200 mg/g dry root, compared with the current majority which range from 50 to 80 mg/g. Also, natural quantitative herbicide tolerance has been identified and selected in several accessions. The TK genome has been generated and used to design schemes of high-troughput genotyping for marker assited selection (MAS) and to identify multiple gene targets for CRISPR gene editing. Current TK lines are not invasive and gene flow via pollen into common dandelion (triploid apomict) has been proven to be impossible. Weediness potential will be carefully monitored as more competitive genotypes, such as recently developed polyploids, advanced selections from improved populations generated through MAS, CRISPR gene editing, and additional germplasm derived from interspecific crosses are obtained. Suppression of weediness is a key trait in our effort of engineered domestication. Cost analyses and crop enterprise budgets have indicated that TK can become a competitive US crop once domesticated.TK produces rubber similar in quality to that from the tropically-grown rubber tree and a robust US domestic crop will allow self-sustainability and resilience in this critical agricultural material. The inulin, sugar and biomass co-products can be converted via relatively simple, pre-existing conversion processes into a suite of products including biofuels. The potential to farm this crop across the northern states, creates opportunities to improve farm gate value and provide an opportunities for product diversification for potato and sugar beet farmers and creates a number of irrigated and rain-fed models for TK crop production which can be used by growers in states throughout the Northern US.The outcomeof this project will be knowledge,agricultural systems and products thatpropel Taraxacum kok saghyz(TK) toward a scale needed to supply US manufacturers with domestically producednatural rubber and supplysignificant amounts of biofuel.We foresee the potential for several million acres of TK for the production ofrubber and biofuel, and an industry that createsjobs throughout the agribusiness value chain across the northern U.S. Products derived from TK roots (rubber, C6 sugars, and bagasse) address the goals of reducing US demand for fossil fuels and increasing bioproduct production by providing a critical bioproduct and displacing oil used for the production of synthetic rubber, by providing a pure C6 sugar stream for further bioconversion into biofuels, and by providing a cellulosic feedstock useful directly, or after conversion, as a biofuel.At the projected planting density of 1.5 million plants/ha and average dry root weight of 6 g, 9,000 kg dw/ha of TK roots would be available for processing. Based on compositional analysis of TK roots field harvested in 2014 in Ohio, and a projected rubber content of 10% (dw), this crop would provide 900 kg of rubber and 2870 kg of inulin/ ha. An additional 1,755 kg of lignocellulosic sugars also would be available. Combined, the inulin and cellulosic sugars could be hydrolyzed and converted into 701 gal/ha of ethanol. The value of these products at current (Sept 2017) prices would be $2,517/ha. This compares to a value of $2,321/ha for ethanol and dry distillers grains made from the average yield (188 bu/acre; 12,643 kg/ha) of Iowa corn in 2016.The project will also deliver integrated agricultural systems, with state-specific protocols, and detailed assessments of TK production impacts on water quality, soil quality, and ecosystems services. ' />
Source: OHIO STATE UNIVERSITY submitted to NRP
BARRAL-BIOENERGY, ADVANCED BIOFUEL, AND RUBBER RESEARCH AGRICULTURAL LINKAGES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1016294
Grant No.
2018-10008-28571
Cumulative Award Amt.
$1,999,995.00
Proposal No.
2018-03699
Multistate No.
(N/A)
Project Start Date
Sep 1, 2018
Project End Date
Aug 31, 2023
Grant Year
2018
Program Code
[BRDI]- Biomass R&D Initiative FY2009 Forward
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
Department of Food, Ag. and Biological Engineering
Non Technical Summary
Natural rubber is a critical natural resource used for countless numbers of products essential to modern life. These include automobile, truck and airplane tires, machine parts, medical supplies and other materials. However the current supply of natural rubber comes almost exclusively from the rubber trees grown in Southeast Asia. As developing countries modernize and areas available for plant cultivation shrink, there is likely to be a shortfall in natural rubber in the future. The goal of the 'Bioenergy, Advanced biofuel, and Rubber Research Agricultural Linkages' (BARRAL) project is to overcome barriers to the commericialization of an alternative natural rubber and industrial sugar crop Taraxacum kok-saghyz (TK) in the northern US. The principal products of this dandelion crop are natural rubber, inulin (a starch-like carbohydrate), sugar, and biomass. The latter three of these have mutiple conversion opportunities to bioenergy and biofuel. The BARRAL Consortium is led by The Ohio State University (OSU) in collaboration with the University of Nebraska and Oregon State University.The project builds upon previous progress funded by public and private sources and an established collaborative group (PENRA) and has the following objectives.Objective 1. Maximize crop establishment and crop production by developing and adapting common and region-specific agricultural equipment and methods.Objective 2.Optimize and demonstrate field production in Ohio, Nebraska and Oregon by translating and adapting recent Ohio successesObjective 3.Maximize extractable rubber yield and purity by optimizing harvest, post-harvest handling and storage methods and/or equipment.Objective 4. Maximize the yield of TK fermentable sugars, and rubber that meets tire manufacturing standards, by optimizing previously developed OSU innovative inulin and root rubber extraction and purification methods.Objective 5.Evaluate technoeconomic and life cycle environmental impacts of a TK rubber and sugar based industry across the Northern United States sufficient to support US natural rubber self-sustainability.In the last five years, TK has been produced on about 50 acres in Ohio and Oregon, including on commercial Ohio farms. Transplanted crop, but not direct seeded crop, had sufficient size to permit efficient weed control using cultivation, hand weeding and limited herbicide application. Direct seeded crops are slow to establish and even hand weeding does not support competitive plant growth nor produce large rooted plants with high rubber and inulin yield at harvest. Outdoor planting box trials demonstrated that commercially competitive yields can be obtained under optimal conditions at high planting density and long growing season. Individual selections among large TK plants have revealed genotypes with >200 mg/g dry root, compared with the current majority which range from 50 to 80 mg/g. Also, natural quantitative herbicide tolerance has been identified and selected in several accessions. The TK genome has been generated and used to design schemes of high-troughput genotyping for marker assited selection (MAS) and to identify multiple gene targets for CRISPR gene editing. Current TK lines are not invasive and gene flow via pollen into common dandelion (triploid apomict) has been proven to be impossible. Weediness potential will be carefully monitored as more competitive genotypes, such as recently developed polyploids, advanced selections from improved populations generated through MAS, CRISPR gene editing, and additional germplasm derived from interspecific crosses are obtained. Suppression of weediness is a key trait in our effort of engineered domestication. Cost analyses and crop enterprise budgets have indicated that TK can become a competitive US crop once domesticated.TK produces rubber similar in quality to that from the tropically-grown rubber tree and a robust US domestic crop will allow self-sustainability and resilience in this critical agricultural material. The inulin, sugar and biomass co-products can be converted via relatively simple, pre-existing conversion processes into a suite of products including biofuels. The potential to farm this crop across the northern states, creates opportunities to improve farm gate value and provide an opportunities for product diversification for potato and sugar beet farmers and creates a number of irrigated and rain-fed models for TK crop production which can be used by growers in states throughout the Northern US.The outcomeof this project will be knowledge,agricultural systems and products thatpropel Taraxacum kok saghyz(TK) toward a scale needed to supply US manufacturers with domestically producednatural rubber and supplysignificant amounts of biofuel.We foresee the potential for several million acres of TK for the production ofrubber and biofuel, and an industry that createsjobs throughout the agribusiness value chain across the northern U.S. Products derived from TK roots (rubber, C6 sugars, and bagasse) address the goals of reducing US demand for fossil fuels and increasing bioproduct production by providing a critical bioproduct and displacing oil used for the production of synthetic rubber, by providing a pure C6 sugar stream for further bioconversion into biofuels, and by providing a cellulosic feedstock useful directly, or after conversion, as a biofuel.At the projected planting density of 1.5 million plants/ha and average dry root weight of 6 g, 9,000 kg dw/ha of TK roots would be available for processing. Based on compositional analysis of TK roots field harvested in 2014 in Ohio, and a projected rubber content of 10% (dw), this crop would provide 900 kg of rubber and 2870 kg of inulin/ ha. An additional 1,755 kg of lignocellulosic sugars also would be available. Combined, the inulin and cellulosic sugars could be hydrolyzed and converted into 701 gal/ha of ethanol. The value of these products at current (Sept 2017) prices would be $2,517/ha. This compares to a value of $2,321/ha for ethanol and dry distillers grains made from the average yield (188 bu/acre; 12,643 kg/ha) of Iowa corn in 2016.The project will also deliver integrated agricultural systems, with state-specific protocols, and detailed assessments of TK production impacts on water quality, soil quality, and ecosystems services.
Animal Health Component
30%
Research Effort Categories
Basic
10%
Applied
30%
Developmental
60%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5012249202025%
6012249202020%
1022249106035%
2132249114010%
6012249107010%
Knowledge Area
601 - Economics of Agricultural Production and Farm Management; 102 - Soil, Plant, Water, Nutrient Relationships; 501 - New and Improved Food Processing Technologies; 213 - Weeds Affecting Plants;

Subject Of Investigation
2249 - Rubber, gum, and resin plants, general/other;

