Progress 06/01/12 to 05/31/17

Outputs
Target Audience:Both academic and industrial audiences were targeted in papers published and news articles reporting on guayule progress during this reporting period Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?See list of publications and presentations listed in "Products" section. Regarding the public release of the genome data The annotated Meraculus assembly and genome sequencing raw reads have been uploaded to the NCBI wgs (whole-genome shotgun contigs) www.ncbi.nlm.nih.govdatabase for public release upon publication. The tetraploid databases are housed in a server at the USDA Western Regional Research Center in Albany, CA, currently being curated by the Genomics and Gene Discovery Unit. They will be uploaded to NCBI at the time of transcriptome analysis publication. The ~40K genes annotation GFF files will also be housed at the USDA-ARS-WRRC-GGD. What do you plan to do during the next reporting period to accomplish the goals?This is the final report, although there still remains a small amount of work on direct seeding to be completed and will be reported in the literature. The reason for the delay is a change in personnel which resulted in a delay.

Impacts
What was accomplished under these goals? 1.Genome sequencing of the heterozygous diploid guayule accession PI 478663. The acquisition of guayule genomic DNA and RNA transcriptome sequence represents baseline data for application of molecular genetics to a guayule improvement. A deep guayule transcriptome database has been generated to serve as both a component of the genome annotation program and an independent resource for characterization of transcriptome profiles and metabolic potential. Genomic data have been uploaded to NCBI pending publication acceptance. 2. Genomic library construction and skim resequencing of 20 guayule accessions.This was replaced by genotype-by-sequencing (GBS) genotyping. The GBS marker discovery protocol for guayule was developed and published. The Cornell group processed and analyzed genotype data for 405 samples representing 69 accessions of guayule and allied species, and obtained approximately 50,000 high quality markers representative of the genetic diversity present in the stocks maintained by NALPGRU at Parlier, CA. Cornell performed phylogenetic analysis of the samples and identified new mariola X guayule hybrid accessions as well as a novel class of hybrid accessions that includes accessions AZ-2 and AZ-3, two of the guayule accessions commonly used by commercial entities. 3.A method to use direct seeding of guayule instead of transplanting. Investigate herbicides for guayule -Direct seeding work is still in progress due to changes in personnel. Four post-emergence herbicides (Gramoxone, Aim EC, Fusilade DX and ET Herbicide/Defoliant) registered previously for guayule were tested. At 1X 1.0pts/20 gallons Gramoxone, the guayule transplants survival rate was 70%, but the rate dropped to 33% when plots were sprayed with 1.5X Gramoxone. This study will continue and be published. 4. Determination of the effect of various irrigation procedures on the guayule plant and rubber production. The most extensive irrigation study to date was conducted and published.The two year study comparing subsurface drip irrigation (SDI) with surface irrigation (SI) showed plant dry biomass was significantly different for all treatments in the SDI study with a nearly four-fold increase from 25% to 125%. The trend of increased rubber percentage with less irrigation (more water stress) did not prevent a significant increase in rubber yield with irrigation level, where the 125% achieved the highest rubber yield. For the SI study, plant dry biomass again increased significantly with irrigation. Dry biomass at the highest irrigation under SI was less than one-half that under SDI at the highest irrigation. A web based application using the results of this study is being completed. 5. Development of a process to make a tire grade guayule rubber. A specification was developed for guayule which limits resin content to 3% for tire compounds. PanAridus delivered thousands of pounds of high quality guayule natural rubber which afforded meaningful tire compounding data, and provided information for all aspects of the LCA work. 6. Development of a process to make epoxidized guayule rubber. A new process was developed and discussed with a toll manufacturer, but due to timing and available rubber, another approach to the tread compound was taken. 7. Building and testing a "Concept Tire" which uses only elastomers derived from domestically grown guayule. One major goal of the grant was to build a concept tire containing all guayule or guayule derived rubber, which would be compared to aCS4 tire. This work showed that an all-guayule tire can be built, but some components are much more amenable to replacement with guayule than others. A key question which had to be answered was whether or not guayule could replace Hevea because of differences in strain induced crystallization (SIC). SIC was determined at SUNY in Prof. Ben Hsaio's lab. The key findings of these studies are:1) Raw guayule natural rubber (GNR) films undergo SIC at -50ºC (strain ~ 3+) similar to synthetic polyisoprene, but, unlike Hevea, not at room temperature.2) Cured GNR undergoes SIC at room temperature; onset strains (~3-4) and extent of crystallinity (10+%) are similar to that observed in Hevea. 3) Cured, carbon black filled GNR also undergoes SIC, at lower strain (~2+) than unfilled and in proportion to the amount of filler (20phr vs. 40phr).4) In almost every case, addition of HNR proteins to GNR resulted in significant increases in extensibility of raw GNR films.5) In some cases, addition of HNR proteins to GNR latex (at 0.1 to over 5 w/w %) improved the % gel and/or the strength of the material and resulted in detectable levels of SIC. SIC appeared to be more pronounced when lower molecular weight proteins (obtained by filtration or digestion) were used. In some cases quite significant levels of crystallinity were measured (~3+ %) but these did not translate to higher strength. After initial compounding work, the compounds were mixed in an industrial mixer and processed in a commercial tire facility. Significant reductions in mixing time and energy could be achieved with the guayule compounds when compared to the control compounds. Tire testing was conducted at Cooper's Pearsall test track. The handling for the 100% guayule concept tire was similar to the CS4 control in both wet and dry conditions. The handling in wet conditions was slightly better for the 75% guayule concept tires, while the handling in dry conditions was slightly inferior to the control. The braking performance in dry conditions was equivalent to the control for both guayule concept tires. In wet conditions, the guayule tires were significantly better than the control. In an extended wear test, the mileage for both the guayule and control tires were equivalent. 8. Determine the suitability of guayule bagasse for use in cogeneration facilities. Oneuse for the bagasse is to produce energy to operate the guayule extraction facility. Surplus bagasse could be sold for bioenergy production, including cogeneration and conversion to liquid biofuels. Currently, Arizona has only one large scale biomass boiler facility and the facility limits chloride levels in feedstocks to 1500 ppm. Guayule produced for this work averaged 3500 ppm. A wash system wasdeveloped which reduces the chloride levels in guayule bagasse to700 ppm so this material should be suitable for use in cogeneration. 9. Determine if guayule bagasse can be used to make fuel precursors via a fermentation process. PanAridus retained LanzaTech to conduct a Techno Economic Analysis (TEA) for conversion of guayule bagasse to ethanol via gasification followed by fermentation. The estimated cost for converting guayule bagasse to ethanol is about $2/gallon of ethanol. The current average price of ethanol is only $1.60/gallon. Thus, without government subsidies, the conversion of guayule bagasse to ethanol is not profitable. The conversion of ethanol to Jet Fuel A was also studied and would not be profitable in the absence of government subsidies for ethanol. 10. Development of a Tool for Sustainability Impact Assessment (ToSIA) to determine the sustainability implications and economic scalability of large scale guayule. The ToSIA tool was changed during the course of the program to an Excel based program. A Life Cycle Assessment (LCA) of a 100% guayule tire was constructed that included tire manufacturing, tire supply-chain and tire use. Inventory data was collected from Cooper, PanAridus and ALARC and modular life cycle analyses were developed over five years. A gate-to-gate comparative LCA of flood vs. drip irrigation using irrigation data collected from ALARC for guayule water use and rubber/ biomass yields was developed. A batch, upgraded batch and commercial-scale rubber processing LCA as developed using data collected from PanAridus.

