermentation and Microbial Electrochemical Process for Volatile Fatty Acid Production from Food Waste

Techno-economic analysis (TEA) and life cycle assessment (LCA) were conducted for an integrated system designed for the production of volatile fatty acid (VFA) from food waste. The TEA estimated a production cost of $\$$3.12/kg VFA, and the LCA predicted negative greenhouse gas (GHG) emissions of -0.4 kg CO 2 e/kg VFA, driven primarily by diverting organic waste from landfills and avoiding methane emissions while producing valuable chemical products. Hotspot analysis showed arrested methanogenesis (AM) fermentation as the largest contributor to costs (37%) and environmental burden (47%), driven by high sodium hydroxide (NaOH) consumption. Distillation and microbial electrosynthesis (MES) units were the next-largest environmental contributors (28% and 18%). Major cost drivers also included residuals management (biosolids and wastewater) and the equipment and operating costs for AM, MES, and sonication pretreatment units. Although the new integrated system is environmentally benign, its costs and environmental impacts can be further reduced by integrating alternative energy sources, minimizing chemical and energy inputs through process optimization, and improving efficiency. In conclusion, this work highlighted the viability of waste-derived VFA production and provided a clear, data-driven strategy to accelerate the commercialization of waste valorization technology.

Carboxylic Acid Production

Upstream considerations for gas fermentation processes

Gas fermentation enables the production of fuels, chemicals, and foods from gaseous carbon sources and could serve as a technology for valorizing carbon that may otherwise be emitted to the atmosphere. In this review, we focus on upstream feedstock considerations: the supply of carbon and the supply of electrical power. Electrical power serves a dual role, providing both process energy and biochemical redox potential (via hydrogen or reduced intermediates). We define gas fermentation as bioprocesses involving gaseous feedstocks metabolized by microbes, distinct from microbial electrosynthesis. Trends in CO 2 point sources and low-carbon electricity systems are analyzed, highlighting opportunities and challenges for future deployment. This review synthesizes current knowledge and identifies key R&D priorities for process integration at industrial scale.

09 BIOMASS FUELS

Techno-economic and life cycle analysis of bio-hydrogen production using bio-based waste streams through the integration of dark fermentation and microbial electrolysis

Hydrogen derived from bio-based sources, or biohydrogen (bioH 2 ), has the potential to reduce GHG emissions from industrial and transportation sectors, owing to the low carbon footprint and myriad applications like refinery operation, ammonia production, steel production, fuel cell, etc. To evaluate the commercialization potential of bioH 2 production, we modeled bioH 2 production and conducted techno-economic analysis (TEA) and life cycle analysis (LCA) of two facilities producing 50 metric tonnes of bioH 2 per day from cheese whey (CW) and solid food waste (SFW) through the integration of dark fermentation (DF) and microbial electrolysis cell (MEC) technologies. LCA results showed that CW and SFW can produce carbon-negative bioH 2 , with emissions of −8.6 and −8.0 kg GHG kg −1 bioH 2 with carbon sequestration and renewable electricity resources, respectively, making bioH 2 potentially eligible for a tax credit of $\$3$ kg −1 H 2 based on provision 45 V of the U.S. Inflation Reduction Act (IRA). In this study, bioH 2 production treats waste streams to generate fresh water, thus, potentially can receive waste water treatment fee that varies with regions. The MEC capital cost dominates the bioH 2 cost, which is mainly determined by current density. With a current density of 20 A m −2 , the production cost for CW input varied between $\$17$ and $\$24$ kg −1 bioH 2 , while that for SFW input ranged from $\$29$ to $\$30$ kg −1 bioH 2 under different operating conditions, considering the 45 V tax credit, waste water treatment fee and production revenue. If the current density increases to 100 A m −2 , the bioH 2 cost decreases to a range of $\$4.0$–$\$6.9$ for CW and $\$5$–$\$6$ for SFW scenarios. This study also shows that low-cost bioH 2 can be produced using CW waste stream as feedstock.

Ganguly, Arna [Argonne National Laboratory (ANL),