Field Of Science
2020 - Engineering; 1060 - Biology (whole systems); 1070 - Ecology; 1140 - Weed science;
Goals / Objectives
The United States has a critical need for both transportation biofuels and a domestic supply of natural rubber (NR). We are developing n Taraxacum kok-saghyz (also known as Russian dandelion andTK) as anatural rubber and industrial sugar crop for the northern US toaddressboth of these needs. Inulin and biomass from TK have mutiple conversion opportunities to biofuel. TK NR has qualities almost identical to the rubber extracted from rubber trees (Hevea brasiliensis), and can be similarly used for a variety of applications, including technically-sophisticated, high performance automobiletires. The goals of the BARRAL Consortium, led by The Ohio State University (OSU), are toovercome remaining key commericialization barriers faced by TK. This projectbuilds upon previous successes in germplasm improvement, agronomy and processing, funded by public and private sources, and an established collaborative group (the PENRA Consortium) and expands the effort to multiple states. The specific ojectives are the following:Objective 1. Maximize crop establishment and crop production by developing and adapting common and region-specific agricultural equipment and methods.Objective 2.Optimize and demonstrate field production in Ohio, Nebraska and Oregon by translating and adapting recent Ohio successesObjective 3.Maximize extractable rubber yield and purity by optimizing harvest, post-harvest handling and storage methods and/or equipment.Objective 4. Maximize the yield of TK fermentable sugars, and rubber that meets tire manufacturing standards, by optimizing previously developed OSU innovative inulin and root rubber extraction and purification methods.Objective 5.Evaluate technoeconomic and life cycle environmental impacts of a TK rubber and sugar based industry across the Northern United States sufficient to support US natural rubber self-sustainability.
Project Methods
Planter EvaluationOhio, Oregon, and Nebraska will comparebroadcast treatment where TK seed is spread over the soil surface toa mechanically driven roller seeder designed for planting pelleted seed (JP-6 3-point seeder, Jang Automation Co.). One to three additional planters or drills will be comparedwith the total treatments at each location ranging from three to five. Treatments will consist of a10 m pass per study replicate, with each method calibrated to deliver a similar amount of seed/ha. The soil will be prepared to provide a vegetation-free, even seedbed ideal for planter comparison. The calibration of each planter type will provide a theoretical planting population, if 100% emergence is realized, adjusted based upon seed viability. Evaluation of the realized stands will occur at 1, 2, 3, and 4 weeks after planting, and every 4 additional weeks until crop harvest. Treatments will be evaluated by comparing theoretical vs realized stand. Root, rubber and inulin yield will also be recorded. Treatments using a planter or drillwill deliver ~1.8 million seeds/ha, while broadcasted seed will be planted at adensity of ~2.2 million seed/ha, to account for seed loss and movement from the soil surface. All treatments will use primed pelletized seed from the same seed lot.?Harvester EvaluationA harvester evaluation study will be conducted at the same three locations. The common treatment across all locations will be a hand-dug check, where roots will be excavated using shovels and forks to ensure that the entirety of roots are obtained. Ginseng, potato, and sugarbeet harvesters will be compared to thishand-dug check, with each location consisting of two to four treatments based upon equipment availability. Each plot will consist of a 10 m long strip planted using the JP-6 roller planter. Plant stand will be recorded throughout the season and final stand counts will be recorded just prior to harvest.Both planter and the harvest evaluation studies will be designed asrandomized complete block designswith five replicates. Trials will be evaluated using ANOVA with significant results compared using Tukey's protected HSD. Harvest, planting treatments, and study location are fixed effects. Soil data will be collected from each location along with real-time weather data using on-site weather stations. Both studies will be planted early (April - June) and harvested late (October - December). Both trials will be maintained weed-free by herbicides and hand.Optimization and demonstration of field production in Ohio, Nebraska and OregonIn year 1 twoseed types will be distributed among the three locations: (a) 10 million primed, pelletized seed (from parent populations selected for rubber content and root size) for the mechanical optimization research. (b) Seed from plants selected for natural resistance to pre-emergence (Pursuit) and post-emergence (imazethapyr) herbicides. Inyear 2 seed from best performing trials will be distributed for the common experiment. Seed from plants selected for natural resistance to pre-emergence (Pursuit) and post-emergence (imazethapyr) herbicides will be distributed for small plot trials. Tetraploid seed also will be distributed for small plot trials. Inyear 3 seed from the most promising trials will be distributed for the common experiments, including broad use of herbicide resistant and tetraploid germplasm.Post-harvest processingA water-based extraction process (PENRA III) will be improved based on compositional analysis of TK roots, kinetic studies and quantification of the effects of process variables. Accelerated solvent extraction, chromatography and gravimetric methods will be used to quantify rubber and inulin extraction efficiency, rubber molecular weight and inulin degree of polymerization (DP). Bioprocessing variables will include root rubber and inulin content, milled root size, pebble milling parameters, inulin extraction conditions, enzymeactivityand downstream rubber and inulin purification. Counter current extraction will be usedto minimize water use, resulting in a syrup with a high inulin sugar concentration for fermentation. Inulin saccharification usingdilute acid or inulinase will be investigated, and fermentation and extraction conditions will be optimized for biofuel yield similar to starch conversion to ethanol at dry mill corn ethanol plants. Alkaline pretreatments that remove lignin, commercially-available cellulase, pectinase, protease, xylanase and arabinase enzymes as well as milling parameters, including grinding media size and shape, sequential milling and washing, will be investigated. Enzyme kinetics, product inhibition and deactivation as a function of reaction temperature and pH will be determined by measuring product formation using liquid chromatography and used in process models to optimize enzyme activity, reaction rate and recycling. The optimization experiments will be done at laboratory scale and best practicesdemonstrated at pilot scale. Bagasse will be analyzed and TEA used to compare potential conversion processes including pretreatment and enzymatic hydrolysis to generate five and six carbon sugar monomers for fermentation, drying and pelletizing for boiler fuel, dilute acid pretreatment followed by simultaneous saccharification and fermentation, hydrothermal liquefaction, combustions and anaerobic digestion. Rubber samples will be provided to industrial partners foranalysis, testing, and product development and validation.Technoeconomic analysis (TEA) and life cycle assessment (LCA) AnalysisTEA and LCA throughout the project willidentify and prioritize the research and development efforts. TEA and LCA of the entire value chain from TK production through conversion to the end products, including prior data on process efficiencies already collected at OSU will be analyzed. A comprehensive TEA model from field to TK conversion using SuperPro Designer (Intelligen Inc.) process simulation software will be used together with pilot data to refine and optimize process models and estimate capital and operating costs of a commercial scale biorefinery. Existing feedstock production and logistics models will be used toanalyzeinfrastructure, equipment, energy, labor, and consumables for different operations of the TK production and logistics system, followed by economic analysis. A TEA for multipleend-products including TK rubber for hevea supplementation and synthetic rubber displacement, and inulin and bagasse conversion to biofuels will be developed. Previous TEA models of bioconversion processes will be adopted and modified. Models for root processing will analyze mass and energy flow at the commercial-scale, followed by resource requirement estimation and economic analysis of costs and return on investment. Estimates of resource requirements, costs, ROI for commercial-scale TK production and processing will be made. Sensitivity analysis will identify production and conversion parameters impacting TEA outcomes. Results will set targets for enhancing technical and economic feasibility of root production, logistics, and conversion. The environmental impacts of TK production focusing on fossil energy consumption and related GHG emission, and carbon releases into the atmosphere will be assessed using LCA including material and energy inputs and outputs into the air, water, and soil, to estimate environmental impact, and can compare cropping systems and locations to determine the most sustainable system. OpenLCA, RUBBEREL, and GREET will be used to model the life cycle from field production-to-logistics-to-conversion. A life cycle impact assessment (LCIA) of the site specific systems will use the Tool for Reduction and Assessment of Chemicals and Other Environmental Impacts (TRACI) model to identify impacts on fossil energy consumption, global warming, acidification, eutrophication, and photochemical smog formation.

