Progress 09/01/08 to 08/31/10
Outputs
OUTPUTS: This project seeks to develop new processing technologies that underpin advanced biofuels from corn and soybean grain as well as lignocellulosic crops and crop residues. New more cost-effective conversion technologies are needed to meet the 2007 Energy Security and Independence Act, which mandates nearly 25% of today's motor fuels (36 billion gallons) be provided from biorenewable resources by 2022. We are contributing towards this goal by developing fungal fermentation of thin stillage, a byproduct produced by dry-grind ethanol plants, to produce value-added feed products; using oleaginous yeast to convert waste glycerol from biodiesel plants to single cell oil for additional biodiesel; using waste glycerol as feedstock for omega-3 fatty acids accumulating yeasts to improve nutrition of bovine milk; and using fungal processes to produce biodiesel from lignocellulose. This project focuses on refining, scaling-up and demonstrating these technologies. To meet these goals, a 500-L fermenter was required. Thus, a 500-L, top-drive, stirred-tank fermenter was put up for bid and a contract was awarded to Applikon Biotechnology (Dover, NJ). The College of Agriculture and Life Sciences contributed $61,863 and the Plant Sciences Institute contributed $20,000. The fermenter was delivered and installed at the BioCentury Research Farm (formerly the New Century Farm) in May 2010. The new BioCentury Research Farm, the first-in-the-world integrated biomass production and processing facility, was completed in July 2009 and dedicated in September. Because of the investment being made in the fermentation at the BioCentury Research Farm, Johnson and Johnson (Allentown, PA) donated 75-L and 250-L fermenters, five processing tanks, two large peristaltic pumps, a large autoclave and other fermentation equipment. These fermenters have been installed at the BioCentury Research Farm and are operational, users have been trained, and large-scale dry-grind corn ethanol fermentation protocols were developed to simulate industry practices. Additional funding ($249,317) was provided by the Iowa Energy Center to support fungal fermentation of thin stillage. Another proposal was submitted with General Atomics (San Diego, CA) to supplement our work on oleaginous yeast fermentation of glycerol; however, the project has not yet received funding because of insufficient commercial partners located in Iowa. Our work on fungal conversion of thin stillage was awarded the Grand Prize for University Research by the American Academy of Environmental Engineers, a project Innovation Award from the International Water Association, and a coveted 2008 R&D 100 Award that recognizes the top 100 innovations during the year. Our work on converting cellulosic feedstocks to produce oil for biodiesel received a 2009 R&D 100 Award. Six research papers were published in peer-reviewed journals. Two graduate students in Civil, Construction and Environmental Engineering received their degrees and three more continue to make progress towards graduation by working on these research objectives. Two more students in Biorenewable Resources Technology and Food Science worked on the project. PARTICIPANTS: Joe P. Colletti (Project Co-administrator), Senior Associate Dean, College of Agriculture and Life Sciences, and Robert C. Brown (Project Co-administrator), Director, Bioeconomy Institute; Lawrence A. Johnson (Project Director), Director, BioCentury Research Farm and Professor, Food Science & Human Nutrition (FSHN); Hans van Leeuwen (Co-principal Investigator), Professor, Civil, Construction and Environmental Engineering (CCEE), responsible for evaluating fungal fermentation of thin stillage and for optimizing biological pretreatment of lignocellulose and conversion to yeast oil and for filamentous fungal cultivation and oil production; and Sam Beattie (Co-principal Investigator), Assistant Professor, FSHN, responsible for yeast production of omega-3 fatty acids and for conversion of glycerol to yeast oil. The following Iowa State University staff worked on the project: John Strohl, Manager, ISU Fermentation Facility; Andy Suby, Manager, BioCentury Research Farm; and Carol Ziel, Research Associate. The following graduate students received research experiences by working on the project: D.R. Iassonova, PhD, FSHN; M.L. Rasmussen, PhD, CCEE; P. Shrestha, PhD, CCEE; and N. Jasti, PhD, CCEE; S. Sankaran, MS, CCEE, Y. Kambam, MS, CCEE; M. Vincent, PhD, BRT/CCEE; D. Mitra, PhD, CCEE/BRT/FSHN. Collaborators and Contacts: The team collaborated with the General Atomics to develop a follow-on project proposal submitted to the Iowa Power Fund. The team also collaborated with Ag Ventures Alliance to submit a competitive proposal to the Biomass Research initiative of USDA and DOE, which just missed funding and was recommended for resubmission. TARGET AUDIENCES: Target audiences include: farm associations, such as the Farm Bureau; and corn farmers and corn grower's associations, such as the National Corn Growers Association; and fuel ethanol producers and biodiesel manufacturers, such as the Renewable Fuels Association. PROJECT MODIFICATIONS: A no-cost extension was approved in 2009 in order to complete pilot-plant trials requiring the 500-L fermenter purchased on the project. The bidding and manufacturing time required 12 months to complete. Delivery occurred in May 2010. One principal investigator, Anthony Pometto III, Food Science and Human Nutrition, resigned from Iowa State University to become Chair, Department of Food Science and Human Nutrition at Clemson University.