Publications

• Type: Journal Articles Status: Other Year Published: 2017 Citation: Hathwaik U., Lhamo D., Su Y., Hsiao B., McMahan C. Effect of rubber biosynthetic pathway related protein additions on properties of guayule natural rubber (in preparation).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zachary Walters "Development of the Guayule Tire" Presentation at 2017 International Tire Technology Expo, Hanover Germany
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Howard Colvin "Securing the future of natural rubber - An American tire and bioenergy platform from guayule" Keynote address at 2016 AAIC meeting Rochester NY
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Nick Sauty "Byproduct differences in guayule prepared by latex and solution processes" Presentation at 2017 International Tire Technology Expo, Hanover Germany
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Pragnya Eranki, Amy Landis, Howard Colvin, Colleen McMahan, "A framework to evaluate the sustainability of guayule rubber tires", ISIE-ISSST Conference (June 2017)
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Pragnya Eranki, Diaa El-Shikkaa, Doug Hunsaker, Kevin Bronson, Amy E. Landis, 2017 "A comparative life cycle assessment of flood and drip irrigation for guayule rubber production using experimental field data", Industrial Crops and Products.
• Type: Conference Papers and Presentations Status: Under Review Year Published: 2016 Citation: Habib Azarabadi, Pragnya Eranki, Amy E. Landis, 2016 "Life cycle impacts of commercial guayule rubber production estimated from batch-scale operation data", The sustainability Collection, OnSustainability conference publication.
• Type: Journal Articles Status: Other Year Published: 2017 Citation: Ranjani Theregowda, Pragnya Eranki, Amy E. Landis, 2017 "Use Phase Energy Analysis for Conventional and Guayule Rubber Tire Compositions", In preparation
• Type: Journal Articles Status: Other Year Published: 2017 Citation: Pragnya Eranki, Amy E. Landis, 2017 "Life cycle assessment of a concept guayule natural rubber automobile tire", In preparation
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Pragnya Eranki, Kullapa Sortana, Daina Rasutis, Habib Azarabadi, Amy E. Landis, "Life cycle analysis of tire production using guayule rubber", Gordon Research Conference for Industrial Ecology, 2016
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Habib Azarabadi, Pragnya Eranki, Amy E. Landis, "Comparative LCA of Commercial and Batch Process for Guayule Rubber Production", OnSustanability Conference, 2016
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Pragnya Eranki, Diaa El-Shikhaa, Doug Hunsaker, Kevin Bronson, Amy E. Landis, "A Comparative Life Cycle Assessment Using Experimental Field Data of Flood vs. Drip Irrigation in Guayule Rubber Cultivation", ACLCA conference 2016, Engineering Sustainability conference 2017
• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: William Belknap and Jos� Arcadio Valdes Franco Modular assembly of transposable element arrays by microsatellite targeting in the guayule and rice genomes describing the guayule genome was prepared and submitted to Nature Genetics. In revision for submission to BMC Genomics.
• Type: Journal Articles Status: Other Year Published: 2017 Citation: William Belknap Differential expression of water stress-associated genes in guayule (Parthenium argentatum) and their impact on rubber synthesis (in preparation)
• Type: Journal Articles Status: Other Year Published: 2017 Citation: DC Ilut et al. A Century of Guayule: Comprehensive Genetic Characterization of the Guayule (Parthenium argentatum A. Gray) USDA Germplasm Collection (journal manuscript in preparation)
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Nick Sauty "Thermal treatment of guayule resin and use in compounding" Presentation at 2017 International Tire Technology Expo, Hanover Germany.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Hunsaker, D.J., and El-Shikha, D.M. Surface irrigation management for guayule rubber production in the US desert southwest. Agricultural Water Management, 185:43-57. 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Elshikha, D.M., Hunsaker, D.J., Bronson, K.F. and Sanchez, P.L. Using RGB-based vegetation indices for monitoring guayule biomass, moisture content and rubber, Paper #162380922, pp. 1-12. In ASABE Annual Int. Meeting, Orlando, FL, un-paginated. 2016. (Conference Proceedings)
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Hunsaker, D.J. and Elshikha, D.M. Guayule biomass and yield under variable water inputs using surface irrigation, Paper #162414845, pp. 1-11. In ASABE Annual Int. Meeting, Orlando, FL, un-paginated. 2016. (Conference Proceedings)
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: McMahan, C., Hathwaik, U., Lhamo, D. 2016. Role of proteins and amino acids in natural rubber: guayule (Parthenium argentatum Gray) rubber addition studies. p.38. In M.T. Berti and E. Alexopoulou Eds. 2016. Industrial Crops Promoting Sustainability. International Conference in Industrial Crops and 28th Annual Meeting of the Association for the Advancement of Industrial Crops (AAIC). Program and Abstracts. September 24-28, 2016, Rochester, NY, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: McMahan, C. and Lhamo, D. 2016. Effect of protein addition on properties of guayule natural rubber. Proceedings of the 186th Technical Meeting of the Rubber Division of the American Chemical Society, Inc. Pittsburgh, PA, October 11-13, 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: McMahan, C., Hathwaik, U., Lhamo, D. 2017 Protein modifiers in guayule rubber compounds. Presented at Tire Technology Expo 2017, Hanover, Germany, February. 14-16, 2017.
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Lhamo, D. and McMahan, C., 2017 Effect of protein addition in properties of guayule natural rubber. Rubber Chemistry and Technology. In press.