Progress 09/01/18 to 08/31/23

Outputs
Target Audience:Target audiences included growers who could benefit from crop diversification by growing TK. This target audience was predominately farmers and landowners who are looking to diversify their operations. For this group it was important to demonstrate the crops economic opportunity and trustworthy management guidelines for production.A second target audience was the scientific community in both academia and business. This audience included the rubber industry, researchers, students, farmers, seed industry, enzyme manufacturers, etc. This audience was reached by publication of research results and exchanging information at scientific exchanges, presentations and meetings. In Ohio, research results were presented to BARRAL cooperators including Goodyear and American Sustainable Rubber, and to other rubber and agricultural industry leaders at semiannual research group and other meetings. There was tremendous interest by the general public and within the business community regarding the potential for a new crop and supporting industry. Changes/Problems:The scope of work was reduced in the 2020, 2021 and 2022 growing seasons due to COVID (2020 and 2021) and severe reductions in irrigation water availability and labor availability at both KBREC and SOREC (2021 and 2022). These multi-year challenges resulted in smaller, less comprehensive studies than were initially planned. Scheduled field days in 2020 and 2021 (including discussion of this research) were canceled due to COVID-19 and in 2022 due to a lack of suitable demonstration fields to show in Oregon. Due to these limitations, some tasks planned at Oregon State University were not accomplished. This reduced effort at Oregon State allowed us to transfer $70,000 of grant funds back to Ohio State University to allow completion of other grant-related TKS research done there. In Nebraska, a severe hailstorm impacted trials in August of 2019, preventing yield results from being collected, however establishment measurements were not impacted. Covid resulted in difficulty finding student workers, and impacted outreach activities. However, planned activities did occur in 2020. A severe drought impacted trials in 2021, limiting early season germination at a time when supplemental irrigation was not available. What opportunities for training and professional development has the project provided?Students developed professionally by learning to conductresearch aspart of MS and PhD research dissertation projects. One student was trained on assessing the techno-economic feasibility and life-cycle assessment of TK production and processing. Project personnel were trained in laboratory safety andcrop production at field and greenhouse scales. Multiple undergraduate and graduate students received trained in writing publications, abstracts and making posters for research conferences. How have the results been disseminated to communities of interest?Presentations were made to BARRAL cooperators at monthly Zoom meetings, semi-annual research update meetings and presentations made by PIs at annual meetings of the Program of Excellence in Natural Rubber Alternatives (PENRA). These included the following presentations. Roseberg, R.J. and T.B. Silberstein. 2019. Agronomic management of Taraxacum kok-Saghyz for natural rubber production in Oregon. PENRA Annual Research Meeting - March 7, 2019. Roseberg, R.J. and T.B. Silberstein. 2020. Agronomic management of Taraxacum kok-Saghyz for natural rubber production in Oregon. PENRA Annual Research Meeting - March 17, 2020. Presentation prepared but meeting was cancelled due to emerging COVID-19 travel restrictions. Roseberg, R.J. and T.B. Silberstein. 2021. Agronomic management of Taraxacum kok-Saghyz for root biomass and natural rubber production in Oregon. PENRA Annual Research Meeting - March 9, 2021. Roseberg, R.J. ,T.B. Silberstein, and E. McLennon. 2022. Agronomic management of Taraxacum kok-Saghyz for root biomass and natural rubber production in Oregon. PENRA Annual Research Meeting - March 15, 2022. Field days scheduled for 2020 and 2021 (including discussion of this research) were cancelled due to COVID-19. and in 2022 due to lack of suitable demonstration fields to show in Oregon. In Nebraska, a number of field days were held to share information and goals of the research project. These included the following: Panhandle Weed Tour, June 2019, 50 Participants. PARTT Field Day, August 2020, 150 Participants.. Panhandle Weed Tour, June 2020, 20 Participants. PARTT Field Day, August 2021, 50 participants. Panhandle Weed Tour, June 2021, 40 Participants. PARTT Field Day, August 2021, 100 Participants. Many presentations were made to interested scientific groups around the world. These included: Cornish, K., Valorization and scalability of alternative rubber through properties and production processes. Proceedings of the International Conference on Development of Alternatives Sources of Natural Rubber, Montpellier, France, November 4-6, 2019. Cornish, K. Valorization and scalability of alternative natural rubber through tailoring properties, Proceedings of the Tire Technology International Conference, Hannover, Germany, February 25-27, 2020. Cornish, K. Minimizing risks around NR through alternative crops. Proceedings of the Tire Technology International Conference, Hannover, Germany, February 25-27, 2020. Ariyaratne, M., Benzle, K., Cornish, K., Increased overall carbon capture in Taraxacum kok-saghyz through the expression of a mutated de-inhibited Rubisco, OSU Plants Science Symposium, July 23, 2020. Cornish, K., Convergent rubber science and engineering, NSF Convergent Accelerator Workshop (virtual), Sept 830, 2020. Cornish, K., Natural rubber security and high-performance biobased materials, International Elastomer Conference (virtual), October 19-23, 2020. Cornish, K., A career in rubber research (virtual), Albert Pye Primary School, Beccles, England., Nov. 5, 2020. Roseberg, R.J. 2020. How Joseph Stalin used dandelions to control the future of air travel (the history and future of natural rubber). Encinitas Coastal Rotary Club, December 15, 2020. Roseberg, R.J. and T.B. Silberstein. 2021. Agronomic management of Taraxacum kok-Saghyz for root biomass and natural rubber production in Oregon. PENRA Annual Research Meeting - March 9, 2021. Description: Research results presented at annual meeting of research consortium. Cornish, K., Considerations around metabolic engineering of rubber biosynthesis, First International Conference of the BrainKorea21, February 25, 2021 (Four Smart Farming in the New Normal Era). Michel, FC. 2021. Improvements in TK Rubber Bioprocessing Yield and Recovery. PENRA Annual Research Meeting - March 9, 2021. Description: Research results presented at annual meeting of research consortium. Lankitus, D., Zhang, Y., Benzle, K., Liu, M., Fresnedo-Ramirez, J., Cornish, K., Analysis of genetically altered Taraxacum kok-saghyz (TK) for Increased Rubber Production, CFAES Research Conference, April 8, 2021. Cornish, K., Biodiversification of natural rubber by metabolic engineering, 27th Anniversary meeting of the Bio-Environmental Polymer Society (BEPS), June 23-25th, 2021. Fresnedo Ramirez, J., Genomics-aided research in the domestication and breeding of outcrossing crops. Genomes of Animals & Plants (GASP2022). Virtual. February 18, 2022. Organized by Cantata Bio former Dovetail Genomics. Cornish, K., Progress on TKS genome editing, germplasm innovation, breeding and its commercial planting in USA, Seminar, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, July 11, 2022. Cornish, K. Biological and geographical diversification of natural rubber: US supply security, rural development and public health, University of Ashland Environmental lecture series - Global Commerce, Biodiversity & Human Health, February 2, 2023, Ashland, OH. Cornish, K. Biological and geographical diversification of natural rubber: US supply security, rural development and public health Summit County Educational Service Center Tour, March 9, 2023, Wooster, OH. Cornish, K, Blakeslee JJ, Sadre, R, Ezeji, TC, Biomanufacturing of natural rubber - a critical imported raw material, Biomanufacturing Opportunities Workshop, April 4-5, 2023, Columbus, OH. Cornish, K, Blakeslee JJ, Sadre, R, Ezeji, TC. Biomanufacturing of natural rubber - a critical imported raw material. OSU meeting with UES, July 14, 2023, Columbus, OH. King-Smith, N., Cornish. K., Ebb and Flood Hydroponic Dandelion Rubber Production, CFAES Research Conference, Wooster, OH. Cornish also presented two live streams at Technobiz, on the need for natural rubber bio and geographical diversification, and an OSU podcast as one of two speakers on "The secret world of supply chains". What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Maximize crop establishment and crop production by developing and adapting common and region-specific agricultural equipment and methods. Field experiments were performed in Ohio, western Nebraska and Oregon using region-specific equipment to identify how cultural practices interact and affect Taraxacum kok-saghyz (TK) root biomass and rubber/inulin yield. In Oregon, two research sites with different climates were used; ORSU-Klamath Basin Research & Extension Center (KBREC) and the Southern Oregon Research & Extension Center (SOREC) that has a warmer and longer growing season. Factors including planting and harvest date, plant density, irrigation, soil fertility, planting methods/seedbed preparation, soil type and treatment, harvest method, and weed control (herbicide testing). Shallow seed depth and access to continuous soil moisture while avoiding poor oxygenation improved germination for both bare and pelleted seed. Natural or amended organic matter generally improved germination, due to improved moisture retention and sufficient oxygenation. Selections and crosses of field grown TK were made for large root size and 1.8 million seed of an advanced population (Foxtrot), were sent to Farmed Materials/Goodyear to meet the requirements of a $6 million grant from the US Air Force to BioMade to produce TK rubber (TNR) for jet plane tires. A high throughput, multi-generation hydroponic system was developed that allowed rare plants with rubber concentrations of 15% or greater to be selected for breeding programs while maintaining vigorous growth. Weed control is essential to assure high yields of TNR. A chemical weed control procedure was developed that involved spraying with a preemergence treatment of Pursuit 6oz/ followed by a Harmony postemergence herbicide 0.25oz/A + Select 12oz/A. Broadleaf weeds were mostly controlled (70-86%) and plant survival ranged from 65 to 81% of seeds sown. Companion-crop or stubble-seeding may be helpful with summer weed competition and TKS stand establishment where soil moisture consistency is an issue during TKS germination and establishment. Objective 2. Optimize and demonstrate field production in Ohio, Nebraska and Oregon by translating and adapting recent Ohio successes. Millions of TK seeds were produced that were used raw, or primed and coated or primed and pelletized. Raw and coated seed seem to produce the best establishment results. In western Nebraska, late March through mid-April resulted in higher TK establishment than later plantings. In contrast, TK planted in early June in Ohio did best. TK roots double in size and rubber content increases in late fall so roots should be harvested as late as possible. Thus, growing season duration is critical-the longer the better. This effect was apparent in all growing sites. In both Oregon and Ohio, TK grew larger roots on lighter soils than heavy ones. The range of plant densities achieved over several seasons ranged from 66,000-1.1 million plant/ha at harvest. Over this range, TK consistently showed a nearly linear response between total root biomass and plant density. TK plant canopy typically "closes" (100% ground area covered by plant stems and leaves) at about 1.6M - 2.0M plant/ha. Maximum economic root production probably occurs at these greater planting densities. In general, root dry biomass was between 3g and 8g per plant, with a mean of about 6g/plant. A few individual plants that survived overwintering (seeded in spring and harvested late the following summer) had root dry biomass in the 40-50g range. Roots from experiments at SOREC typically were about half the size of those grown at KBREC. Plant size and yield was greatly enhanced in the following summer. However, the unpredictability of this overwinter survival requires better understanding to reduce the risk to growers of such weather-related responses. At 8% rubber (dw), and the higher density of 2.0M plant/ha, this would yield 320 kg/ha of TK rubber. Objective 3. Maximize extractable rubber yield and purity by optimizing harvest, post-harvest handling and storage methods. Post-harvest experiments to determine possible effects of ethylene, ethephon and acid pH during a week post-harvest, demonstrated that rubber concentration and extractability increased in ethephon and acid conditions. However, gaseous ethylene did not have the same effect - possibly due to insufficient time or too low a concentration. A new method was developed that doubles the amount of extractable latex (versus solid rubber) from greenhouse grown roots by increasing rubber particle buoyancy. A provisional patent was filed on this technology. Objective 4. Maximize the yield of TK fermentable sugars, and rubber that meets tire manufacturing standards, by optimizing previously developed OSU innovative inulin and root rubber extraction and purification methods. Alkaline pretreatment of TK roots and simplified processing methods were developed to increase recovery and purity of solid rubber from TK. Alkaline pretreatment followed by enzymatic digestion was efficient, but negatively impacted rubber quality. To improve yield and quality of rubber without pretreatment, a simultaneous enzyme digestion ball milling process was developed. Rubber yields reached 82% with 97% purity. Rubber yield and purity were similar when digestion time was reduced from 48 to 3 hours, cellulase dosage was reduced by 70% and hydrolysis performed at 50 °C. This drastically reduced the time and projected capital costs for full scale recovery and purification of TK rubber. To increase the yield of latex versus solid rubber, a method which doubles the amount of extractable latex from greenhouse and field-grown roots by increasing rubber particle buoyancy was developed. Objective 5. Evaluate technoeconomic and life cycle environmental impacts of a TK rubber and sugar- based industry across the Northern United States sufficient to support US natural rubber self-sustainability. Techno-economic analysis (TEA) for Ohio TK field production for commodity ($1.6/kg) and premium rubber ($10/kg) prices were conducted for field production alone at direct seeding planting densities of 0.15 -2,4M plants/ha. Field production costs were $915-$1176/ha, and net revenue for commodity rubber at market selling price of $1.6/kg, including inulin (selling price of $4/kg) and rubber, was in the range -$595 to $634/ha. For premium rubber selling prices at $10/kg, net direct-seeded revenue was -$426 to $1,866/ha. Planting density was one of the most influential factors affecting the net revenue. Sensitivity analysis with planting density excluded found that net revenue per hectare of direct-seeded TK would be most positively affected by increasing inulin content, fresh root yield, inulin recovery rate and root recovery. While transplanting TK seedlings yields larger roots and more rubber per hectare, high greenhouse costs limit its use. Key findings were that root yield and rubber concentration must increase for direct seeded crop, and inulin valorization will be critical to establish an economically viable TK crop. Calculated total GHG emissions for the TK supply and processing were 15.1 kg CO2 eq/kg rubber compared to 13 kg and 34 kg CO2 eq/kg rubber for Hevea and guayule, respectively.

Publications

  • Type: Other Status: Published Year Published: 2023 Citation: Cornish, K, Sustainable Alternative Natural Rubber. The U.S. Arid Land Agricultural Research Center, June 9th, 2023 (paid travel).
  • Type: Other Status: Published Year Published: 2023 Citation: Cornish, K. Alternative Natural Rubber and Sustainable Additives and Fillers  the art and practice of biobased technology in tire/rubber applications. 63rd Annual General Meeting of the International Institute of Synthetic Rubber Producers, Boston. MA, May 16-19, 2023 (paid travel)
  • Type: Other Status: Published Year Published: 2023 Citation: Cornish, K. Biological and geographical diversification of natural rubber: US supply security, rural development and public health, University of Ashland Environmental lecture series - Global Commerce, Biodiversity & Human Health, February 2, 2023, Ashland, OH.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Cornish, K., Biological and geographical diversification of the global natural rubber supply  a critical agricultural material, HCS seminar, The Ohio State University. Nov, 15, 2023.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Keener, HM, A Shah, M Klingman, S Wolfe, D Pote, R Fioritto. Progress in Direct Seeding of an Alternative Natural Rubber Plant, Taraxacum kok-saghyz (L.E. Rodin) Agronomy 8 (9), 182. 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Marrero, O., Cornish., K., Barker, D., Rubber dandelion performance on contaminated soils. CFAES Research Conference, Mar 28, 2023, Columbus, OH (Won first place)
  • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: McNulty, SK. Accelerated Crop Domestication through Identification of Phenotypic Characteristics of Taraxacum kok-saghyz Relevant to Rubber Yield. MS Thesis, The Ohio State University. http://orcid.org/0000-0002-8232-9357. 2019.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang, L, A Shah, FC Michel Jr. Synthesis of 5-hydroxymethylfurfural from fructose and inulin catalyzed by magnetically-recoverable Fe3O4@SiO2@TiO2HPW nanoparticles. Journal of Chemical Technology & Biotechnology 94 (10), 3393-3402. 2019.
  • Type: Websites Status: Published Year Published: 2019 Citation: Ostdiek, D. Can Nebraska Grow Rubber-Producing Dandelions on a Farm Scale? CropWatch. 221 Page Views. May 17th, 2019. https://cropwatch.unl.edu/2019/dandelion-rubber.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: David A. Ramirez-Cadavid, Jesse Rossington, Frederick C. Michel. 2019. Effects of pretreatment on the properties of rubber extracted from Taraxacum kok saghyz, the rubber dandelion. Conference: 2019 ASABE Annual International Conference, Proceedings paper. Paper # 1901187. Boston, MA.
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Mofidi, SSH, M Reza Naghavi , M Sabokdast, P Jariani, M Zargar , K Cornish. Effect of drought stress on natural rubber biosynthesis and quality in Taraxacum kok-saghyz roots. Plus One (submitted, in review, PONE-D-23-31319).
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Puskas, JE, K Cornish, B Kenzhe-Karim, M Mutalkhanov, K Molnar and G Kaszas. Natural Rubber  an Irreplaceable Renewable Resource. Submitted to Helyion Journal (in review)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Roseberg, R.J., T.B. Silberstein, and E. McLennon. 2023. Improving germination of rubber dandelion (Taraxacum kok-saghyz Rodin) for improved stand establishment and root rubber yield. Abstr. P. 27, Assoc. for the Advancement of Industrial Crops Annual Meeting, Corvallis, OR, Aug. 27-30, 2023.
  • Type: Other Status: Published Year Published: 2019 Citation: Stephen, K. Dandelions as a Cash Crop. Scottsbluff Star Herald November 24, 2019. (weekly Distribution of 15,000. https://starherald.com/townnews/agriculture/dandelions-as-a-cash-crop/article_34a6d9d3-f2eb-5f95-8d1e-00bd109b2154.html