Impacts
We have shown that the oleaginous yeast Candidia curvatus grows well and accumulates triglycerides (oil) when cultivated on glycerol, a byproduct of biodiesel manufacture. The yeast accumulated 63% fat and the obese yeast would float due to the low density of oil enabling the yeast to be easily harvested. We discovered that ultra-sound sonication can break the cell walls and release the oil. These advances make glycerol conversion to oil feasible. We demonstrated proof-of-concept at pilot-plant scale by using the newly acquired 500-L fermenter. The yeast grew on a simple, commercially viable medium comprised of water, yeast extract and glycerol but growth and use of glycerol were sluggish. Adding urea increased growth rate and utilization of glycerol. Both refined glycerol and unrefined glycerol obtained from a local biodiesel plant gave good results. These advances enable recycling glycerol to produce oil that can be converted into more biodiesel, thereby increasing biodiesel yield and eliminating a low-value byproduct. We successfully encapsulated heath-promoting fish and linseed oils and conjugated linoleic acid. Yeast encapsulation protects the oil from rumen hydrogenation, which is important to improving the fat composition of cow's milk. These fat sources (rich in omega-3 fatty acids) may be important to cardiovascular health, preventing cancer, promoting brain function, enhancing immune function, and treating rheumatoid arthritis. Increasing the levels of omega-3 fatty acids in diets of dairy cows may make milk more healthy. We showed that saccharification of cellulosic materials with our wood-rot fungal process is feasible. Corn fiber, a byproduct from wet milling, was best degraded to sugars by using aerobic solid-state fermentation with the soft-rot fungus Trichoderma reesei. Both white-rot fungus Phanerochaete chrysosporium and brown-rot fungus Gloeophyllum trabeum produced additional useful enzymes indicating a consortium of these fungi would be best. We demonstrated proof-of-concept at pilot-plant scale where P. chrysosporium organisms grew well with minimal contamination on unsterilized corn stillage. In all cases, a subsequent anaerobic yeast process under submerged conditions is required to ferment the released sugars to ethanol using Saccharomyces yeasts. We also demonstrated it is possible to convert these sugars to oil by using a filamentous fungi, Mucor circinnelloides. This oil could be converted to biodiesel. We demonstrated improved energy and water balances in corn ethanol plants and production of a high-protein feed product for non-ruminants by cultivating Rhizopus microsporus on thin stillage. The fungi removed waste products from yeast fermentation making possible direct recycle of the water recovered (and enzymes). This greatly reduced energy input into the ethanol process by avoiding the need for evaporating thin stillage. We demonstrated proof-of-concept at pilot-plant scale. We found Rhizopus oligosporus grew well with minimal contamination in the pilot-scale reactor on unsterilized corn stillage. We are seeking industry partners to adopt fungal conversion of thin stillage into commercial practice.
Publications
- Jasti, N., S.K. Khanal, A.L. Pometto III, and J. (Hans) van Leeuwen. 2009. Influence of Selected Operating Parameters on Fungal Production from Corn-Ethanol Wastewater. J. Environ. Eng. 35 (11):1106-1114 .
- Rasmussen, M.L., P. Shrestha, S.K. Khanal, A.L. Pometto III, and J. (Hans) van Leeuwen. 2010. Sequential Saccharification of Corn Fiber and Ethanol Production by the Brown-rot Fungus Gloeophyllum trabeum. Bioresource Tech. 101(10):3526-3533.
- Sankaran, S., S.K. Khanal, A.L.Pometto III, and J. (Hans) van Leeuwen. 2010. Use of Filamentous Fungi for Wastewater Treatment and Production of High Value Fungal By-products: A Review. Critical Rev. Environ. Science & Biotech. 40(5):1-49.