Progress 06/01/15 to 05/31/16

Outputs
Target Audience:Both the academic and industrial sectors have been targeted during this reporting period. The publication section of this report shows several papers targeted at potential guayule breeders and farmers. Changes/Problems:Professor Landis moved from Arizona State University to Clemson. Tires made with one specific 100% guayule component passed all Department of Transportation tests, but did not pass Cooper's extended testing suite. NIFA was petitioned to allow the use of some non-guayule rubber, if necessary, in tire components so as to demonstrate the utility of guayule blends. Permission was granted. What opportunities for training and professional development has the project provided?José Arcadio Valdes Franco, from Universidad Autónoma de Nuevo León, Monterrey, Mexico, has been working on bioinformatic aspects of the guayule program under the BRDI grant for the past year and a half in William Belknap's laboratory (Albany, CA). Arcadio, who previously obtained a Masters degree in bioinformatics from the University of Glasgow, has been trained in computational analytical practices, particularly with respect to plant genomes. Arcadio is planning to extend this training by entering a PhD program in plant genomics at Cornell University this fall. Daniel C. Ilut, the primary research support specialist assigned to this project in the Gore group at Cornell, attended the Tucson Plant Breeding Institute workshop in January 2016 as part of his training through this grant. The goal of the Tucson Plant Breeding Institute (TPBI) is to offer state-of-the-art instruction and training in modern plant breeding tools such as statistics, molecular breeding, and computation. Olivia Tonge, an undergraduate researcher in the Gore group at Cornell, was directly involved in the phylogenetic analysis of the guayule germplasm collection during the 2015-2016 academic year. She received training and mentoring in computational and data analysis skills, population genetics, phylogenetic reconstruction, and statistical analysis. Greg Leake, USDA-ARS technician- pesticide handling certificate. Quinn Waltz, CAC spring intern- agronomic practices for growing guayule. Stephanie Myak - Cooper intern - Organic synthesis/chemical processexperience. Upul Hathwaik attended a gas chromatography mass spectrometry (GC/MS) training offered by Bio-products research unit in Albany, CA . Dhondup Lhamo attended: Gas Chromatography - Mass Spectrometry (GC-MS) training, United States Department of Agriculture, Agriculture Research Services, Albany, CA. Dhondup Lhamo attended: Plant Metabolomics and Stable Isotopic Labeling Workshop 2015. Mass Spectrometry Informed by Stable Isotope Methods. University of Minnesota, Saint Paul, MN. Dr. Landis Incorporated aspects/concepts from the BRDI project into her undergraduate classes at Clemson Fall (A civil engineering class: Creatively Applying Science for Sustainability) and Spring (CU 2010 Sustainability Leadership). How have the results been disseminated to communities of interest?The Cooper Ride and Drive event, which was the first ride and drive where modern tires containing multiple components consisting of 100% guayule rubber, attracted both technical and popular media. Numerous articles appeared regarding the event,and the implications for the future of guayule rubber. A paper was published in the technical literature, and an oral presentation was made to a technical audience. Numerous technical exchanges took place between BRDI researchers and their coworkers at organizations taking part in the program. What do you plan to do during the next reporting period to accomplish the goals?The Gantt chart will be followed to complete the program by June 2017

