Source: TEXAS A&M UNIVERSITY submitted to NRP
ADVANCING CELLULAR AGRICULTURE THROUGH GUT MICROBIAL COMMUNITIES FERMENTATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032609
Grant No.
2024-67017-43835
Cumulative Award Amt.
$596,727.00
Proposal No.
2023-10620
Multistate No.
(N/A)
Project Start Date
Sep 15, 2024
Project End Date
Sep 14, 2027
Grant Year
2024
Program Code
[A1364]- Novel Foods and Innovative Manufacturing Technologies
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
(N/A)
Non Technical Summary
Developing an innovative approach to establish a circular economy platform for lignocellulosic agricultural wastes and materials holds the potential to revolutionize alternative protein production while simultaneously addressing the pressing issues of waste reduction. Cellular agriculture combined with fermentation, two emerging and transformative fields, hold the potential to address these challenges by harnessing cellular and biotechnological breakthroughs to produce animal-derived products while circumventing traditional farming practices. This proposal presents a comprehensive strategy to propel cellular agriculture forward, focusing on the conversion of lignocellulosic wastes into postbiotics (include a wide range of beneficial substances such as short-chain fatty acids, enzymes, organic acids, and peptides) using gut microbial communities and generating microbial protein sources through methane-oxidizing bacteria. Our main goal is to develop new biomanufacturing technologies for the conversion of lignocellulosic agricultural feedstocks into valuable postbiotics and microbial proteins to support cell cultures. The successful outcomes from this proposed research would improve costs and reduce the environmental impact associated with fermentation and postbiotics production, through comprehensive objectives including Aim 1: Isolate, characterize and identify microbial communities from Bovine rumen, insects gut, and herbivorous fish species gut for utility in waste conversions; Aim 2: Transform lignocellulosic agricultural waste materials into postbiotics through the utilization of the gut microbial communities; Aim 3: Produce and apply microbial protein biomass by converting biogas (e.g. Carbon dioxide and methane) from Aim 2 to microbial biomass; Aim 4: Downstream processing of postbiotics and microbial protein and their utilization in cell culture media.
Animal Health Component
80%
Research Effort Categories
Basic
0%
Applied
80%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5011599103060%
5013320104040%
Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
3320 - Meat, beef cattle; 1599 - Grain crops, general/other;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology;
Goals / Objectives
Aim 1: Isolate, characterize and identify microbial communities from Bovine rumen, insects gut, and herbivorous fish species gut for utility in waste conversions; Aim 2: Transform lignocellulosic agricultural waste materials into postbiotics through the utilization of the gut microbial communities; Aim 3: Produce and apply microbial protein biomass by converting biogas (e.g. Carbon dioxide and methane) from Aim 2 to microbial biomass; Aim 4: Downstream processing of postbiotics and microbial protein and their utilization in cell culture media.
Project Methods
Gut collection. Selected organisms gut bacteria will be collected for DNA sequencing. Once removed, the guts will be immediately suspended in a physiological saline solution (PBS-buffer) and maintained on ice [28]. Once completed, gut samples will be frozen at −80°C. Gut collection from insects will be conducted over three months due to the size and the number of required samples for gut collection.Gut microbial community analysis. The gut will be homogenized with a tissue homogenizer for 4 min at 12,000 rpm. Identification and quantification of bacterial genus in samples will be performed at TxGen, the Texas A&M AgriLife Genomics and Bioinformatics Service. The microbiome total DNA will be extracted using the QIAamp DNA Microbiome Kit according to the manufacturer's protocol. The quality and concentration of the extracted DNA will be measured using a ND-1000 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States).Developing and characterizing postbiotics through gut microbial communities and cost-effective feedstock (Fermentation process) without pretreatment. Different feedstock and gut microbial communities (Table 1) will be used for fermentation and producing postbiotics.Table 1: Feedstock and microbial community sources for fermentation and producing postbiotics.Feedstock for digestionFeedstockAlfalfa; Corn byproducts; Brewers' spent grain, sorghum seeds, and byproductsMicrobial communities from the different animals for producing postbioticsInsect gut biomesBlack soldier fly (Hermetia illucens)Termite (Nasutitermes ephratae)Herbivorous fish specieTilapia (Oreochromis niloticus)BovineForegutFor