Jay Cheng

Lemnaceae is a family of small floating aquatic plants more commonly known as duckweed. Lemnaceae species are some of the fastest growing plants on the planet and have the potential to produce many times more biomass per acre than any existing crop species, making them ideal candidates for bioenergy applications. Lemnaceae species are also extremely efficient at absorbing nutrients from the water they grow in, making them ideal candidates for wastewater remediation. North Carolina is the second largest hog producer in the United States. There are a huge amount of swine wastewater that needs to be properly managed to avoid negative environmental impacts. In this proposal, we view swine wastewater as a nutrient rich resource. If funded, this study will examine the potential of growing 3 different Lemnaceae species (duckweed) with swine lagoon water followed by subsequent anaerobic digestion of the duckweed biomass for biogas production. The 3 Lemnaceae species were selected for their excellent performance of growth with swine lagoon water in North Carolina in our previous research projects. We propose to: determine an optimum continual-harvest protocol for the Lemnaceae species, determine nutrient removal rates from swine lagoon water, determine the optimal Carbon: Nitrogen ratio of mixing duckweed biomass with swine manure for biogas production, and characterize the composition of the resulting digestate for potential usage of growing duckweed biomass.

Utilization of various organic waste materials for low-carbon energy production has made significant contributions to environmental protection and sustainable development. Novozymes and North Carolina State University (NCSU) have a common interest in promoting and optimizing bioenergy production from organic waste materials with enzymes. Specifically, we will work collaboratively to develop and optimize an enzyme enhanced anaerobic digestion (2E-AD) bioprocess from source separated organics (SSO) and municipal solid waste (MSW) in this proposed project.

If funded, this early-stage research will establish the groundwork to ultimately develop an innovative bioprocess that decouples land- and ocean-use from protein feed production by converting air-derived CO2 and N2 into an alternative source of protein to supplement, or possibly replace, certain animal and fish feed products that are currently made in an unsustainable fashion. The potential for economic impact is profound given that animal agriculture and associated industries generate revenues of ~$1.25 trillion each year in the US.21 Electrification of protein via A+B systems has exciting potential to address critical issues in agriculture and aquaculture, and, if successful, this particular project will help realize this potential.

It is hypothesized that the biodegradability of synthetic, natural and emerging bio-based polymer product are not fully understood in aerobic or anaerobic conditions in surface waters and may accumulate in the environment. The recalcitrance of some of these natural materials may be due to the chemical or compositional modifications to impart desired product properties. For instance, many naturally based nonwoven materials have additives incorporated to develop water, oil, or UV resistance. The overall goal of this study is to determine the factors that determine the aquatic biodegradability in surface waters (fresh and sea water) of non-woven products used in disposable applications. These factors will include the fiber structure, chemistry and assembly in the non-woven structure. The objectives of the study are the following: (1) To benchmark common disposable non-woven products with regard to their aquatic biodegradation in fresh and sea waters. (2) To benchmark natural fibers, semi-natural biobased fibers, and synthetic fibers used in non- wovens for their aquatic biodegradation in fresh or sea waters. (3) To understand how chemical, physical, and polymeric characteristics of non-woven fibers affect the aquatic biodegradation. (4) To determine how chemical and physical treatments of the fibers affect the aquatic biodegradation. (5) To model and predict the fate of such fibers in the environment, including the lifetime, fate and adsorption of toxic organic chemicals. The results of the study will allow non-woven manufacturers, researchers, suppliers, and consumers to better understand how the choice of materials will affect expected aquatic degradation, allowing all stakeholders to make more informed and better material choice decisions

Lignocellulosic materials, such as switchgrass and coastal Bermuda grass, have been extensively studied as potential feedstock alternatives to corn for the production of bioethanol. Considerable research has been devoted to the development of rapid pre-treatment methods to enhance the hydrolysis of cellulose to fermentable sugars by removing lignin and/or hemicellulose, in order to reduce the crystallinitiy of cellulose, and increase the porosity of the feedstock. These pretreatment schemes include the use of chemicals (acids and bases), electromagnetic heating technologies (microwave and RF), ultrasonication, steam explosion, or a combination of these methods. Continuous flow microwave processing at high power levels (100 kW) and industrial frequency ranges (915 MHz) allowing, for higher throughputs (semi-industrial capacity of 1-2 liters per minute to industrial capacities of 1-10 gallons per minute), has proven to deliver an economically viable processing technology for efficient and uniform heat treatment of various biomaterials. This patented technology, along with process validation tools, has resulted in the creation of 5 businesses (presently providing 40 new jobs with an estimated 250 additional jobs within 5 years in North Carolina). For this project, the main goal is to generate data for (i) scale-up, (ii) optimization, and (iii) progress to commercialization of continuous flow microwave-assisted alkaline (1% NaOH) pretreatment of lignocellulosic feedstock (switchgrass or coastal Bermudagrass) using a prototype continuous flow microwave system. The data will potentially establish increased surface area of the lignocellulosic substrate and improved enzymatic hydrolysis, thereby increasing the yield of alcohol (using less energy) and. Once the continuous microwave technology is optimized for maximal ethanol yield, scale-up and regeneration loops will be designed in a manner similar to that for systems currently in place for commercial processing of biomaterials.