Impacts
What was accomplished under these goals? Program significance - This program looks at improving the entire guayule natural rubber (GNR) technology spectrum from identification of the genome, plant agriculture, development of tire technology and a sustainability analysis of the entire supply chain. Program success will result in tools which potentially can be used to improve yields, improved methods for isolation of GNR, and establishment of where GNR can most effectively be used in tires. Guayule Genome The objective of GNR genome sequencing is to lay the foundation for molecular breeding in guayule. Two assemblies have been developed and uploaded to the project database. One of the assemblies covers essentially the entire genome. A public GNR transcriptome derived from a presumptive tetraploid was used for the initial assembly. Eight Illumina HiSeq transcriptome libraries have been constructed to provide deep transcriptome coverage for more accurate gene annotation. Molecular Breeding Tools The large-scale SNP discovery project from year three was leveraged to genotype all available accessions of guayule from the germplasm collection maintained by the National Arid Land Plant Genetics Resources Unit (NALPGRU). The pilot study results and the genotype-by-sequencing (GBS) marker discovery protocols for guayule were finalized and published. These protocols were further refined to genotype 414 samples representing 66 accessions of guayule and allied species. From this information we obtained approximately 35,000 high quality markers representative of the genetic diversity present in the stocks maintained by NALPGRU. Phylogenetic analysis identified a preliminary guayule diversity panel for this collection, consistent with a history of two distinct rounds of wild population sampling. The two diploid guayule accessions (PI 478663 and PI 478664) appear genetically indistinguishable. Guayule Cultivars To develop a segregating population of the diploid accessions, twenty crosses were made between two 6-years old plants of PI478663 and PI478664; none of the F1 seeds germinated. 100 seeds of each accession were planted in the greenhouse. Seedlings are growing and leaf tissue will be harvested for genotyping (GBS sequencing) to select the most diverse plants to be used for population development. Direct Seeding A field study was initiated where seeds of two cultivars (AZ-2 and AZ-6) were planted under three treatment conditions. Four post-emergence herbicides previously registered for guayule will be tested. Seeds were planted manually on the surface or under 0.5 cm of soil. Survival rates, rubber content, and efficacy of herbicides against various kinds of weeds will be recorded Irrigation Studies The two year study comparing subsurface drip irrigation (SDI) with surface irrigation (SI) was completed. Plant dry biomass was significantly different for all treatments in the SDI study with a nearly four-fold increase from I25% to I125%. The trend of increased rubber percentage with less irrigation (more water stress) did not prevent a significant increase in rubber yield with irrigation level, where the I125% achieved the highest rubber yield. For the SI study, plant dry biomass again increased significantly with irrigation. Dry biomass at the highest irrigation under SI was less than one-half that under SDI at the highest irrigation. Irrigation scheduling software for guayule is being developed by ARS and the University of Arizona, using data collected in the above field trials. This will provide information to determine when irrigation is required and how much water to apply. Two models, one for SI and one for SDI will be developed. Guayule Latex Blends: Proteins The addition of commercial proteins to guayule latex could potentially improve polymer properties such as green strength, gel and strain induced crystallization. A series of commercially available proteins were added to guayule latex. Results: Gel content increased in most cases, Most proteins serve as plasticizers at 100°C, providing ease of processability of the rubber. Addition of proteins generally improved thermo-oxidative stability. Green strength was improved except with gliadin, tensile strength improvement depended on the antioxidants used. Production of tire grade guayule rubber - Important polymer characteristics for tire grade rubber have been identified, and specifications have been set for GNR. High quality rubber is being delivered for tire builds. Important factors for producing high quality GNR are low residual resin, high molecular weight, a minimum level of antioxidant and low level of biomass. Studies have been conducted which have demonstrated methods for lowering the cost of production by improvement of solvent recovery. The extraction process has also been optimized to improve rubber yields. Compounding guayule rubber for tire components and tire building programs - Compounding for all tire components has either been completed or is at an advanced stage. Four tire builds were conducted during the reporting period, with multiple components consisting of 100% guayule or models for guayule derived rubber. Plant processing issues were encountered with some of the components, but were solved so that tires could be constructed and tested. All components passed Department of Transportation testing. The highlight of the tire program was the BRDI annual meeting which was held at the Cooper test track. A ride and drive event was conducted where BRDI scientists and administrators were able to ride on tires containing multiple 100% guayule components, and compare them to conventional tires. Green Power and Biofuel PanAridus has established the energy value of extracted guayule bagasse, and it is competitive with natural gas. Currently, Arizona has only one large scale biomass boiler facility and the facility limits chloride levels in bagasse to 1500 ppm. A wash system has been developed which will reduce the chloride levels in guayule bagasse to 700 ppm. LanzaTech (LT) has a unique fermentation process that utilizes syngas (from guayule bagasse gasification) for conversion of carbon to ethanol. A Techno Economic Analysis (TEA) for conversion of guayule bagasse to ethanol via gasification followed by fermentation has been conducted by LT. The estimated cost for this conversion is $2/gallon of ethanol. Since the average price of ethanol is $1.60/gallon, the conversion of bagasse to ethanol is uneconomical without significant government subsidies. The TEA also included conversion of ethanol to jet A fuel using conventional catalyst technology. This approach also would not be profitable in the absence of government credits for ethanol. Sustainability studies Irrigation data collection for guayule water use, rubber and biomass yields of guayule from ARS is completed and being used to develop an irrigation comparative LCA model. On the rubber-processing side, batch and upgraded batch process data collection from PanAridus is completed and being used to develop a comparative processing LCA model. Iterative data collection from Cooper for the material supply chain and new tire tests is ongoing.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Azarabadi, H.A., Eranki, P.L., Landis, A.E., Comparative LCA of Commercial and Batch Process for Guayule Rubber Production, International Conference on Environmental, Cultural, Economic, and Social Sustainability (On Sustainability) Conference, Portland, OR, 2016.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: DC Ilut, PL Sanchez, DE Costich, B Friebe, TA Coffelt, JM Dyer, MA Jenks, MA Gore Genomic diversity and phylogenetic relationships in the genus Parthenium (Asteraceae) Industrial Crops and Products 2015, vol 76, pp 920929 doi: 10.1016/j.indcrop.2015.07.035