- Rasmussen, M.L., S.K. Khanal, A.L. Pometto III, and J. (Hans) van Leeuwen. 2010. Water Reclamation and Value-added Animal Feed from Corn Ethanol Stillage by Fungal Processing. Biomass and Bioenergy.
- Khanal, S.K., M.L. Rasmussen, P. Shrestha, B.P. Lamsal, J. (Hans) van Leeuwen, C. Visvanathan, and H. Liu. 2008. Bioenergy and Biofuel from Wastes/Residues of Emerging Biofuel Industries. Water Environ. Res. 80(10):1625-1647. Shrestha, P., S.K. Khanal, A.L. Pometto III, and J. (Hans) van Leeuwen. 2009. Corn Fiber Induced Extracellular Enzymes Production by Wood Rot and Soft Rot Fungi for Subsequent Fermentation of Hydrolyzate to Ethanol. J. Agric. Food Chem. 57:4145-4161.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: This project seeks to develop new processing technologies that underpin advanced biofuels from corn and soybean grain as well as lignocellulosic crops and crop residues. New more cost-effective conversion technologies are needed to meet the 2007 Energy Security and Independence Act, which mandates nearly 25% of today's motor fuels (36 billion gallons) be provided from biorenewable resources by 2022. We are contributing towards this goal by developing fungal fermentation of thin stillage, a byproduct produced by dry-grind ethanol plants; using oleaginous yeast to convert waste glycerol from biodiesel plants to single cell oil (SCO) for additional biodiesel; using waste glycerol as feedstock for omega-3 fatty acids accumulating yeasts to improve nutrition of bovine milk; and using fungal processes to produce biodiesel from lignocellulose. This project focuses on refining, scaling-up and demonstrating these technologies and to meet this goal, a 500-L fermenter is required. Thus, a 500-L, top-drive, stirred-tank fermenter was put up for bid and a contract was awarded to Applikon Biotechnology (Dover, NJ). The College of Agriculture and Life Sciences contributed $61,863 and the Plant Sciences Institute contributed $20,000 towards the fermenter. The fermenter will be delivered and installed at the BioCentury Research Farm (formerly known as the New Century Farm) in February 2010. The new BioCentury Research Farm, the first-in-the-world integrated biomass production and processing facility, was completed in July 2009 and dedicated in September. Because of the investment being made in the fermentation processing train at the BioCentury Research Farm, Johnson and Johnson (Allentown, PA) donated 75-L and 250-L fermenters, five processing tanks, two large peristaltic pumps, and other fermentation equipment. These fermenters have been installed at the BioCentury Research Farm and are operational, users have been trained, and development of large-scale dry-grind corn ethanol fermentation protocols is underway. This project was extended for an additional year to complete the proposed pilot-plant testing. Additional funding ($249,317) was provided by the Iowa Energy Center to support fungal fermentation of thin stillage. Another proposal was submitted with General Atomics (San Diego, CA) to supplement our work on oleaginous yeast fermentation of glycerol; however, the project has not yet received funding because of insufficient commercial partners located in Iowa. Our work on fungal conversion of thin stillage was awarded the grand Prize for University Research by the American Academy of Environmental Engineers, a project Innovation Award from the International Water Association and a coveted 2008 R&D 100 Award that recognizes the top 100 innovations during the year. Two research papers were published in peer-reviewed journals. Two graduate students in Civil, Construction and Environmental Engineering received their degrees and three more continue to make progress towards graduation by working on these research objectives. Two more students in Biorenewable Resources Technology and Food Science are working on this project. PARTICIPANTS: Individuals: Joseph P. Colletti (Project Co-administrator), Senior Associate Dean, College of Agriculture and Life Sciences, and Robert C. Brown (Project Co-administrator), Director, Bioeconomy Institute; Lawrence A. Johnson (Project Director), Director, BioCentury Research Farm; Hans Van Leeuwen (Co-principal Investigator), Professor, Civil, Construction and Environmental Engineering (CCEE), responsible for evaluating fungal fermentation of thin stillage and for optimizing biological pretreatment of lignocellulose and conversion to yeast oil and for filamentous fungal cultivation and oil production; and Sam Beattie (Co-principal Investigator), Food Science and Human Nutrition (FSHN), responsible for yeast production of omega-3 fatty acids and for conversion of glycerol to yeast oil. The following Iowa State University staff worked on the project: John Strohl, ISU Fermentation Facility Manager; and