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Target audiences includegrowers who could benefit from crop diversification by growing TK. This target audience is predominately farmers and landownerswho are looking to diversify their operations. For this group it is important to demonstrate the cropseconomic opportunity and trustworthy management guidelines for production. This involves publishing Extension guides and conducting field demonstrations/field days. A second target audience is the scientificcommunity in both academia and business. This audience includes the rubber industry, researchers, students, farmers, seed industry, enzyme manufacturers, etc. This audience can be reached by publications of research results and exchanging information at scientific exchanges. In Ohio, results of research hasbeen presented to BARRAL cooperators including Goodyear and American Sustainable Rubber, and to other rubber and agricultural industry leaders at semiannual research group and othermeetings. Additionally, there is tremendous interest by the general public, and within the business community, regarding the potential for a new crop and supporting industry. Changes/Problems:Numerous challenges were encountered due to the ongoing COVID-19 pandemic. Restrictions on field, lab, and greenhouse work until spring 2022 have resulted in smaller, less comprehensive studies. Hiring freezes by Universities resulted in difficulties in hiring appropriate seasonal labor and student interns that affected timeliness of some research operations. An irrigation water shortage at KBREC and SOREC also negatively affected research operations in 2021 and 2022. At KBREC no regular irrigation district water delivery occurred at all for the 2021 or 2022 growing seasons. Very small experiments planted in spring 2021 were maintained by a small amount of emergency well water or a very small amount of city water. At SOREC the regular irrigation district water deliveries were cut off in July for both 2021 and 2022. Existing plantings were maintained with the small amount of emergency well water available, but no new plantings were made in spring 2022. Overwintering plant survival is good at KBREC in some years, but significant mortality can occur depending on winter weather and plant vigor in fall. When overwinter survival is good, resulting plant size and yield is greatly enhanced the following summer. However, the unpredictability of this survival warrants better understanding of such weather-related responses. Weed control continues to be a significant challenge. Because TK grows slowly, a better herbicide program is required if the goal is a clean field to minimize competition. However, based on TK's native growth environment, there may be situations where companion-cropping might provide better weed competition while not hindering TKS growth and yield. Companion crop composition, timing, and other management issues have yet to be worked out. Planned extension events to showcase TKS during the reporting year were also canceled due to COVID-19 restrictions. A no cost Extension was sought and granted to be able to address some of these challenges. At Oregon State, the long-term Faculty Research Assistant who was responsible for nearly all day-to-day operations retired at the end of 2021. Some of his effort is now done by a post-doctoral researcher stationed at KBREC. In Ohio, multiple grant proposals were prepared and submitted but no funds were forthcoming apart from a new equipment grant. John Cardina, the weed scientist PI on the project, retired during COVID leaving a gap in expertise on the project. What opportunities for training and professional development has the project provided?Project personnel continueto keep up with laboratorysafety training. Students receivedtrained in making posters for research conferences. How have the results been disseminated to communities of interest?On April 7, 2022, the Goodyear Tire & Rubber Companyannounced a multi-year, multi-million-dollar program supported by the U.S. Department of Defense (DoD), the Air Force Research Lab (AFRL)to work with Ohio-based Farmed Materials to develop a domestic source of natural rubber from TK germplasm developed at OSU as part of this project. Several meetings with the USDA led Domestic rubber strategy group and otherconference presentations and press releases were made. What do you plan to do during the next reporting period to accomplish the goals?Optimization of latex extraction methods and TK root production using amultiple harvest hydroponics approach will be developed. In Nebraska, Surviving accessions may be well adapted, seed increases are currently ongoing to continue breeding efforts into 2023. A publication is planned on the role of soil porosity in controlling TK seed germination. In Oregon, BARRAL-related plots will be harvested. Aplanned sequential field planting at KBREC and SOREC will be continued on a reduced scale in spring 2023, pending availability of irrigation water and appropriate seasonal labor. For each harvest sampling, roots will be washed and air-dried before shipment to Ohio for rubber and inulin analysis. The series of growth chamber germination studies will continue to further understand the effects of soi moisture, native or amended organic matter, and related factors on germination success. Completing a publication describing the effect of various seedbed factors on germination is planned for 2022-23.Publication of peer-reviewed papers describing the research outcomes and yet more attempts to obtaingrant funding and industrial support will continue the last year of the project is also planned for the last year of the project.

Impacts
What was accomplished under these goals? Objective 1. Maximize crop establishment and crop production by developing and adapting common and region-specific agricultural equipment and methods. New advanced TK populations were characterized following selection among field grown plants. The Golf population has nearly 1.5x the root size of Foxtrot. The mean rubber content the top 15 plants was 103 mg/g dwt, with the highest individual containing 137 mg/g dwt. Work at Oregon State (ORSU) was focused at ORSU-Klamath Basin Research & Extension Center (KBREC), and the Southern Oregon Research & Extension Center (SOREC). The ongoing series of growth chamber experiments were conducted with Taraxacum kok-sagyz (TKS) to identify factors that enhance seed germination. Soil texture and organic matter, soil amendments, seed depth, seed type/age, moisture duration, oxygenation, and seed pelleting were examined. Shallow seed depth and access to continuous soil moisture while avoiding poor oxygenation all improved germination for both bare and pelleted seed. Presence of natural or amended organic matter generally improved germination, but this could be a function of improved moisture retention along with sufficient oxygenation rather than simply organic matter content per se. Objective 2. Optimize and demonstrate field production in Ohio, Nebraska and Oregon by translating and adapting recent Ohio successes InOhio, 1.8 million seed of Foxtrot, an advanced population selected for root size, were transferred to Farmed Materials/Goodyear so that they could establish the first crop they need to meet the initial requirements of their $6 million grant from BioMade (Air Force funding) to produce TK rubber for jet plane tires. In Oregon, portions of several field experiments that were seeded at KBREC and SOREC in fall 2020 and/or spring 2021 were sampled in fall 2021. These continued to demonstrate the trend that significant root growth and rubber production occurs in the last few weeks before fall harvest for spring-seeded crops as well as for crops that survived overwintering and harvested the following summer. However, the scope of work was reduced in the 2021 and 2022 growing seasons due to COVID (2021) and severe reductions in irrigation water availability and labor availability at both KBREC and SOREC (2021 and 2022). Weed control was also an issue affecting some of these experiments. In the range of plant densities we have achieved in field plantings (ranging from 30,000 plant/acre to 500,000 plant/acre at harvest), TKS showed a nearly linear response between total root biomass and plant density, confirming previous estimates that mature plant densities need to be well above this range to maximize root yield. Root mass dry weights at KBREC were usually between 3g and 8g per plant, with a mean of about 6g/plant, translating to a dry matter root yield of about 1200 lb/acre. However, a few individual plants that survived overwintering (seeded in spring and harvested late the following summer) have had root biomass in the 40-50g range. Roots from experiments at SOREC tended to be about half the size of those grown at KBREC. This "root vigor" effect has been seen in other crops grown commercially in the Klamath Basin such as sugar beets, horseradish, potatoes, and non-root crops such as strawberry plantlets and peppermint. In Nebraska, field trials continued to screen advance germplasm. 2022 was an extremely dry and hot year and consequently the majority of lines screened failed to germinate. However, those that did germinate under extreme conditions are currently undergoing seed increases and will be used to develop future Nebraska specific accessions in planned future experiments. Additionally, previous lines which have shown promise in Nebraska field trials are currently undergoing greenhouse testing to compare germination potential at various depth under controlled conditions. Results of greenhouse experiments will be used to better screen accessions prior to 2023 field trials. Objective 3. Maximize extractable rubber yield and purity by optimizing harvest, post-harvest handling and storage methods and/or equipment. New processing methods were developed to extract and purify latexfrom Taraxacum kok-saghyz roots. This form can be used in high margin specialty dipped product markets and can be dried to solid rubber of high purity. Objective 4. Maximize the yield of TK fermentable sugars, and rubber that meets tire manufacturing standards, by optimizing previously developed OSU innovative inulin and root rubber extraction and purification methods. Rubber yields using the simultaneous enzyme digestion ball milling process and biological treatmentreached 82% and 99% respectively in lab scale experiments. Rubber yield and purity were found to be similar when digestion time was reduced from 48 to 3 hours, cellulase dosage was reduced by 70% and hydrolysis was performed at 50 C. An invention disclosure was filed on this process at Ohio State. Objective 5. Evaluate technoeconomic and life cycle environmental impacts of a TK rubber and sugar based industry across the Northern United States sufficient to support US natural rubber self-sustainability. A preliminary assessment of life cycle greenhouse gas (GHG) emissions was conducted for the production, supply chain, and processing of field-grown TK roots to extract rubber. Inputs to the system included diesel, fertilizer, herbicide, electricity, and processing chemicals and enzymes. Total emissions for the TK supply and processing was 15.1 kg CO2 eq/kg rubber which is comparable to guayule rubber. The projected rubber yield based on field grown Golf plants is 720 kg/ha in Ohio. New yield values in Oregon and Nebraska will be also used to reassess the life cycle assessment.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Roseberg, R.J. ,T.B. Silberstein, and E. McLennon. 2022. Agronomic management of Taraxacum kok-Saghyz for root biomass and natural rubber production in Oregon. PENRA Annual Research Meeting - March 15, 2022.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Salehi, M., Bahmankar, M., Naghavi, M.R., Cornish, K. Rubber Extraction Processes for Taraxacum kok-saghyz, Industrial Crops and Products, 178: 114562, 2022.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ramirez-Cadavid, D.A., Cornish, K., Hathwaik, U., McMahan, C.M., Michel, Jr., F.C., Alkaline Pretreatment of Taraxacum kok-saghyz (TK) Roots for the Extraction of Natural Rubber (NR), Biochemical Engineering Journal 181: 108376, 2022.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: King-Smith, N, Cornish, K., Taraxacum kok-saghyz latex quantification and extraction from roots. Rubber World. November 2022. (editor reviewed)
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Lankitus, D., Zhang, Y., Ariyaratne, M., Barker, D.D., McNulty, S.L., Amstutz, N., Zhao, L., Iaffaldano, B.J., Cornish, K., Agrobacterium rhizogenes-induced altered morphology and physiology in Taraxacum kok-saghyz (rubber dandelion) after genetic transformation, Journal of the American Horticultural Society
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ren, X., Barrera, C.S., Tardiff, J.L., Cornish, K., Sustainable epoxidized guayule natural rubber, blends and composites with improved oil resistance and greater stiffness. Materials 15: 3946, 2022, https://doi.org/10.3390/ma15113946
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ren, X.; Barrera, C.S., Tardiff, J.L., Gil, A.; Cornish, K. Liquid guayule natural rubber, a sustainable processing aid, enhances the processability, durability and dynamic mechanical properties of rubber composites. Materials 15: 3605, 2022. https://doi.org/10.3390/ma15103605
  • Type: Books Status: Published Year Published: 2022 Citation: Barrera, C.S., Cornish, K. Fly ash as a potential filler for the rubber industry, Chapter 25, 765-792, In: Handbook of Fly Ash, Kamal K. Kar (ed). Pub. Butterworth-Heinemann, an imprint of Elsevier, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Cornish, K., Progress on TKS genome editing, germplasm innovation, breeding and its commercial planting in USA, Seminar, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, July 11, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Fresnedo Ramirez, J., Genomics-aided research in the domestication and breeding of outcrossing crops. Genomes of Animals & Plants (GASP2022). Virtual. February 18, 2022. Organized by Cantata Bio former Dovetail Genomics.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Cornish, K., Herkins, A., Davis, S., Latex gloves provide frontline protection for healthcare workers and patients  but how effective are they really? Pathogens-eCon2022, November 30, 2022.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Ramirez-Cadavid, DA, S Valles-Ramirez, K Cornish, FC Michel JrSimultaneous quantification of rubber, inulin, and resins in Taraxacum kok-saghyz (TK) roots by sequential solvent extraction. Industrial Crops and Products 122, 647-656
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Cornish, K., Sustainable alternative fillers for latex and rubber. Rubber Industry News Hour, May 23, 2022.
  • Type: Books Status: Published Year Published: 2021 Citation: Cardina, J. Lives of Weeds: Opportunism, Resistance, Folly. Cornell University Press. 296 pp. ISBN:9781501759000, 1501759000