Progress 06/01/14 to 05/31/15

Outputs
Target Audience: Both the academic and industrial sectors have been targeted during this reporting period. The publication section of this report shows several papers targeted at potential guayule breeders and farmers. In addition, there are several publications dealing with LCA issues surroundingcultivating and using guayule rubber in the tire industry. Cooper has had several press releases on various aspects of grant progress whichhas beenof great interest to the tire industry. Changes/Problems: The major change in the program has been to replace Yulex with PanAridus as the supplier of the guayule rubber. This was taking place at approximately the same time the last reporting period ended. PanAridus has worked in a very collaborative fashion and has successfully supplied rubber for the tire builds in this reporting period. There was a great deal of difficulty in getting permissionto make this change, which adversely affected the program. Without this change, it is doubful that the program objectives could be achieved.A reallocation of funds was proposed to and accepted by NIFA. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Dr. Landis discussed preliminary results from the BRDI grant in her talk at the Denison University Department of Chemistry graduation banquet. April 2015. She also presented the BRDI work at the Gordon Research Conference on Green Chemistry Invited Plenary, "Sustainability Assessment of Polymers: Advancing sustainability methods and tools for bio-based polymers." July 28, 2014, Hong Kong, China. She also presented the work at USDA ARS Berkeley "Life Cycle Analysis as a tool for environmental sustainability assessment of agricultural feedstocks for plastic and rubber products." May 22, 2014, Berkeley, CA. Finally Dr. Landis presented the work to ASU's Dean's Freshman Lecture "Sustainable Engineering of the Next-Gen Auto Tire" December 6, 2013. Numerous press releases to the tire/rubber community have been made discussing the progress that the consortium is making toward program goals. What do you plan to do during the next reporting period to accomplish the goals? The team will continue to work to the Gantt chart to achieve all program goals.

