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%
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].