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:One target audience is growers who could benefit from crop diversification by growing TK. This target audience is predominately farmers and landowners, who are looking to diversify their operations. For this group it is important to demonstrate the crop's economic opportunity and trustworthy management guidelines for production. This involves publishing Extension guides and conducting field demonstrations/field days. A second target audience is the scientific community. This audience includes the rubber industry, researchers, students, farmers, seed industry, enzyme manufacturers. This audience can be reached by publications of research results and exchanging information at scientific exchanges. In Ohio, results of research have been presented to BARRAL cooperators including Goodyear and American Sustainable Rubber, and to other rubber and agricultural industry leaders at semiannual research group meetings. Additionally, there is tremendous interest by the general public, and within the business community, regarding the potential for a new crop and supporting industry. Changes/Problems:Numerous challenges were encountered due to the ongoing COVID-19 pandemic. Restrictions on field, lab, and greenhouse work in 2020 and 2021 have resulted in smaller, less comprehensive studies. Hiring freezes by Universities resulted in difficulties in hiring appropriate seasonal labor and student interns that affected timeliness of some research operations. all PIs from NE were extremely short-staffed on TK-related projects due to a University-wide hiring freeze, which prevented the planned hiring of multiple undergraduate students. COVID restricted the ability to conduct all aspects of field, greenhouse, and laboratory research. In spite of this, field studies were established but began at a later than optimum date. An irrigation water shortage at KBREC and SOREC also negatively affected research operations. At KBREC no regular irrigation district water delivery occurred at all for the 2021 growing season. Experiments planted in Fall 2020 and very small experiments planted in spring 2021 have been maintained by a small amount of emergency well water or a very small amount of city water. At SOREC the regular irrigation district water deliveries were cut off in mid-July. Existing plantings are being maintained with the small amount of emergency well water available, but no new plantings will be made in 2021. The reduced field operations resulted in a lack of updated experimental data for TEA and lifecycle modeling. The lack of harvest material and reduced plot sizes also severely impacted Objective 3. Overwintering plant survival is good at KBREC in some years, but significant mortality can occur depending on winter weather and plant vigor in fall. When overwinter survival is good, resulting plant size and yield is greatly enhanced the following summer. However, the unpredictability of this survival warrants better understanding of such weather-related responses. Weed control continues to be a significant challenge. Because TK grows slowly, a better herbicide program is required if the goal is a clean field to minimize competition. However, based on TK's native growth environment, there may be situations where companion-cropping might provide better weed competition while not hindering TKS growth and yield. Companion crop composition, timing, and other management issues have yet to be worked out. In summer, problems occurred with hot and humid greenhouse conditions preventing seed set. In addition, a seed store overheated and everything had to removed and replaced after the repair. Some students related to the project work have found it very difficult to work under COVID-19 restrictions and two left prematurely. Planned extension events to showcase TK during the reporting year were also canceled due to COVID-19 restrictions. Use of antioxidants will be used to investigate methods to reduce rubber quality degradation during pretreatment, extraction and purification. A no cost Extension was sought and granted to be able to address some of these challenges. What opportunities for training and professional development has the project provided?Two graduate students were trained on conducting systems-level techno-economic and life cycle analyses of TK based natural rubber production. One graduate student has graduated with an M.S. degree. One postdoc was trained in the use of FTIR, GPC and other analytical methods for the analysis of TK rubber. Project personnel continue to keep up with pesticide training. Students participated in virtual conferences, including presentations at the OSU Plant Sciences Symposium and the OSU-CFAES Research Conference. One Postdoc on the project obtained a position at Kultevat, a company commercializing TK rubber production. He will design and develop their rubber extraction process. How have the results been disseminated to communities of interest?Results have been disseminated mostly virtually due to COVID restrictions. Zoom presentations of TK TEA model results have been made to principal investigators and others working on the project. Research results by all investigators were made to industry cooperators at full day semi-annual research meetings. Results were also disseminated at the (virtual) annual meeting of the Program of Excellence in Natural Rubber Alternatives academic/industrial consortium. Field days scheduled for 2020 and 2021 (including discussion of this research) were cancelled due to COVID-19. Investigators have also contributed to public press stories written as rubber supply problems in the US become more critical. Reports include BBC, CNBC, Rubber and Plastics News, Marketplace Morning Report, Chemistry World, and OSU's Alumni Magazine. What do you plan to do during the next reporting period to accomplish the goals?In the final year of the project, TEA models will be improved and further dveloped with updated experimental data, as it becomes available. BARRAL-related plots will be harvested in fall 2021 at both KBREC and SOREC. The sequential field plantings will be continued as outlined in the scope of work for spring/fall 2021. For each harvest, roots will be washed and air-dried before shipment to Ohio for rubber and inulin analysis. Harvest field evaluations of improved Ohio State germplasm in Oregon is planned in spring/fall 2022 pending lifting of COVID restrictions and availability of irrigation water and appropriate seasonal labor. Two reps of the OH selection nursery have been planted. One will be harvested at 4 months on Oct. 1, and then vernalized to induce flowering. The other will be harvested in December. Advancements from the two reps will be crossed in the greenhouse independently from one another, and then planted in the 2022 field for comparison. If selections made at 4 months maturity are comparable to full term selections, then it may be possible to reduce selection periods to enable an increase in the number of selections per year. In greenhouse experiments, field selections from both Ohio and Nebraska will be crossed in the greenhouse this winter. Once the rubber concentration analysis is complete, previously collected seed from the rubber concentration advancements will be planted in the greenhouse and the inbreeding cycle will continue while monitoring for deleterious effects. An experiment will be conducted regarding the logistics of backcrossing offspring to parent, a valuable breeding technique that could prove useful when introducing traits such as self-seeding or herbicide tolerance into more robust germplasm. Primary concerns are flower synchronization and inbreeding depression. The experiment will use the NE Golf plants (currently in vernalization) and their NE Hotel offspring. The non-flowering potential biennial screen of F1 and F2 plants will continue for 6 months, culling all plants that flower and monitoring the ratio of flowering to non-flowering plants. Advancements will be crossed and screened again for 7 months, again culling those that flower and monitoring segregation ratios. This process will continue until the non-flowering trait is fixed. The same screening process will happen for Washington and Golf potential biennials. Efforts will be Increased on germplasm development so that a more robust plant with more rapid establishment can be used in field settings. Field experiments conducted during the 2020 / 2021 field season will continue next year as proposed. Additionally, further greenhouse and field studies investigating germplasm and soil amendments are planned for the 2021-2022 field season to take advantage of discoveries in the past year. A manuscript detailing the role of soil porosity, or available oxygen, is planned for publication in 2022. To improve rubber recovery, further tests of simultaneous pebble milling and enzymatic hydrolysis without pretreatment, will be made to improve rubber recovery and purity. Studies on the use of antioxidants to reduce rubber quality degradation during pretreatment will also be done.