fermentation, 10 mL of gut will be homogenized and will be directly added onto the feedstock and incubated in anaerobic conditions at 29°C (insects and tilapia) and 37°C (for cow) and 150 rpm for 24 h, under the anerobic chamber. During the anaerobic digestion, the condition will be achieved by using GasPak™ EZ Gas Generating Pouch Systems containing anaerobic blue/white indicators and double bagging each culture with plastic zip bags based on our developed method. Our previous studies indicated that there was no difference between established gut biome (cultured under the anerobic conditions on enriched media for three days), and direct application of gut biome for fermentation, on postbiotics quality. Harvest of the fermentation products and non-fermented controls will be performed by centrifugation of 5 min at 5,000 rpm, and sequential filtrations of the liquid phase through 100 μm, 70 μm and 40 μm cell strainers. Samples for analysis of bacterial communities will be taken after fermentation and DNA will be sequenced according to Task I. Fermentation will be conducted using freshly harvested microbial communities (109 CFU/mL), and same bacteria consortium will be used for fermentation of new batch, and this step will be repeated five times. Postbiotics and bacterial DNA will be collected from each step to study the microbiome shift during the fermentation after five times. PI, Ovissipour, will oversee the fermentation process.Aerobic biogas-oxidizing bacteria production using Aim 2 biogas. Two bacteria including methane-oxidizing (Methylococcus capsulatus) bacterium, and monoxide-oxidizing (Pseudomonas carboxydohydrogena) bacterium will be grown in an aerobic environment, using biogas as carbon source. These bacteria will be provided through American Type Culture Collection (ATCC). Nitrogen source will be provided for them using peptone water. Bacterial growth, and kinetics of growth will be monitored during the production. Bacteria will be harvested, inactivated using eBeam technology, and thermal processing, and will be freeze dried. Protein content, and functional properties (solubility), will be evaluated.Removing bacteria from postbiotics. We will employ a triad of distinct methodologies to achieve the sterilization of postbiotics, encompassing filtration, thermal processing, and Electron Beam technologies. Filtration will be conducted through a series of filtration from 100 µm, to 0.22 µm; thermal processing will be conducted at 70°C for 2 min (after come-up time), and samples will be cooled on iced-water immediately; We will also evaluate the impact of Electron Beam technology on postbiotics microbial inactivation through Texas A&M University National Center for Electron Beam Research. Treated samples using these three methods will be used for cell culture media formulation and will be used for growing cells to evaluate the impact of these three technologies on postbiotics metabolites. Furthermore, an in-depth analysis will encompass the evaluation of metabolites, chemical constituents, and microbial profiles on the treated samples. Filtration, and heat treatment will be led by PI, Ovissipour, and EBeam treatment, will be conducted by Co-PI, Pillai, the director of the EBeam Center at Texas A&M University.Cell culture and maintenance: Different cells lines including Zebrafish (Danio rerio) embryo fibroblast ZEM2S cell line obtained from ATCC (Manassas, VA, USA) [19]; Bovine satellite cell lines and Mackerel myogenic cells line, developed in Co-PI, Kaplan's lab at Tufts University will be studied. Cells will be grown in T75 flasks with 10 mL of cell maintenance media composed of Leibovitz L-15 media (L-15) with 10% fetal bovine serum (FBS) and 1% antibacterial-antimycotic. Aquatic cells will be incubated at 27°C without providing carbon dioxide, and Bovine cells will be incubated in a carbon dioxide incubator at 37°C. Subculturing will be performed when cells reached a confluency of 80-85% by removing media, rinsing cells with phosphate-buffered saline (PBS) and detaching cells from the flask surface with trypsin for 3-4 min. Seeding density for cell maintenance is approximately 10,000 cells/cm2. Cell growth and morphology will be routinely assessed using a phase-contrast microscope [19].?Cell differentiation study: Differentiation will occur using differentiation media, with media changes every three days for 3-4 weeks. After two weeks of treatment, cultures will be screened for myogenic related gene expression and immunocytochemistry every three days, as appropriate. Immunostaining for myosin heavy chain will be conducted following the protocol outlined in Saad et al. [21]. Following culture, cells will be fixed with 4% paraformaldehyde at room temperature for 30 minutes (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the cells will be rinsed with Phosphate Buffered Saline (PBS, Sigma Aldrich, Burlington, MA, USA) and permeabilized for 