Impacts
What was accomplished under these goals? Major progress has been made in all areas of the program. Information gathered during this reporting period will lead to publication of a reference guayule genome. Data have been generated to allow for more efficient irrigation- critical to the economic sustainability of guayule. A solvent process to isolate rubber from the plant has been developed and continues to be improved. Guayule rubber from this process has been used to build components of a modern tire, and tire testing results are good thus far. Sustainability analysis continues in the three defined areas - economic, environmental and social. Guayule Genome Sequencing Illumina short-read runs employing genomic DNA from diploid guayule were combined with Roche 454 reads and subjected to assembly. The current draft genome contains ~85% of the expressed genes. Syntenic relationships displayed in the guayule contigs and scaffolds relative to tomato and citrus indicates accurate assembly. Molecular breeding tools A genotype-by-sequencing (GBS) pilot study of 62 plants from 15 Parthenium accessions developed a protocol and bioinformatics tool for genotyping plants with variable ploidy. Several thousand single-nucleotide polymorphism (SNP) markers were identified, phylogenetic relationships elucidated, and several cryptic interspecific hybrids identified in germplasm from USDA. . Cultivar evaluations and direct seeding studies of guayule The effect of temperature and salinity on guayule seed germination was evaluated using raw and pelleted seeds. Collection of soil and water samples confirmed the type of soil and water salinity, which was used in a Spring 2014 field trial. Seedling emergence and survival were monitored, and plants harvested for rubber quantification. Irrigation and Nitrogen Management Two types of irrigation were compared - Subsurface Drip Irrigation (SDI) and Surface Irrigation. In both studies, whole plants for wet/dry biomass weight, plant height, and plant cover were collected four times in 2014. Plant dry weight increased as a function of irrigation treatment for the four sample dates in 2014 for both fields. In the surface-irrigated field lower water use efficiency was found at the two highest irrigation levels. Plant dry-mass increased in the SDI study in a ~linear manner to a maximum of 2.0 kg/plant at the 125% level indicating water use efficiency did not change with irrigation level. Polymer-protein-amino acid-lipid interactions Compounding studies focused on the impact of addition of 1) commercial proteins, 2) extracted Hevea proteins, and 3) chemical additives, on the physical and chemical properties of GNR. Addition of proteins to GNR improved gel content, viscosity, thermo-oxidative stability, as well as green and tensile strength to various extents. Extracted Hevea proteins improved some properties and imparted measureable increases in crystallinity, but not to the extent of Hevea NR. Chemical modification of GNR TMTD/ZnO Tetramethylthiuram Disulfide and Zinc Oxide added to GNR improved physical propoerties after 1 month storage. It appears chemical additives can be used to generate polymer crosslinks during storage. Epoxidation of Guayule rubber Epoxidation of guayule latex would require further purification of the rubber An alternative approach is to epoxidize the rubber in solution after the resin has been removed, producing rubber which can be used without the need for further purification. A solution method was developed in the laboratory which can produce enough epoxidized guayule rubber for laboratory studies. The rubber from the solution process is chemically different than that from the latex process and the effect of this change on compounded rubber is being explored. Development of Tire Grade Guayule Rubber A greater understanding of the rubber extraction process has resulted in internal specifications for GNR rubber quality. Important factors include acetone extractables and molecular weight. An antioxidant package has been developed for tire builds. A major advance in producing larger quantities of GNR is the extraction reactor design. The ability to recover solvent from the extraction process is a limiting factor. Process modifications reduced the amount of solvent used, and new equipment allows higher solvent recovery rates. Advances in the rubber isolation process have yielded a new method for incorporating the antioxidant and consistent reduction of acetone extractables. Green Power and Biofuels from Guayule Bagasse The energy content of guayule whole shrub and bagasse creates a desirable bioenergy feedstock. Bagasse requirements to produce thermal /cogen energy to operate a GNR extraction pilot plant facility are being estimated. A Minnesota company has evaluated PanAridus guayule bagasse for conversion to biofuel, and plans a pilot plant evaluation to establish conversion efficiency. Currently, Arizona has only one large scale biomass boiler facility. Maximum chloride levels in the bagasse cannot exceed a specific level. Typical feedstocks such as switchgrass, and cotton stocks exceed this level. Bagasse from guayule produced in the soils of central Arizona also exceeds this limit. We are evaluating systems to reduce the halide content of the bagasse. Development of the Guayule based tire Compounding guayule rubber: Major progress has been made in compounding GNR to replace all compounds in the CS4 tire. A key issue with compounding GNR in place of HNR is a cure adjustment required to match the modulus of the Hevea compound. In some compounds a change in oil type or filler level is also needed. In the tread and innerliner compound, epoxidized natural rubber is being used as a model for epoxidized guayule. Compound development is complete for innerliner, bead filler, sidewall, rim cushion and bead insulation. Compound development is continuing for belt coat, polyester coat and overwrap in preparation for future tire builds. Tire builds Cooper has agreed to build a concept tire containing all guayule or guayule derived rubber, which will be compared to a 225/60R16 T rated CS4 tire. One tire build was completed in 2014, which evaluated an epoxidized natural rubber (ENR) innerliner, an ENR tread, a GNR bead filler, and GNR bead insulation. Wheel testing was successful for all features. The initial liner compound did not have sufficient resistance to air loss, so was modified for the latest build. Track results showed the ENR tread provided improved rolling resistance (better fuel economy), better wear (longer mileage) and better wet traction than the control tire. Environmental Life Cycle Assessment: The LCA of feedstock development technologies is in progress. .Agriculture data collection from PanAridus suppliers is completed. The PanAridus agriculture data is on a commercial scale and gives a better sense of full scale than parallel ARS data. For the LCA of biobased product development technologies, ASU is collecting process data from PanAridus and Cooper. Preliminary data collection from the PanAridus rubber extraction process is completed. Data from a Cooper guayule tire build was collected during a 10 day visit in April 2015. Also, data for the environmental impacts of the lifetime of guayule tire (rolling resistance) was collected. The Sustainability team at ASU reported preliminary findings in October with recommendations for agricultural practices, rubber extraction, and tire manufacture data collection.LCA data collection and LCA model creation will be iterative: the team will have opportunities at each project meeting to provide feedback and update the model. Sustainability Impact Analysis: The development of a system model (ToSIA) is in progress based on process data collected. The Sustainability team is also identifying and assessing opportunities for sustainable waste management and coproducts.

Publications

• Type: Theses/Dissertations Status: Accepted Year Published: 2014 Citation: Daina Rasutis advised by Amy E. Landis. Comparative Life Cycle Assessment of Conventional and Guayule Automobile Tires. Masters thesis in Civil Engineering, Arizona State University (2014)
• Type: Journal Articles Status: Published Year Published: 2014 Citation: 1. Sanchez, P. L., Chen, MK., Pessarakli, M., Hill, H. J., Gore, M. A., Jenks, M. A. 2014 Effects of temperature and salinity on germination of non-pelleted and pelleted guayule (Parthenium argentatum A. Gray) seeds Industrial Crops and Products 55, 9096
• Type: Journal Articles Status: Published Year Published: 2014 Citation: 2. Sanchez, P.L., Costich, D. E., Friebec, B., Coffelt, T. A., Jenks, M. A., Gore, M. A., 2014 Genome size variation in guayule and mariola: Fundamental descriptors for polyploid plant taxa, Industrial Crops and Products 54, 1 5
• Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: 3. Paul L. Sanchez, Mei-Kuang Chen, Brenda Singleton, Nicholas Laforest, Matthew A. Jenks, and John M. Dyer. Temperature, light, and seeding depth effects on guayule (Parthenium argentatum A. Gray) germination and seedling emergence
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Daina Rasutis, Kullapa Soratana and Amy E. Landis. A New Feedstock for Tire Production: Comparative LCA of Guayule and Hevea Natural Rubbers. Proceedings of the ACLCA Life Cycle Assessment XIV Conference. October 6-8, 2014. San Francisco CA.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Daina Rasutis, Kullapa Soratana, Colleen McMahan, Amy E. Landis. A sustainability review of domestic rubber from the guayule plant. Industrial Crops and Products. Published (2015)
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Kullapa Soratana, Daina Rasutis, Habib Azarabadi and Amy E. Landis. Guayule as an Alternative Source of Natural Rubber: A Comparative Life Cycle Assessment with Hevea and Synthetic Rubber. The International Journal of Life Cycle Assessment. Under review (2015)