Carol Ziel, Research Associate. The following graduate students received research experiences by working on the project: M.L. Rasmussen, PhD, CCEE; P. Shrestha, PhD, CCEE; and N. Jasti, PhD, CCEE; S. Sankaran, MS, CCEE, Y. Kambam, MS, CCEE; M. Vincent, PhD, BRT/CCEE; D. Mitra, PhD, CCEE/BRT/FSHN. COLLABORATORS AND CONTACTS: The team collaborated with the General Atomics to develop a follow-on project proposal submitted to the Iowa Power Fund. The team also collaborated with Ag Ventures Alliance to submit a competitive proposal to the Biomass Research initiative of USDA and DOE, which just missed funding and was recommended for resubmission. TARGET AUDIENCES: Target audiences include: farm associations, such as the Farm Bureau; and corn farmers and corn grower's associations, such as the National Corn Growers Association; and fuel ethanol producers and biodiesel manufacturers, such as the Renewable Fuels Association. PROJECT MODIFICATIONS: A no-cost extension was approved in order to complete pilot plant trials requiring the 500-L fermenter purchase on the project. The bidding and manufacturing time required 12 months to complete. Delivery is expected February 2010. One principal investigator, Anthony Pometto, Food Science and Human Nutrition, resigned from Iowa State to become Chair, Department of Food Science and Human Nutrition at Clemson University.
Impacts
In our laboratory work, we have shown that the oleaginous yeast Candidia curvatus grows well and accumulates triglycerides (oil) when cultivated on glycerol, a by-product of biodiesel manufacture. Oleaginous yeast accumulated as much as 63% fat on a dry matter basis. When this level is achieved the obese yeast float due to the low density of oil and can be easily separated from the fermentation broth. We have discovered that ultra-sound sonication can break the cell walls of oleaginous yeast and release the oil, which can then be converted to more biodiesel. These advances make glycerol conversion to oil feasible, but we must yet demonstrate this technology on large scale before encouraging commercial adoption. These advances will enable recycling glycerol to manufacture more oil that can be converted into more biodiesel, thereby increasing the yield of biodiesel per unit of feedstock and eliminating a low-value byproduct. We have successfully encapsulated fish and linseed oil and conjugated linoleic acid. In-vitro tests indicated yeast encapsulation protects polyunsaturated fatty acids from rumen hydrogenation, which is important to our goal of improving the fatty acid composition of bovine milk. Omega-3 fatty acids may be important to cardiovascular health, preventing cancer, promoting brain function, enhancing immune function, and treating rheumatoid arthritis. Increasing the levels of omega-3 fatty acids in bovine milk may make milk even more healthy for consumers. We showed that saccharification of lignocellulosic material with a wood-rot fungal process is feasible. Corn fiber, a byproduct from wet milling, was best degraded to sugars by using aerobic solid-state fermentation with the soft-rot fungus Trichoderma reesei. Both white-rot fungus Phanerochaete chrysosporium and brown-rot fungus Gloeophyllum trabeum produced additional useful enzymes indicating a consortium of these fungi would be best. In all cases, a subsequent anaerobic yeast process under submerged conditions is required to ferment the released sugars to ethanol using Saccharomyces yeasts. We also demonstrated it is possible to convert these sugars to oil by using a filamentous fungi, Mucor circinnelloides. This oil could be converted to biodiesel. We were just informed that this innovation will receive a 2009 R&D 100 Award this fall for being one of the 100 most important research discoveries. We demonstrated the opportunity to improve the energy and water balances in dry-grind ethanol plants and to produce a high-protein feed product for non-ruminants by cultivating Rhizopus microsporus on excess thin stillage using 10-L fermenters. The fungi removed waste products from yeast fermentation (glycerol, lactic and acetic acids) making possible direct recycle of the water recovered (and enzymes). This greatly reduces energy input into the ethanol process by avoiding the need for evaporating thin stillage. We are seeking industry partnerships to adopt fungi conversion of thin stillage into commercial practice and the Iowa Energy Center provided additional funding to enable moving the technology towards commercialization.
Publications
- Khanal, S.K., M.L. Rasmussen, P. Shrestha, B.P. Lamsal, J. (Hans) van Leeuwen, C. Visvanathan, and H. Liu. 2008. Bioenergy and Biofuel from Wastes/Residues of Emerging Biofuel Industries. Water Environ. Res. 80(10):1625-1647.
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