10 minutes using 0.1% Triton-X (Sigma Aldrich, Burlington, MA, USA). Subsequent to permeabilization, the cells will be blocked for 30 minutes using 1× blocking buffer (Abcam, Cambridge, UK), followed by an additional PBS wash. The primary antibody solution, MF-20 (4 μg/mL), will be applied to the cells and allowed to incubate overnight at 4°C. After a subsequent PBS wash, the cells undergo an additional 30-minute blocking step using 1× blocking buffer and were then incubated for 1 hour with secondary antibodies - Goat Anti-Mouse IgG H&L (Alexa Fluor® 594, Abcam, Cambridge, UK), and Phalloidin-iFluor 488 Reagent (Abcam, Cambridge, UK) - each diluted at 1:1000 in 1× blocking buffer. After a final wash with PBS, the cell nuclei will be stained with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL, Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 15 minutes at room temperature. Imaging will be conducted using a fluorescent microscope equipped with an LED Illumination system. Cell culture, proliferation, and differentiation, as well as media formulation, and adjusting, will be conducted at PI, Ovissipour, and Co-PI, Kaplan labs [19, 21].

Progress 09/15/24 to 09/14/25

Outputs
Target Audience:1. Academic Researchers and Students in Cellular Agriculture and Food Science Our project directly serves researchers and graduate students who are developing serum-free or cost-effective media formulations for cultivated seafood and meat. By integrating insights from the gut microbiome into media development, we provide a novel, biologically informed framework that can enhance cell growth, improve nutrient utilization, and reduce reliance on animal-derived components. This knowledge is highly relevant to scientists in cell culture, microbiology, biomanufacturing, and food systems engineering. The project also engaged students in laboratory instruction and experiential learning, providing hands-on exposure to innovative approaches in food biotechnology. 2. Cellular Agriculture Industry and Start-ups Emerging companies in the cultivated protein sector face significant bottlenecks in scaling production due to high media costs and reliance on undefined supplements. Our work targets this audience by generating science-based solutions that directly address cost, scalability, and food safety concerns. By aligning our research outputs with industry needs, we provide pathways to accelerate commercialization and broaden adoption of cultivated proteins in the U.S. and globally. These firms benefit from validated media optimization strategies that are informed by gut biome-derived nutrients and metabolites. 3. Agricultural Stakeholders and Producers Texas and U.S. agricultural producers are increasingly interested in biomanufacturing opportunities that complement traditional production systems. By valorizing agricultural inputs and connecting them to high-value cultivated protein markets, our work matters to farmers, producers, and cooperatives who are exploring diversification strategies in a rapidly evolving food system. This audience benefits from knowledge of how microbial metabolites and agricultural by-products may serve as functional media components, thus creating new value chains and market opportunities. 4. Regulatory and Food Safety Communities Because cellular agriculture products must ultimately be approved by regulatory bodies (USDA, FDA), our project provides foundational data relevant to safety and nutritional quality assessments. Gut biome-assisted media development contributes to understanding metabolic flux, potential contaminants, and nutritional equivalence, all of which are critical considerations for regulators. This audience matters because our outputs inform both the regulatory approval process and broader public trust in cultivated protein products. 5. Workforce Development and Educational Communities Future workforce capacity in cellular agriculture is limited, and our project plays an important role in training the next generation of scientists. Through laboratory instruction, mentoring, and workshops, we targeted undergraduate and graduate students in food science, nutrition, microbiology, and engineering. These efforts build technical expertise in advanced cell culture, metabolomics, and bioinformatics, positioning students for careers in academia, industry, or government service. 