Biogas generation and capture using covered lagoons is a promising renewable energy technology that can be utilized by North Carolina swine farmers to increase on-farm income and mitigate rising energy costs. Covering lagoons would also reduce ammonia and greenhouse gas emissions, particularly methane, and have potential to obtain Carbon Credits. Currently, swine producers in the state have reservations about covering lagoons for biogas production as the economic recovery associated with energy generation equipment is unknown for NC and concerns about nutrient management have lead state officials to exclude covered lagoon projects from state conservation agricultural cost-share programs. An overlooked aspect of anaerobic digestion is the use of the nutrients in the production of crops that can be converted into value-added agricultural products. Algae and duckweed are examples of energy crops that could be grown in a media similar to digested swine wastes and then used in the production of biodiesel or bioethanol. The ultimate goals of this project is to exhibit effective, economically-feasible energy generation technologies utilizing biogas, demonstrate utilization of biogas combustion by-products (i.e. heat and CO2), and to show a variety of processes that can be implemented to manage swine effluent nutrients. The technologies to be demonstrated in this project include: 1.Anaerobic digestion of swine manure for biogas production and utilization of methane for electricity generation through an internal combustion engine, 2.Utilization of CO2 from methane combustion to enhance tomato production in greenhouse, 3.Nitrification biofiltration for ammonia emission control, very low cost denitrification in pits for nitrogen removal, and nutrient utilization for greenhouse tomato production, 4.Growing duckweed to recover nutrients from swine wastewater and convert duckweed into bioethanol, and 5.Struvite crystallization for phosphorus removal from swine wastewater. Also, technologies and extension education materials will be transferred to interested stakeholders. Workshops, field days, extension publications, and website spreadsheets and other materials will be developed from the knowledge obtained from this project.

The goal of this proposed research is to develop a technology that efficiently converts genetically engineered switchgrass with less lignin and more cellulose to bioethanol. Lower costs will be expected for the technology, compared to the current ones. The specific objectives are to: 1. Clone cDNA of a key lignin biosynthesis gene 4CL (4-coumarate:coenzyme A ligase) from switchgrass (Panicum virgatum L.); 2. Genetically transform switchgrass for reduced lignin and increased cellulose; and 3. Improve efficiencies of transgenic switchgrass to ethanol through pretreatment, hydrolysis and fermentation. In this multi-disciplinary research team, we have expertise in grass transformation, lignin/cellulose synthesis biochemistry and molecular biology, and fermentation for ethanol production from cellulose. Collaborative experiments will be conducted to achieve the goal and objectives of this project. This proposed project will have significant economic benefits for renewable energy production. Although at the present there is very little switchgrass grown in the US, many states, e.g. North Carolina, are very suitable for its growth. If the price is right the acreage would rapidly grow. An estimate by NCDA at 2004 indicates that switchgrass could be grown on some 4.4 million acres in the state, involving 11,000 farms, and by our calculation, the switchgrass-to-ethanol industry revenue could reach $4 billion/year. Moreover, hundreds of jobs will be created by the switchgrass-to-ethanol plants. At this scale, a reduction of each one percentage unit of lignin and conversion of it to cellulose would increase the revenue by $95 million/year without any more input due to the increased ethanol production.

The goal of this proposed research is to develop a technology that efficiently converts genetically engineered switchgrass with less lignin and more cellulose to bioethanol. Lower costs will be expected for the technology, compared to the current ones. The specific objectives are to: 1. Clone cDNA of a key lignin biosynthesis gene 4CL (4-coumarate:coenzyme A ligase) from switchgrass (Panicum virgatum L.); 2. Genetically transform switchgrass for reduced lignin and increased cellulose; and 3. Improve efficiencies of transgenic switchgrass to ethanol through pretreatment, hydrolysis and fermentation. In this multi-disciplinary research team, we have expertise in grass transformation, lignin/cellulose synthesis biochemistry and molecular biology, and fermentation for ethanol production from cellulose. Collaborative experiments will be conducted to achieve the goal and objectives of this project. This proposed project will have significant economic benefits for renewable energy production. Although at the present there is very little switchgrass grown in the US, many states, e.g. North Carolina, are very suitable for its growth. If the price is right the acreage would rapidly grow. An estimate by NCDA at 2004 indicates that switchgrass could be grown on some 4.4 million acres in the state, involving 11,000 farms, and by our calculation, the switchgrass-to-ethanol industry revenue could reach $4 billion/year. Moreover, hundreds of jobs will be created by the switchgrass-to-ethanol plants. At this scale, a reduction of each one percentage unit of lignin and conversion of it to cellulose would increase the revenue by $95 million/year without any more input due to the increased ethanol production.