Progress 06/01/13 to 05/31/14

Outputs
Target Audience: The guayule agricultural community was presented withtwo publications by members of ARS. The LCA community was presented with with a paper introducing the concept of comparing conventional tires with tires made from guayule rubber. The tire and rubber community has been targeted with selective press releases outlining the importance of this work to the tire industry. Changes/Problems: The delays in the program caused by Yulex' issues in supplying rubber, lack of Yulex progress in the bioenergy field and Yulex reluctance to attend team meetings led to their replacement with PanAridus. We have already observed a great deal more cooperation and anticipate this will carry over into technical progress. There will be some delay from the rubber delivery schedule worked out with Yulex since PanAridus is just ramping up production, but a new schedule is in place and will allow for guayule tire builds either late in 2014 or early in 2015. After being asked to leave the grant, Yulex has been reluctant to disengage from grant related agreements, and this has delayed a full team meeting. Progress is being made in spite of this, but until this issue is fully resolved, there will be delays and the full effect of the delays will not be known until later. What opportunities for training and professional development has the project provided? Several co-op students at Cooper tire have been used on ancillary guayule projects needed to make progress on the grant work. This has allowed them to become familiar with guayule rubber and how it is processed. Daina Rasutis has used her work on this project as partial fulfilment of her MS degree at ASU. She worked at Cooper for a month gathering information, and communicating LCA related information to Cooper staff. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We intend to stay as close to the Gantt chart as possible, but there have been delays. Many of the delays in the program have been the result of Yulex actions or inactions. Compounding studies have been delayed because of lack of rubber from Yulex, but this will be corrected now that PanAridus is supplying rubber. LCA information from Yulexwas sparse, so this information will be collected from PanAridus beginning in the second half of2014. Epoxidation studies have been delayed, but should be completed in the next annual reporting period. Once these studies are complete, the process of identifying a toller for epoxidized guayule will be started.