6. Extension and Public Outreach Audiences Public perception of cellular agriculture remains uncertain, and transparent communication is vital. We targeted extension professionals, K-12 educators, and community groups interested in sustainable food systems. By providing science-based knowledge through outreach events and workshops, we contributed to increasing awareness of cultivated protein technologies, their sustainability benefits, and their alignment with U.S. agricultural priorities. This audience matters because informed communities are more likely to support innovation, participate in workforce development, and engage in constructive dialogue about the future of food. Changes/Problems:1- Having access to specific organisms, and isolating the gut biome, specifically from insects. This challenge was addressedproperlyby engaging with insect farmers, and lamb slaughterhouse. A proper number of samples were collected, and the biotic samples were isolated. What opportunities for training and professional development has the project provided?1. Graduate Student Training Graduate students were directly involved in experimental design, laboratory research, and data analysis related to gut biome-assisted media optimization. They gained hands-on expertise in advanced cell culture, metabolomics, and proteomics, as well as training in experimental design of media formulations using statistical and AI-driven optimization approaches (RSM, ANN). This experience enhanced their technical and analytical skills and prepared them for careers in cellular agriculture and food biotechnology. 2. Undergraduate Experiential Learning Undergraduate students participated in structured laboratory rotations where they learned foundational techniques in sterile culture, metabolite extraction, and data handling. These experiences provided early exposure to research and innovation in cellular agriculture and supported workforce development in biomanufacturing. 3. Professional Development Through Workshops and Seminars Project team members and students attended interdisciplinary workshops and seminars related to cellular agriculture, microbial biotechnology, and sustainable food production. These events facilitated networking with industry stakeholders and regulatory representatives, strengthening participants' understanding of commercialization pathways, safety assessments, and broader industry needs. 4. Mentorship and Collaborative Training The project fostered a mentoring environment where senior researchers guided students in laboratory practices, grant writing, and manuscript preparation. This mentorship contributed to professional skill-building in communication, project management, and career planning. 5. Skill Development in Data Science and Bioinformatics Students were introduced to computational tools for analyzing multi-omics datasets derived from gut biome research. Training in bioinformatics pipelines, statistical modeling, and visualization enhanced their ability to interpret large-scale biological data, a critical skill for modern biomanufacturing research. 6. K12 education on biomanufacturing Trainings were provided under the Summer Bootcamp for 12 K-12 students with special needs in Houston. How have the results been disseminated to communities of interest?We have several manuscriptsunder review and under preparation, additionally we attended several conferences, and organizeda symposium. Under Review/In Preparation Peer-reviewed Articles Okehie, I. D., Riaz, M. N., Pillai, S., Ovissipour, R. (2025). Enhanced nutritional quality, digestibility, and flavor of Grasshopper through Solid State Fermentation. Under Review, Scientific Reports. Pakbin, B., Ovissipour, R. (2025). Postbiotics as a Functional Supplement to Develop Cell Culture Media for Cultivated Meat Production. In Preparation. Rease, M., Thilakarathna, W., Pillai, S., Ovissipour, R. (2025). Multi-Organ Approaches to Cultivated Meat Biomanufacturing: Conceptual Applications of Ruminal Fermentation and Co-cultures. In Preparation. Conferences-Graduate Students as the Presenter 1.BEYOND BUGS: EXPLORING EDIBLE INSECTS AS SUSTAINABLE ALTERNATIVES FOR FUTURE FOOD SYSTEMS - 2024 IFANCA-Food Diversity Innovation Program Symposium on "Alternative Proteins" - October 2024 - Texas A&M - Poster 2. Enhancing the nutritional and functional properties of grasshoppers through traditional fermentation methods - Insect plus 2025 - May 2025 - Cloppenburg, Germany - Oral Keynote/Invited Speaker-Reza Ovissipour 2024-30-10, Cellular Agriculture: Transforming Protein Production for a Sustainable Future, Food Diversity symposium on "Alternative Protein", Texas A&M University, College Station, TX, Role: Keynote Speaker, National. 2025-09-06, The Role of Cellular Agriculture in Food Production for Space Exploration, In Vitro Biology Conference,Norfolk, VA, Role: Invited Speaker, National. 2025-03-04, Cellular Agriculture and Biomanufacturing, Penn State University Seminar, State College, PA, Role: Invited Speaker, National. Symposium and Workshops Organized by PI, Ovissipour 2024-19-09, Cellular Agriculture and Food Biomanufacturing Symposium, Texas A&M University, College Station, TX, Role: Organizer, National. What do you plan to do during the next reporting period to accomplish the goals?1- Metagenomics analysis of the gut biome isolated from target organisms, 2- Metabolomicsanalysis of the metabolites generated with these biome grown on different waste, 3- Developing cell culture media for target cells.