Impacts
What was accomplished under these goals? Objective 1. In Ohio, growth chamber experiments were conducted with Taraxacum kok-sagyz (TK) to identify methods to enhance seed germination. Soil temperature, soil texture and organic matter, soil amendments, seed depth, moisture, and seed pelleting were examined. Seed depth and access to good and continuous soil moisture were essential for dependable germination for both bare and pelleted seed. Over 4 million TK seed from OSU production greenhouses were primed, pelletized and returned for project test plots. The Ohio 2020 field selection nursery was a complete loss due to COVID, but Golf (G) and Washington (W) germplasm plants grown to maturity were received from the Nebraska nursery. Over winter, top performers were selected, vernalized, moved to the greenhouse, and crossed to create NE Hotel (H) and NE Adams (A) lines. In 2021, the Ohio field selection nursery was direct seeded and has been successful to this point. Growing are G and W plants (advancements to form H and A), Foxtrot (F) and A as controls and each of the NE H advancements (23) and NE A advancements (11) to compare with the same material being grown in Nebraska. Work at Oregon State (ORSU) was focused at ORSU-Klamath Basin Research & Extension Center (KBREC), and the Southern Oregon Research & Extension Center (SOREC). Several field experiments were seeded in fall 2020 and spring 2021, with first harvest planned for fall 2021. However, the scope of work was reduced in the 2020 and 2021 growing seasons due to COVID and severe reductions in irrigation water availability and labor availability at KBREC and SOREC. Seed production in 2020, resulted in 4.8 million seeds from the greenhouses (both F and G). This year, estimates indicate that numbers will surpass last year's (all G). W source material was increased in the OSU Madison Greenhouses over the winter. During the 2020/21 winter, previous rubber concentration advancements were grown in the greenhouse and crossed (half-sibbed) to form an S2 generation in rubber concentration beta selections (derived from Hi x Hi), and S1 generation in RCAs (derived from A). Seed have also been collected from TK plants that germinate at greater depths. Approximately 12,000 seed were collected which geminated from soil depths of 0.5 inches and deeper and 1500 seed were sent to NE for screening. Field establishment of TK was successful by seeding with a commercial shallow depth Jang seeder. Broadcast seeding has also been successful. Soil moisture must be maintained for at least 7 days to assure germination and early establishment as germination and seedling growth are slow resulting in competitive disadvantage relative to weeds. Mechanical cultivation is difficult without seedling injury. Therefore, pre-emergence herbicides are being used. Of over 30 herbicides tested, those safest on TK seedlings are imazethapyr and sulfentrazone at their lowest labeled rates. All others caused excessive TK injury at lowest useful rates of application. The only broadleaf herbicide that can be applied without injury is thifensulfuron. All post-emergence grass herbicides appear to be safe. Initial efforts at recurrent selection for herbicide tolerance have produced a line of TK with increased tolerance to imazethapyr, but this did not translate to tolerance to other ALS herbicides. No reliable tolerance to other herbicide classes has been found. To increase establishment success, various land preparation methods have been tested. Optimum planting date corresponded to the last week in May and first week in June. Overwintered plants seeded on a range of dates did not survive. Raised beds amended with compost gave slight increases in establishment and allowed for heavier root growth. In Nebraska, greenhouse trials indicated that soil porosity, or available oxygen, and available soil water, are limiting factors in TK germination. These trial results were confirmed through field experiments where soil porosity and water availability in the top 15 cm was improved using composted manure and surface-drip irrigation. Experiments are ongoing to leverage soil amendments to increase soil porosity in the top few cm of soil, to provide a scalable methodology to improve TK germination in a field setting. Objective 2. At KBREC, fall 2020 harvests occurred for some experiments that were seeded in fall 2019 and spring 2020. Due to variable summer weed pressure and variable winter survival, plant density in individual plots ranged from 30,000 plant/acre to 400,000 plant/acre. This range of plant densities showed a nearly linear response between total root biomass and plant density, confirming previous estimates that mature plant densities need to be well above this range to maximize root yield. Root mass dry weights were usually between 3g and 8g per plant, with a mean of about 6g/plant, translating to a dry matter root yield of about 1200 lb/acre. Field scale plantings of TK were established on silt loam soils in northern Ohio with mixed results. Successful establishment corresponded to sites with adequate rainfall immediately after planting, however puddling and poor drainage were not tolerated by TK seedlings. The imazethapyr weed control approach was successful for the limited number of weeds susceptible to that herbicide. Weeds that could not be controlled included yellow nutsedge and Canada thistle. Field TK breeding trials conducted at the Panhandle-REC have found several lines which germinate well with minimal irrigation and no use of soil amendments in coarse low-organic matter soil. Given that the success in increasing establishment through germplasm improvements and the use of soil amendments have been independent, we have two different processes to leverage to adapt TK as a production crop in Western Nebraska, greatly improving the chance of success among stakeholders. Objective 3. A series of experiments were conducted using ethylene exposure at different concentrations to replace previously successful soaking of harvested roots in ethephon as a processing aid. However, gaseous ethylene exposure did not improve extractability of rubber from fresh roots. Objective 4. Alkaline pretreatment of TK roots was further investigated in the past year. Alkaline pretreatments of TK roots, after inulin extraction, were tested at a range of sodium hydroxide loading rates from 33 to 132 mg NaOH/g TK roots and temperatures of 25°C to 160°C. After pretreatment, the material was enzymatically digested with pectinase and cellulase. As NaOH loading rate and temperature increased, rubber yield increased from a low of 67.6% to a maximum of 82.6%, but rubber molecular mass and rubber gel content decreased. Rubber impurity content and thermal properties were unaffected by loading rate or temperature. The pooled mean of dirt contents among treatments was 2.6±0.4% w/w. The untreated control yielded 63.0% with a dirt content of 3.1±0.2% w/w. FTIR analysis indicated that alkaline pretreatments modified non-rubber components. SEM images revealed dramatic structural changes in embedded root tissues as a function of reaction temperature. Thus, while alkaline pretreatment followed by enzymatic digestion was efficient, at temperatures above 120°C and NaOH loadings above 66 mg NaOH/g TK roots, the process negatively impacted rubber quality. A simultaneous pebble milling and enzyme hydrolysis process was developed that reduces processing time from 48 hours to 3 hours, drastically reducing the capital costs of processing. Objective 5. An assessment of life cycle greenhouse gas (GHG) emissions was conducted for the production, supply chain, and processing of field-grown TK roots to extract rubber. Inputs to the system included diesel, fertilizer, herbicide, electricity, and processing chemicals and enzymes. Total emissions for the TK supply and processing were 15.1 kg CO2 eq/kg rubber which is comparable to guayule rubber. Comparisons to Hevea rubber are underway.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: *Roseberg, R.J. and T.B. Silberstein. 2021. Agronomic management of Taraxacum kok-Saghyz for root biomass and natural rubber production in Oregon. PENRA Annual Research Meeting - March 9, 2021. Description: Research results presented at annual meeting of research consortium.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: *Roseberg, R.J. 2020. How Joseph Stalin used dandelions to control the future of air travel (the history and future of natural rubber). Encinitas Coastal Rotary Club, December 15, 2020.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Salehi, M., Cornish, K., Bahmankar, M. Naghavi, M.R., Natural rubber-producing sources, systems, and perspectives for breeding and biotechnology studies of Taraxacum kok-saghyz, Industrial Crops and Products: Volume 170, 15 October 2021, 113667
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Michel, FC. 2021. Improvements in TK Rubber Bioprocessing Yield and Recovery. PENRA Annual Research Meeting - March 9, 2021. Description: Research results presented at annual meeting of research consortium.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ramirez-Cadavid, D.A., Cornish, K., Michel, F.C.,Jr., Alternative NR Production: An investigation has been carried out into the processing of Taraxacum kok-saghyz roots to produce natural rubber (NR), inulin and co-products, Tire Technology International 2020 Annual Review, p 60-63, 2020.
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Abdul Ghaffar, M.A., Cornish, K. New Developments in rubber particles biogenesis of rubber-producing species. Chapter 10: 153-168, In: The Rubber Tree Genome, Compendium of Plant Genomes, Eds: Minami Matsui and Keng-See Chow, Springer Nature Switzerland AG, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Cornish, K., Natural rubber security and high-performance biobased materials, International Elastomer Conference (virtual), October 19-23, 2020.
  • Type: Other Status: Published Year Published: 2020 Citation: Cornish, K., A career in rubber research (virtual), Albert Pye Primary School, Beccles, England., Nov. 5, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ariyaratne, M., Benzle, K., Cornish, K., Increased overall carbon capture in Taraxacum kok-saghyz through the expression of a mutated de-inhibited Rubisco, OSU Plants Science Symposium, July 23, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Cornish, K., Convergent rubber science and engineering, NSF Convergent Accelerator Workshop (virtual), Sept 830, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Cornish, K., Biodiversification of natural rubber by metabolic engineering, 27th Anniversary meeting of the Bio-Environmental Polymer Society (BEPS), June 23-25th, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Lankitus, D., Zhang, Y., Benzle, K., Liu, M., Fresnedo-Ramirez, J., Cornish, K., Analysis of genetically altered Taraxacum kok-saghyz (TK) for Increased Rubber Production, CFAES Research Conference, April 8, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Cornish, K., Considerations around metabolic engineering of rubber biosynthesis, First International Conference of the BrainKorea21, February 25, 2021 (Four Smart Farming in the New Normal Era).
  • Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Ramirez-Cadavid, D, K Cornish, U Hathwaik, C McMahan, FC Michel Jr. 2021. Alkaline Pretreatment of Taraxacum kok-saghyz (TK) Roots for the Extraction of Natural Rubber (NR). Biological Engineering Journal. Accepted for publication 12/2021.


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:The Target Audience includes the rubber industry, researchers, students, farmers, seed industry, enzyme manufacurers. In Nebraska the target audience is predominately farmers and landowners, who are looking to diversify their operations. Additionally, there is tremendous interest by the general public and within the business community regarding the potential for a new crop and supporting industry in western NE. In Ohio presentations of weed results have been made at meetings with rubber industry partners. Results of research have been presented to BARRAL cooperators including Goodyear, Cooper Tire, American Sustainable Rubber, Atlantic Biomass LLC and to other rubber and agricultural industry leaders at semi-annual research group meetings (All) Public press stories included: Wayne and Holmes County editions of the Bargain Hunter publications Ohio COuntry Journal, The Wooster Weekly, The OSU Lantern, Tire technology International, Automotive Magazine Presentations included: September 19-22, 2019, Review of Heilongjiang Academy of Sciences led rubber dandelion program. Katrina Cornish, Seminar on rubber dandelion at the Biotechnology College of Heilongjiang University, September 25-26, 2019, ITEC Green Tire Conference, Cuyahoga Falls, OH, Katrina Cornish, Alternative and Sustainable Natural Rubbers and Additives October 3-5, 2019, Seminar at Michigan State University, East Lansing MI, Katrina Cornish, Alternative rubber crops - increasing yield and viability. November 4- 6, 2019, International Conference on Development of Alternatives sources of Natural Rubber, Montpellier, France, Plenary talk, Katrina Cornish, Valorization and scalability of alternative rubber through properties and production process. November 15, 2019, TEDxColumbus, SPARK, Invited, and presented to (i) high school students (morning) (ii) then again to paying public (afternoon). One of 13 selected of 179 submissions. Katrina Cornish, Natural rubber. November 26, 2019, Seminar to the Department of Food Agricultural and Biological Engineering, Wooster, OH, (Cornish), Valorization and Scalability of Domestic Natural Rubber through Properties and Agricultural, Processing and Production Engineering. TK Technoeconomic modelling results were also presented to Graduate Students in FABE (Shah) Changes/Problems:The project has been significantly impacted by the OSU response to COVID-19 as OSU campuses were depopulated except for essential maintenance and COVID-19 research. This prevented our OSU personnel from accessing laboratory space and completing laboratory and greenhouse-based research. OSU began a Phased repopulation in June and Phase 5 begins on August 15th. However, building use is restricted to 25% normal occupancy and requires masks and six feet of separation. In Ohio, irrigation failed at planting; by the time seedlings emerged, yellow nutsedge was established and cannot be controlled without harm to the TK crop. Some experiments were repeated as a result. Lack of enough experimental data for TEA and LCA models has been another resullt due to COVID-19. Overwintering plant survival is good in some years, but significant mortality can occur depending on winter weather and plant vigor in fall. Some aspects of studies involving over-wintering plants may need to be modified based on weather-related responses. We will likely continue the companion study at SOREC in Oregon to further examine weather/climate effects. Weed control continues to be a significant issue. Because TKS grows slowly, a better herbicide program is required if the goal is a clean field to minimize competition. However, based on TKS's native growth environment, there may be situations where companion-cropping might provide better weed competition while not hindering TKS growth and yield. Companion crop composition, timing, and other management issues have yet to be worked out. In Nebraska planned extension events to showcase TK during the reporting year were canceled due to COVID-19 restrictions. Additionally, all PIs from NE were extremely short staffed on TK related projects due to a University-wide hiring freeze, which prevented the planned hiring of multiple undergraduate students. What opportunities for training and professional development has the project provided?Project personnel continue to keep up with pesticide training. Advanced Excel training for staff. Participation in virtual conferences, including making posters for the POSU Plant Sciences Symposium Two graduate students are being trained in conducting systems-level techno-economic and life cycle analyses of TK based natural rubber production. One undergraduate completed the Ohio Research Internship Program, authoring a report and making a scientific presentation. How have the results been disseminated to communities of interest?Presentations of weed results have been made at meetings with industry partners. Presented to BARRAL cooperators at semi-annual research group meetings. Public press stories included: Tire technology International, Automotive Magazine, The Wooster Weekly, The OSU Lantern Presentations included: September 19-22, 2019, Review of Heilongjiang Academy of Sciences led rubber dandelion program. Katrina Cornish, Seminar on rubber dandelion at the Biotechnology College of Heilongjiang University, September 25-26, 2019, ITEC Green Tire Conference, Cuyahoga Falls, OH, Katrina Cornish, Alternative and Sustainable Natural Rubbers and Additives October 3-5, 2019, Seminar at Michigan State University, East Lansing MI, Katrina Cornish, Alternative rubber crops - increasing yield and viability. November 4- 6, 2019, International Conference on Development of Alternatives sources of Natural Rubber, Montpellier, France, Plenary talk, Katrina Cornish, Valorization and scalability of alternative rubber through properties and production process. November 15, 2019, TEDxColumbus, SPARK, Invited, and presented to (i) high school students (morning) (ii) then again to paying public (afternoon). One of 13 selected of 179 submissions. Katrina Cornish, Natural rubber. November 26, 2019, Seminar to the Department of Food Agricultural and Biological Engineering, Wooster, OH, Katrina Cornish, Valorization and Scalability of Domestic Natural Rubber through Properties and Agricultural, Processing and Production Engineering. What do you plan to do during the next reporting period to accomplish the goals?Weed control and agronomic practice experiments will be repeated. Additional experiments will be conducted to better identify optimum planting time. We will evaluate establishment and weed control using improved seed genetics. Postharvest ethylene treatments to enhance rubber yield and extractability will be repeated and extended to longer time periods. Samples of putative polyploids will be sent to a testing company in the EU because our internal ploidy analyzer is no longer operating. Two accelerated solvent extractors will be brought back into operation to quantity tuber and inulin and tackle the backlog of samples built up through the COVID restrictions. Additional post harvest cool and cold storage experiments will be performed on fall-harvested fresh roots. Seed will be produced, collected, cleaned and distributed from our latest population - which has a 2x increase in field grown root size. Seed scale up from plants grown from deep plantings (two generations of selection) will be collected, cleaned and sent to U Nebraska which has field conditions that require deeper plantings than Ohio and Oregon. Oregon will conduct BARRAL-related plot harvest in fall 2020. Continue sequential field plantings as outlined in scope of work in fall 2020 and spring/fall 2021. Second BARRAL-related harvest in summer and fall of 2020. For each harvest, roots will be washed and air-dried before shipment to Ohio St Univ. for rubber/inulin analysis. Harvest field evaluation of the improved Ohio State germplasm is planned in spring/fall 2021. Nebraska field experiments conducted during the 2019 / 2020 field season will continue next year as proposed. Rubber processing experiments will to examine the potential for enzyme recycling to reduce costs. Additional sources of cellulase, pectinase and hemicellulase enzymes tested. Experiments with lignin modifying enzymes such as laccase and manganase and lignin peroxidases will be conducted. Antioxidant effects on rubber deterioration during thermochemical pretreatments will be examined. TEA models will be improved with updated experimental data, and process data as available.Life cycle assessment based on comprehensive findings of the plant production and process research will be developed.

