Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates

Applied Microbiology and Biotechnology, Aug 2015

Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-products. We irradiated Y. lipolytica NRRL YB-567 with UV-C to enhance ammonia (for fertilizer) and lipid (for biodiesel) production on low-cost protein and carbohydrate substrates. The resulting strains were screened for ammonia and oil production using color intensity of indicators on plate assays. Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various substrates. Strains were identified among these mutants that had a faster doubling time, produced higher maximum ammonia levels (enzyme assay) and more oil (Sudan Black assay), and had higher maximum soluble protein levels (Bradford assay) than wild type. When grown on plates with substrates of interest, all mutant strains showed similar results aerobically to wild-type strain. The mutant strain with the highest oil production and the fastest doubling time was evaluated on coffee waste medium. On this medium, the strain produced 0.12 g/L ammonia and 0.20 g/L 2-phenylethanol, a valuable fragrance/flavoring, in addition to acylglycerols (oil) containing predominantly C16 and C18 residues. These mutant strains will be investigated further for potential application in commercial biodiesel production.

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Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates

Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates Mitch R. Lindquist 0 1 2 3 4 5 6 Juan Carlos López-Núñez 0 1 2 3 4 5 6 Marjorie A. Jones 0 1 2 3 4 5 6 Elby J. Cox 0 1 2 3 4 5 6 Rebecca J. Pinkelman 0 1 2 3 4 5 6 Sookie S. Bang 0 1 2 3 4 5 6 Bryan R. Moser 0 1 2 3 4 5 6 Michael A. Jackson 0 1 2 3 4 5 6 Loren B. Iten 0 1 2 3 4 5 6 Cletus P. Kurtzman 0 1 2 3 4 5 6 Kenneth M. Bischoff 0 1 2 3 4 5 6 Siqing Liu 0 1 2 3 4 5 6 Nasib Qureshi 0 1 2 3 4 5 6 Kenneth Tasaki 0 1 2 3 4 5 6 Joseph O. Rich 0 1 2 3 4 5 6 Michael A. Cotta 0 1 2 3 4 5 6 Badal C. Saha 0 1 2 3 4 5 6 Stephen R. Hughes 0 1 2 3 4 5 6 0 South Dakota School of Mines & Technology, Chemical and Biological Engineering , 501 East Saint Joseph Street, Rapid City, SD 57701-3995 , USA 1 National Coffee Research Centre - Cenicafe, National Federation of Coffee Growers of Colombia - FNC , Cenicafé Planalto Km 4 vía Antigua Chinchiná, Manizales, Caldas , Colombia 2 United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit , 1815 North University Street, Peoria, IL 61604 , USA 3 Mitsubishi Chemical, USMC Research & Innovation , 410 Palos Verdes Blvd, Redondo Beach, CA 90277 , USA 4 USDA, ARS, NCAUR, Bacterial Foodborne Pathogens and Mycology Research Unit , 1815 North University Street, Peoria, IL 61604 , USA 5 USDA, ARS, NCAUR, Bioenergy Research Unit , 1815 North University Street, Peoria, IL 61604 , USA 6 USDA, ARS, NCAUR, Bio-oils Research Unit , 1815 North University Street, Peoria, IL 61604 , USA Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-products. We irradiated Y. lipolytica NRRL YB-567 with UV-C to enhance ammonia (for fertilizer) and lipid (for biodiesel) production on low-cost protein and carbohydrate substrates. The resulting strains were screened for ammonia and oil production using color intensity of indicators on plate assays. Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer. - * Stephen R. Hughes substrates. Strains were identified among these mutants that had a faster doubling time, produced higher maximum ammonia levels (enzyme assay) and more oil (Sudan Black assay), and had higher maximum soluble protein levels (Bradford assay) than wild type. When grown on plates with substrates of interest, all mutant strains showed similar results aerobically to wild-type strain. The mutant strain with the highest oil production and the fastest doubling time was evaluated on coffee waste medium. On this medium, the strain produced 0.12 g/L ammonia and 0.20 g/ L 2-phenylethanol, a valuable fragrance/flavoring, in addition to acylglycerols (oil) containing predominantly C16 and C18 residues. These mutant strains will be investigated further for potential application in commercial biodiesel production. One of the major challenges facing commercial production of biofuels and bioproducts is cost-effective utilization, detoxification, and processing of biomass and other inexpensive carbon sources such as coffee and fruit processing wastes and other agricultural and food waste. The efficient conversion of lowcost substrates to advanced biofuels requires development of improved microbial catalysts (Hughes and Riedmuller 2014; Koutinas et al. 2014; Peralta-Yahya et al. 2012). Economic feasibility of biosynthetic fuel and chemical production depends on optimization of these biocatalysts to achieve high yields of the desired products. Saccharomyces cerevisiae is currently the most employed microbial catalyst in the biotechnology industry, but this yeast is limited in its range of substrates for producing fuel ethanol, and although genetic engineering has improved its utilization of the constituent pentose sugars of lignocellulosic materials, development of a recombinant S. cerevisiae strain capable of efficient pentose utilization remains a challenge (Casey et al. 2013; Garcia Sanchez et al. 2010; Hughes et al. 2009a, b; Kim et al. 2013a, b; Matsushika et al. 2014; Nielsen et al. 2013; Oreb et al. 2012; Zhou et al. 2012). Other microbial catalysts are being investigated for the production of biofuels and value-added bioproducts. One cand (...truncated)


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Mitch R. Lindquist, Juan Carlos López-Núñez, Marjorie A. Jones, Elby J. Cox, Rebecca J. Pinkelman, Sookie S. Bang, Bryan R. Moser, Michael A. Jackson, Loren B. Iten, Cletus P. Kurtzman, Kenneth M. Bischoff, Siqing Liu, Nasib Qureshi, Kenneth Tasaki, Joseph O. Rich, Michael A. Cotta, Badal C. Saha, Stephen R. Hughes. Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates, Applied Microbiology and Biotechnology, 2015, pp. 9723-9743, Volume 99, Issue 22, DOI: 10.1007/s00253-015-6852-2