Impacts
What was accomplished under these goals? Aim 1: Gut biome was isolated from five different insects, and lamb rumen. Metagenomics analyseswere conducted, data is under processing for developing manuscriptsand use for furtheranalysis as well as metabolomicsproduction. Aim 2: Different waste streams including insects, corn stover were prepared and will be used for biome-assisted fermentation. Aim 3: Microbial biomasses were generated, and lysates were producedfrom targeted microorganisms and were tested as media supplements. These following manuscriptshave been prepared so far: Under Review/In Preparation Peer-reviewed Articles Okehie, I. D., Riaz, M. N., Pillai, S., Ovissipour, R. (2025). Enhanced nutritional quality, digestibility, and flavor of Grasshopper through Solid State Fermentation. Under Review, Scientific Reports. Pakbin, B., Ovissipour, R. (2025). Postbiotics as a Functional Supplement to Develop Cell Culture Media for Cultivated Meat Production. In Preparation. Rease, M., Thilakarathna, W., Pillai, S., Ovissipour, R. (2025). Multi-Organ Approaches to Cultivated Meat Biomanufacturing: Conceptual Applications of Ruminal Fermentation and Co-cultures. In Preparation. Conferences-Graduate Students as the Presenter 1.BEYOND BUGS: EXPLORING EDIBLE INSECTS AS SUSTAINABLE ALTERNATIVES FOR FUTURE FOOD SYSTEMS - 2024 IFANCA-Food Diversity Innovation Program Symposium on "Alternative Proteins" - October 2024 - Texas A&M - Poster 2. Enhancing the nutritional and functional properties of grasshoppers through traditional fermentation methods - Insect plus 2025 - May 2025 - Cloppenburg, Germany - Oral Keynote/Invited Speaker-Reza Ovissipour 2024-30-10, Cellular Agriculture: Transforming Protein Production for a Sustainable Future, Food Diversity symposium on "Alternative Protein", Texas A&M University, College Station, TX, Role: Keynote Speaker, National. 2025-09-06, The Role of Cellular Agriculture in Food Production for Space Exploration, In Vitro Biology Conference, Norfolk, VA, Role: Invited Speaker, National. 2025-03-04, Cellular Agriculture and Biomanufacturing, Penn State University Seminar, State College, PA, Role: Invited Speaker, National. Symposium and Workshops Organized by PI, Ovissipour 2024-19-09, Cellular Agriculture and Food Biomanufacturing Symposium, Texas A&M University, College Station, TX, Role: Organizer, National.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: BEYOND BUGS: EXPLORING EDIBLE INSECTS AS SUSTAINABLE ALTERNATIVES FOR FUTURE FOOD SYSTEMS - 2024 IFANCA-Food Diversity Innovation Program Symposium on Alternative Proteins - October 2024 - Texas A&M - Poster
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Enhancing the nutritional and functional properties of grasshoppers through traditional fermentation methods - Insect plus 2025 - May 2025 - Cloppenburg, Germany  Oral
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Cellular Agriculture: Transforming Protein Production for a Sustainable Future, Food Diversity symposium on Alternative Protein, Texas A&M University, College Station, TX, Role: Keynote Speaker, National.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Keynote Speaker: The Role of Cellular Agriculture in Food Production for Space Exploration, In Vitro Biology Conference, Norfolk, VA, Role: Invited Speaker, National.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Invited Speaker: Cellular Agriculture and Biomanufacturing, Penn State University Seminar, State College, PA, Role: Invited Speaker, National.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Organized Symposium by PI, Ovissipour Cellular Agriculture and Food Biomanufacturing Symposium, Texas A&M University, College Station, TX, Role: Organizer, National.