Impacts
What was accomplished under these goals? Objective 1 Several field experiments were begun in fall 2018 in Oregon with first harvest in fall 2019. The goals were to examine how seeding date, harvest date, plant density, irrigation rate, soil fertility, seeding methods/seedbed preparation, harvest method, and weed control (herbicide testing) affect rubber and inulin yields. In winter 2018-19, stand counts (plant density) were generally good. Early winter provided good snow cover but in late winter lack of snow cover and widely varying temperatures resulted in widespread soil frost heave, killing many plants. In spring 2019 poor weed control also damaged the plant stand in some plots. As a result, the plant stand was not good enough to provide dependable yield data in many cases by late summer 2019. However, in some good plots the resulting range of plant densities showed a nearly linear response in root biomass at mature plant densities ranging from 30,000 plant/acre to 300,000 plant/acre. This confirms previous estimates that mature plant densities need to be well above this range to maximize root biomass yield. Root mass dry weight was usually between 4 and 7 g/plant, with a mean of 5.5 g/plant, translating to a root yield of 1200 lb dry/acre. Roots were harvested at various times to measure changes in inulin and rubber content. New field experiments were seeded in fall and spring 2020, with first harvest planned for fall 2020. A separate field evaluation of improved Ohio State germplasm in Oregon's climate is ongoing at KBREC in 2020. A companion study at the ORSU-Southern Oregon Research & Extension Center (SOREC) was seeded in 2020, examining density X cultivar effects under a different climate. The interaction between seedbed preparation and planting method was investigated. Soil was plowed and disked, then reworked using a stale-seedbed approach to stimulate weed germination and reduce the weed seedbank. Soil was worked immediately before planting to produce either a flat smooth surface or a raised bed. Compos was applied before final soil preparation; or one before and after final soil preparation. The target planting density was 2 million seeds/ac. Seeds were either broadcast followed by a cultipacker or planted with a Jang seeder. Imazethapyr was sprayed one day after planting; thifensulfuron + clethodim herbicides were sprayed postemergence 16 days later. Plant establishment was highest where two applications of compost were applied. Final plant densities were ~80% of seeds sown on the raised bed and 65% for flat soil. The Jang seeder resulted in 88% establishment compared with 70% for broadcast seeds. Root weight was higher for plants grown on raised beds. Weed control averaged 82%. In other experiments, coated seeds established 20% more successfully than uncoated seeds and the optimum seed depth was 0.6-1.2 cm. In Nebraska, germination continues to be low but was more successful in the 2019/2020 season due to improved TK germplasm and knowledge gained from the previous cropping season. The most consistent herbicide treatments that suppress weeds without injury to TK were preemergence applications of Pursuit 6oz/A or Spartan 12oz/A. Weed control ranged from 60 to 88% percent and crop injury was 5-15%. Objective 2 Seed supplies from Ohio (>5 million) were pelleted and sent to Oregon, Nebraska and British Columbia. Germination chamber experiments were conducted in Oregon to identify methods to enhance seed germination, examining factors such as soil temperature, soil texture and organic matter, soil amendments, seed depth, moisture, and seed pelleting. Seeds from the most 'advanced' TK lines available from each generation were planted with a common planter (Jang) at all locations at a seed density of 2 M/ha. Stands ranged from 70% to 82% of seeds sown. Rubber yield in the 5th generation seed-lot was 46% higher and that of the 6th generation field-grown plants was 206% higher than rubber in the 2nd generation seed-lot. Rubber yield increases were due to increased biomass as rubber concentration did not change. Top performers based on root biomass from 2020 will again be selected and crossed in the greenhouse over the winter in the Ohio TK Selection Nursery. In Ohio the optimum planting-time window was May 25 to June 8, corresponding to 500 to 700 Growing Degree Days (GDD) and 10-cm soil temperatures over 17 C. Success depended on consistent soil moisture between seeding and early establishment and pointed to the need to develop germplasm that can be planted deeper and is tolerant of dry conditions. TK planting time trails in Nebraska showed that late March through mid-April planting resulted in higher TK establishment. In the second year, greater success in TK establishment was observed in mid to late-April. Using straw as a ground cover to increase available soil moisture at the time of TK establishment resulted in greater germination, compared to other soil covers. Objective 3 Various approaches were evaluated to increase rubber concentration. A post-harvest experiment was conducted with ethylene or ethephon. Results showed that the ethephon treatments had the highest rubber concentrations. Another approach is to induce polyploidy. Currently there are 35 living plants from 4n x 4n crosses and 31 from 3n x 3n crosses that have been isolated and are growing in planting beds. Seeds have been obtained including 2535 from crosses between 3n plants and 1485 collected from crosses of 4n plants. Verification of ploidy number is underway. This is in addition to 3375 4n seeds collected from crosses of confirmed 4n plants and 7395 collected from 2019-3n, 2019-4n and 4n confirmed groups. A CRISPR/cas9 binary vector with two guide RNA (gRNA) expression cassettes targeting two adjacent sites of the TK 1-FFT gene was successfully generated and confirmed with Sanger sequencing. Rubisco enhancement is also being studied via particle bombardment. A variety of potentially high-rubber transgenic TK plants were planted September 2019 in a greenhouse and were harvested in July 2020. Rubber concentration and photosynthetic rates are being measured. Fourth generation seeds are prepared for a Fall (2020) planting. Objective 4 A novel TK rubber extraction process was developed. It uses simultaneous wet-milling and enzymatic digestion of TK roots, after inulin extraction, resulting in yields greater than 82 representing an increase of 34% compared to the Eskew process. Optimization reduced the extraction from 48h to 3h and enzyme dose by more than 80% with minimal impact on rubber yield or quality. The novel process has been scaled up to kilogram quantities at the PENRA pilot plant at purities and yields comparable to those obtained at laboratory scale. Alkaline and acid pretreatments increased rubber yield by an additional 10% and slightly improved purity. However, mechanical and molecular properties of TK rubber from pretreated roots were adversely affected. Antioxidants added prior to pretreatment is being investigated to reduce these effects. Objective 5 A TEA model for Ohio TK field production has been developed for both commodity ($1.6/kg) and premium rubber ($10/kg) prices. These projections relate to field production and not associated processing costs or logistics. Planting densities from 150,000 -2,400,000 plants/ha for direct seeding and transplanting scenarios were modeled. For direct seeding, fresh roots of 3-10 g/plant, with 70-120 mg/g rubber dry weight were used. The estimated direct seeding rubber field production costs were $915.4-$1175.6/ha, and net revenue for commodity rubber at market selling price of $1.6/kg, including inulin (selling price of $4/kg) and rubber, is (-$595.2) to $634.1/ha. For premium rubber selling prices at $10/kg, net revenue ranged from (-$425.8)/ha to $1,865.7/ha. Key findings are that root yield and rubber concentration must increase, and inulin valorization will be critical to establish an economically viable TK crop.

Publications

  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Abdul Ghaffar, M.A., Cornish, K. New Developments in rubber particles biogenesis of rubber-producing species. Chapter 10: 153-168, In: The Rubber Tree Genome, Compendium of Plant Genomes, Eds: Minami Matsui and Keng-See Chow, Springer Nature Switzerland AG, 2020.
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Cornish, K. Perspectives and on-going challenges. Chapter 11: 169-175. In: The Rubber Tree Genome, Compendium of Plant Genomes, Eds: Minami Matsui and Keng-See Chow, Springer Nature Switzerland AG, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Cornish, K. Valorization and scalability of alternative natural rubber through tailoring properties, Proceedings of the Tire Technology International Conference, Hannover, Germany, February 25-27, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Cornish, K. Minimizing risks around NR through alternative crops. Proceedings of the Tire Technology International Conference, Hannover, Germany, February 25-27, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Cornish, K., Valorization and scalability of alternative rubber through properties and production processes. Proceedings of the International Conference on Development of Alternatives Sources of Natural Rubber, Montpellier, France, November 4-6, 2019.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Cornish, K., Rubber dandelions. Natural History Magazine. October 2019, 15-19, 2019.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Cornish, K., Kopicky, S.E., Madden, T., Hydroponic cultivation of rubber dandelion has high annual rubber yield potential. Rubber and Plastics News, Technical Note Oct 7: 23-25, 2019
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: DA Ramirez?Cadavid, K Cornish, U Hathwaik, R Kozak, C McMahan, FC Michel Jr. 2019. Development of novel processes for the aqueous extraction of natural rubber from Taraxacum kok?saghyz (TK). Journal of Chemical Technology & Biotechnology. 94:24522464. https://doi.org/10.1002/jctb.6027.