The Goal of this one-year proposal is to evaluate the feasibility of ethanol production using duckweed biomass as the carbohydrate source. The Objectives are: (1) Create a scaled up version of the duckweed cropping system to finalize cropping management protocols and biomass harvesting; (2) Develop procedure for biomass post-harvest handling to produce dry duckweed with stabilized carbohydrate; (3) Determine ethanol yield from dry duckweed biomass at pilot plant scale; and (4) Perform an economic analysis of the overall process. Our partners include (1) Hog producer, Mr. Julian Barham, to build and test the duckweed cropping system utilizing hog wastewater. Our work also has the support of Murphy Family Farms (See supporting letter). (2)We will partner with Aeroglide, Inc., an international leader in industrial drying, to develop a low-cost drying process for duckweed biomass. (3) We will partner with Novozymes in our work with the NCSU Ethanol Pilot Facility at the Lake Wheeler Experimental Farm to determine ethanol yield from duckweed biomass. The beneficiaries of our work will be North Carolina?s agricultural and industrial sectors and rural counties because of the following positive impacts: ? Microbial fermentation is a major industrial process including, but not limited to, ethanol production. Developing duckweed as an industrial feedstock for microbial fermentation will provide a major incentive for location of microbial biomanufacturing facilities in NC which could become a new economic driver and result in an increase in high-paying jobs in rural NC. We estimate that up to one billion gallons of ethanol could be produced annually from duckweed produced from the State?s hog wastewater. Extending our technology to utilize municipal wastewater would increase production further. ? Our work provides a treatment system for swine wastewater that can meet environmental concerns and provide additional revenue to farmers thru sale of duckweed biomass. Our system transforms hog wastewater from pollution to productive asset and removes a major obstacle and risk to swine industry presence and expansion in NC. Industry expansion would increase agricultural revenue and employment for rural North Carolina counties. ? Developing a new, non-food, industrial fermentation feedstock will ease price pressure on grain commodities and the negative impact price increases have on the swine and poultry industry of NC, while providing a source of added revenue to the farm bottom line. ? Duckweed ponds can utilize land that is unsuitable for crop production, and therefore does not compete for limited, high-value crop land. We estimate that approximately one-half million acres of land would be needed to fully utilize all hog wastewater for duckweed production. Wastewater provides the water and nutrients needed for duckweed production. ? Existing starch-based hydrolysis and fermentation technologies work with duckweed biomass, providing lower technological risk relative to ligno-cellulosic biomass alternatives. Lower risk provides a favorable climate for capital investment needed for industrial scale commercialization. ? The limitation of fresh water resources in NC looms as a challenge to NC economic expansion and is a key consideration for large-scale ethanol production. Our wastewater system cleans wastewater sufficiently to allow for reuse thus significantly impacting the availability of water needed to grow the State?s economy. To achieve our Objectives we are requesting financial support from teh new NC Biofuels Center for our one-year project. Our projects effectiveness will be determined by its success in generating intellectual property (from results of Objectives One-Three) and from the outcomes of Objective Four which will serve as technological and economic results that can be used to initiate discussions with commercialization investment partners.

The ultimate goal of this proposed project is to develop an economically competitive ethanol production system based on the utilization of coastal Bermuda grass (CBG) (scientific name: Cynodon dactylon). The basic idea is to use genetically engineered cellulase enzymes to convert cellulose in CBG to fermentable sugars and to further ferment the sugars to ethanol. The project will lead to the development of a cost-effective technology for biofuel production from a lignocellulosic material. A multi-disciplinary research team with expertise in biological & agricultural engineering, wood & paper science, and enzyme biochemistry & fermentation from North Carolina State University (NCSU) and Novozymes North America, Inc. (Novozymes) will collaborate in the proposed research project. To achieve the project goal, the specific objectives of the project are to: (1) Develop and optimize genetically engineered cellulase enzymes for lignocellulose in CBG; (2) Identify the most efficient pretreatment method to remove lignin and hemicellulose from coastal Bermuda grass; (3) Test the genetically engineered cellulase enzymes for hydrolysis of the CBG cellulose and optimize the enzymatic hydrolysis process; (4) Evaluate ethanol production potential of CBG through fermentation; and (5) Integrate overall CBG-to-ethanol process for feasibility analysis