Progress 09/01/18 to 08/31/19

Outputs
Target Audience:OSU TK BARRAL research was presented to researchers and industry contacts at the following conferences by Dr. Cornish: Bioenvironmental Polymer Society, Gordon Research Conference, Biomass to Biobased Chemicals and Materials, Gordon Research Conference, Plant Metabolic Engineering, International Conference on Biopolymers and Bioplastics, Rubber and Plastics in Automotive Conference Industry executives and other researchers were contacted by Dr. John Cardina. Dr. Ramirez presented a paper to members of the American Society of Agricultural and Biological Engineers at the 2019 ASABE Annual International Conference in Boston, MA on results of research on TK rubber extraction and purification. The presentation was entitled, Effects of pretreatment on the properties of rubber extracted from Taraxacum kok saghyz, the rubber dandelion. Nebraska For the first year, the BARRAL project was exhibited at a field day event at the University of Nebraska-Lincoln (UNL) Panhandle Research and Extension Center June 27, 2019. A total of 47 stakeholders, predominantly area agronomists, crop consultants, and farmers, attended the event. The field day event updated stakeholders on the potential of TK production in western Nebraska, and on the progress of ongoing TK research. Oregon State Presented to BARRAL cooperators at semi-annual research group meetings. Changes/Problems:As stated above, TK establishment was less successful when planting occurred after mid-April. Several planned field experiments in Western Nebraska were established past this date, with poor results, including field herbicide trials. While this is problematic for 2019, these establishment failures are useful data moving forward, and will help reduce established failures in future years of the project. In Oregon, no major changes were made. The spring was very rainy, so some anticipated planting dates were delayed, and subsequent planting times were adjusted accordingly. Overwintering plant survival is minor in some years, but significant mortality can occur depending on winter weather and plant vigor in fall. Some aspects of studies involving over-wintering plants may need to be modified based on weather-related responses. We may also conduct companion studies at another Oregon St. Univ. research station site that is only 80 miles from primary research site but has a warmer and longer growing season (due to 3000 ft elevation difference), to compare climate/weather effects. What opportunities for training and professional development has the project provided?Excel training for 2 researchers in Oregon. Project personnel continue to keep up with pesticide training. How have the results been disseminated to communities of interest?Three peer reviewed publications (see above). Two newspaper articles and one blog post were written about BARRAL research in Western Nebraska. Can Nebraska Grow Rubber-Producing Dandelions on a Farm Scale? CropWatch. 221 Page Views. May 17th. https://cropwatch.unl.edu/2019/dandelion-rubber Can Nebraska Grow Rubber-Producing Dandelions on a Farm Scale? Star Herald. Weekly Distribution of 15,000. May 19th. Project could breathe new life into old crop. Business Farmer. Weekly distribution of 1,500. May 24th. Two newspaper articles and associated video were published about BARRAL-related TKS research in Oregon. Russian dandelion needs help to get rooted. Medford Mail Tribune. Distribution of 120,000. Nov. 28, 2018. https://mailtribune.com/news/top-stories/russian-dandelion-needs-help-to-get-rooted This dandelion could one day make high-grade rubber. Medford Mail Tribune. Distribution of 120,000. Nov. 1, 2018. https://mailtribune.com/news/top-stories/where-the-rubber-meets-the-oregon-crops Presentations of weed results have been made at meetings with industry partners. What do you plan to do during the next reporting period to accomplish the goals?Weed control experiments will be repeated. Additional post harvest experiments in fall 2019 as described in the project plan. These include cooling and temperature tracking in natural storage environments (barn and root cellar) We will also begin the first BARRAL-related plot harvest in fall 2019. Continue sequential field plantings as outlined in scope of work in fall 2019, spring/fall 2020. Second BARRAL-related harvest in summer and fall of 2020. For each harvest, roots will be washed and air-dried before shipment to Ohio St Univ. for rubber/inulin analysis. Further analysis of the rubber samples is planned for the near future, such as, rheology measurements and GC/MS of TK rubber and its on-isoprene fraction. Evaluation of the use of ligninolytic enzyme along with cellulases for the extraction on enzymatic hydrolysis of TK roots for rubber extraction and production of sugars. Development of a process for extraction and recovery and separation of inulin and protein from Taraxacum kok saghyz roots as coproducts of the rubber extraction process. We are currently collecting data related to these different operations from sources, such as published literature and databases. These data will be supplemented with results collected from the field tests at different locations, as outlined in detail in Objs. 1-4, and will be the key inputs to the technoeconomic and life cycle assessments. A full techno-economic analysis of natural rubber and inulin production from Taraxacum kok-saghyz roots using a water-based process will be developed.

Impacts
What was accomplished under these goals? Objective 1. Several field experiments were begun in fall 2018 in Ohio, Nebraska and Oregon with first harvest in fall 2019. These sequential plantings continue into 2019, following the scope of work outline. They examine and identify how the following cultural practices interact and affect root biomass and rubber/inulin yield: Planting date, harvest date, plant density, irrigation rate, soil fertility, planting methods/seedbed preparation, harvest method, and weed control (herbicide testing).In addition, germination chamber experiments, continuing pre-BARRAL work, were conducted to identify methods to enhance seed germination, examining factors such as soil temperature, soil texture and organic matter, soil amendments, seed depth, moisture, and seed pelleting. In the previous two years, planting of TK in western Nebraska failed, with no plants surviving germination. However in the first year of BARRAL, 13.3% of planted TK survived for five months after planting, and greater than 95% of transplanted TK survived. Yield will be assessed after this annual progress report is submitted. In Oregon and Ohio a number of growth trials have been conducted. Planter Evaluation x land preparation study. Goal was to evaluate the interaction between seedbed preparation and planting method. Soil was plowed and disked, then re-worked shallowly to stimulate weed germination. Plots were 6'x20'with four replications per treatment. Soil was worked immediately before planting to give the following; flat smooth surface, heavy compost application before final preparation lightly worked into soil, raised bed, heavy compost application before making raised bed. For 5/22/2019 the target density was 2 Million/Acre using two methods; broadcast TK seeds over the soil surface followed by cultipacker and Jang roller seeder for pelleted seed (JP-6 3-point seeder, Jang Automation Co.). All Plots were sprayed with a preemergence treatment of Pursuit on 5/22/2019 and Harmony postemergence + Select on 6/24/2019.Plants are growing well and weeds mostly controlled; no persistent flooding effect evident. Plant survival ranges from 65 to 81% of seeds sown. Weed control is 70 to 86%. Germplasm evaluation Goal was to evaluate field performance of most 'advanced' TK lines available in each year. Seeds available in 2019 will be the basis for subsequent comparisons. TK seeds were planted with a common planter (Jang) at all locations. The target viable seed density was 2 M/ha. Plots were prepared as described above with four replications in a RCB design. Six seed lots were evaluated from 2016 to 2018. All Plots were planted 5/21/2019 and sprayed as described above. The planting density was 1.5 Million/ Acre. Stands are not impressive, 51 to 67% of seeds sown, possibly due to flooding during springtime; Weed control is marginal, 68 to 77%. Some hand weeding might be necessary. Planting Time Study. (Ohio only) Goal was to determine optimum time of planting for successful TK establishment. Treatments included raw vs pelleted seeds broadcast at six planting times from 5/9 to 7/15. The target seed density was 2 M/ha. Land preparation was the same as described above. All plots were sprayed with Pursuit on day of planting, followed by Harmony + Select 1 month after planting. The goal was to cause as little injury as possible. Actual planting density was 1.3 Million/Acre. TK is growing well, with variation depending on planting time. Estimated survival is 66 to 87% of seeds sown. Weed control is excellent, ranging from 79 to 85%. Preemergence herbicides for TK establishment. Goal was to evaluate treatments that suppress weeds without injury to the TK crop. Treatments were chosen from those that performed well after several years of testing including Pursuit 6oz/A, Spartan 12oz/A, Weed Check, Hand-Weed Check. The plots were planted on 5/21/2019, with a planting density of about 1.5 Million/A. The stand is not outstanding due to flooding which affected not just seedling emergence but also variable effects of herbicide injury. Estimated stands are 53 to 76% of seeds sown. Weed control ranges from 46 to 86 percent. Postemergence herbicide evaluation for TK weed control. The goal was to evaluate postemergence herbicide treatments that suppress weeds without injury to the TK crop. Treatments were chosen from those that showed some promise in previous tests. Eight treatments were evaluated. TK was planted on 5/21/2019. All plots were sprayed with a preemergence treatment. Overall good crop stands where seedlings survived. Weed control generally excellent. Unfortunately, some treatments caused severe injury to the crop. Objective 2. In the first year of BARRAL, progress has been made in identifying optimal TK planting times which differ from other production regions. Late March through mid-April resulted in higher TK establishment, compared to later plantings in western Nebraska. Many of the planned agronomic studies have yet to be established, as they do not begin until after this progress report is submitted. Objective 3. Rubber and latex accumulation were investigated post-harvest in greenhouse-grown roots of diploid and triploid plants, planted in June (2018) and harvested the following February (2019). The tops were removed and the plants stored in a cold box (5-8°C) in the dark. All plants had roots greater than 7g Fwt at harvest. In general, rubber may have increased slightly during the first 33 days after harvest but this was much less that previously observed in fall-harvested large roots . Longer storage times were detrimental to rubber yield as the roots started to rot after this time. Sugar/inulin data will be added and the rubber levels confirmed with ASE, and reported later. Additional cold storage experiments will be performed on field harvested plants in late fall, 2019. Objective 4. Mechanical and molecular properties of TK natural rubber extracted from TK roots at pilot scale using acid and base pretreatments along with enzymatic hydrolysis were evaluated. TK rubber was extracted from TK roots using three different processes: 1. Acid pretreatment at 120?C followed by simultaneous ball milling and enzymatic hydrolysis; 2. Base pretreatment at 120?C followed by simultaneous ball milling and enzymatic hydrolysis; 3. Simultaneous ball milling and enzymatic hydrolysis of TK roots (no pretreatment). The rubber extracted using the processes mentioned above was dried at 50?C under vacuum, and rubber yield and purity were measured. Rubber samples were sent to industry and government collaborators for mechanical testing and molecular weight distribution analysis. These included molecular weight distribution, tensile test, extension cycling fatigue test, plasticity retention index, bulk viscosity, and molecular weight distribution and tensile testing of aged samples. A new process was evaluated wherein rubber was extracted using simultaneous ball milling and enzyme digestion. The optimum parameters for the extraction of rubber and production of sugars from Taraxacum kok saghyz roots were determined. The extraction of natural rubber and production of sugars from TK roots using cellulase was studied using two commercial cellulose hydrolytic enzymes: Cellic Ctec 2 (Novozymes) and Accellerase 1500 (Genencor, now Dupont). Different parameters were investigated using both enzymes, such as, milling time (3h, 6h, 12h, 24h, and 48h), size of grinding media (small vs medium vs big size), grinding media volume, and milling velocity. Rubber yield, rubber purity, and sugar are being analyzed and based on the results optimum parameters for simultaneous ball milling and enzymatic hydrolysis will be determined. Objective 5. Key components of the overall TK production, harvest and conversion systems have been identified.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Bates, G.M., McNulty, S.K., Amstutz, N.D., Pool, V.K., Cornish, K., Planting density and harvest season effects on actual and potential latex and rubber yields in Taraxacum kok-saghyz. Hort Science, accepted May 14, 2019. David A. Ramirez-Cadavid, Jesse Rossington, Frederick C. Michel. 2019. Effects of pretreatment on the properties of rubber extracted from Taraxacum kok saghyz, the rubber dandelion. Conference: 2019 ASABE Annual International Conference, Proceedings paper. Paper # 1901187. Boston, MA. Cherian, S., Ryu, SB, Cornish, K., Natural rubber biosynthesis in plants, the rubber transferase complex, and metabolic engineering progress and prospects. Plant Biotechnology Journal (2019), pp. 121 doi: 10.1111/pbi.13181.