Impacts
What was accomplished under these goals? Program Area – Feedstocks Tools for molecular breeding Preliminary ploidy variation and nuclear genome size analysis of Parthenium argentatum A.Gray (Guayule) andParthenium incanum Kunth (Mariola) has been completed and published (Sanchez et al. 2014), laying the foundation for genomics-informed breeding. Ploidy level characterization of the Guayule germplasm collection continues at the Maricopa, AZ facility. Sequencing for a pilot project to develop and optimize genotype-by-sequencing based markers is underway on a selected set of 72 accessions of Guayule and allies. Current status and future directions of the molecular breeding portion of the project have been presented at the Plant and Animal Genome meeting in January 2014. Guayule genome sequencing – We were unsuccessful in our attempts to generate a dihaploid guayule for genomic DNA sequence acquisition and are currently sequencing a diploid line. To date have completed 13 454 FLX+ runs on this diploid, and have acquired approximately 12 gigabases of sequence (11x coverage) with 900 bp modal reads. We are currently acquiring MiSeq Illumina mate-paired library reads (800 bp fragments, 250 bp end reads) to correct 454 homopolymer errors. 1.5 runs (10x coverage) have been completed, 5 additional runs are scheduled for the coming month (50x coverage). Initial genome assembly will immediately follow the Illumina sequence acquisition. The 454 reads have been employed to hand assemble genes encoding proteins associated with rubber biosynthesis. To date, 18 genes have been assembled and allelic variation noted, including: Cis-PrenylTransferase, CPT (4 genes, 8 alleles) Small Rubber Particle Protein, SRPP(2 genes, 4 alleles) Farnesyl Diphosphate Synthase, FPPS (2 genes, 4 alleles) Allene Oxide Synthase, AOS (4 genes, 8 Alleles). Guayule Direct Seeding - Direct seeding field trial was planted out at UA, MAC field 108 in October 2013. Use of cover crops (barley, wheat and camelina) did not result in successful seedling emergence in all cases. Initial results suggest that cover crops are not important for seed germination. However, a laboratory study revealed guayule germination rate is more sensitive to temperature than previously recognized. Spring planting of direct seeded guayulewas conducted1Q14 and will focus on providing sufficient light, water and an optimum temperature for success. Guayule irrigation – Irrigation treatment field trial scheduling was initiated in May 2013 Target irrigation treatments (100% water use replacement) were given about 1150 mm of water (May through Nov.) under flood irrigation versus 1100 mm under drip. The range in treatment irrigation levels varied from about 450 to 1380 mm under flood and from 350 to 1350 mm in drip. Soil water content measurements were made weekly from June through Sept. and every 3 to 4 weeks from Oct. to Jan. 2014. Soil water balance calculations show obvious irrigation treatment differences starting as early as July 2013 under both irrigation methods. Similarly, the effects of irrigation on guayule height and canopy sizes were apparent starting in mid-July. In addition to irrigation and soil water content monitoring, measurements of plant biomass, rubber, and resin contents were made in July, Aug., Nov., and Jan. Harvesting/Logistics – Yulex has been replaced in the grant by PanAridus who will be responsible for the harvesting research. 2. Program Area – Biobased products and bioenergy a. Extraction/stabilization/drying guayule -PanAridus has replaced Yulex for this responsibility. Stabilization of the guayule natural rubber after isolationhas been an issue with Yulex produced material and is being addressed by PanAridus. Guayule compounding – Limited rubber has been available for compounding, but using a micro mixer, major progress has been made on bead filler, bead insulation, liner and rim cushion compounds for the concept tire. While Yulex was a member of the consortium, there was insufficient rubber to do all testing and compounding needed for all tire builds. This has been addressed by replacing Yulex with PanAridus. Tire builds with guayule rubber – Using novel approaches, a tire build was conducted on bead filler and bead insulation Tire testing is complete. All DOT specifications were met during wheel testing. Polymer-protein interactions – Techniques for isolation and purification of Hevea NR proteins were developed; addition of those proteins to guayule resulted in a slight but measureable increase in strain-induced crystallization (SIC). SIC measurements of model guayule compounds varying in filler, plasticizer, and cure state were performed. Higher filler and lower plasticizer concentrations favored crystal formation in guayule and Hevea. Energy from guayule bagasse There has been no progress reported by Yulex since the lastannual report. PanAridus is now responsible for ensuring that the biomass to energy component of the grant is successfully completed. 3. Program area – Sustainability Guayule tire LCA model - The model is currently under development Inventory data has been collected from Cooper Tire on the guayule tire prototype relating to the raw material composition, transportation of thesupplychain and energy consumption of the manufacturing process. Data on guayule agriculture has been collected throughreview of the literature and discussed with members from the U.S. Arid Lands Agricultural Research Center (ALARC).\Current tasks include calculating the life cycle impact assessment and incorporating the information into the LCA model Near future tasks include collecting agricultural data from ALARC at the end of their guayule harvest, collecting of supply chain data from Cooper, and collection of agricultural data; all of which will be integrated into the LCA model. Daina Rasutis has successfully defended her Master’s thesis which involved the LCA of a guayule tire and comparison to a standard broadline tire.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Sanchez,P.L., Costich, D.E., Friebec, B., Coffelt, T.A., Jenks, M.A., Gore, M.A., 2014 Genome size variation in guayule and mariola: Fundamental descriptors for polyploid plant taxa, Industrial Crops and Products 54, 1-5.
• Type: Other Status: Published Year Published: 2014 Citation: Rasutis, D., Soratana,K., Landis,A.E., 2014 Comparative Life Cycle Assessment of conventional and guayule rubber automobile tires. Proceedings of the International Symposium on Sustainable Systems and Technologies, Oakland, CA. May 18-24, 2014.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Sanchez, PlL., Chen, M.K., Pessaraklic, M., Hill,H.J., Gore, M.A., Jenks, M.A., 2014 Effects of temperature and salinity on germination of no-pelleted and pelleted guayule (Parthenium argentatum A. Gray) seeds Industrial Crops and Products 55, 90-96

Progress 06/01/12 to 05/31/13

Outputs
Target Audience: The guayule scientific community was formally informed of the grant details in a presentation delivered to AAIC by Howard Colvin. The Rubber industry has been informed of the significance and scope of the project by various media outlets such as Rubber and Plastics News. Changes/Problems: Anthony Halog was initially responsible for the sustainability analysis, but he has left the University of Maine and is now in Australia. Dr. Amy Landis of Arizona State University was chosen to replace Dr. Halog after an extensive interview process. We anticipate the sustainability analysis will be successfully completed by the end of the program. Due to extremely cold weather in Arizona, Yulex will not be able to provide rubber in 2013 for tire builds above the 170 lbs provided thus far. There should be sufficient rubber to complete compound studies on a small mixer for all tire components, assuming there are no process issues. There is enough rubber available for a small tire build in the 4th quarter of 2013. Tire builds in 2014 and 2015 will be rearranged to attempt to accomodate the interruption in supply. There has been a delay in the bioenergy program - both in the use of bagasse in cogeneration and as a feedstock for jet fuel, but these areas will be addressed prior to the completion of the grant. What opportunities for training and professional development has the project provided? This project has supplied opportunities in professional development in a number of areas. Cooper tire uses "real world" programs such as this to train co-ops and one co-op has used this opportunity to learn about the isolation and stabilization of guayule. Amy Landis' has two students gathering process data from both Yulex and Cooper to develop a life cycle assesment of the entire process. This is a new process for both of them, and has furthered their education. How have the results been disseminated to communities of interest? This program has been discussed in the media by Jeff Martin CEO of Yulex and Chuck Yurkovich, VP/Global Technology of Cooper. A wide range of media has covered the grant, so the audience has included people in the fields of agriculture, rubber production and tires in addition to the general public. What do you plan to do during the next reporting period to accomplish the goals? Work is planned to continue in line with the Gantt chart developed, with the exception of the rubber deliveries. There have been some delays in the bioenergy portion of the program, but these will be addressed in the future.

Impacts
What was accomplished under these goals? 6/1/2012 – 6/1/2013 Annual report for BRDI program 2012-10006-19391 An overview of the program is shown in the above figure.After a brief discussion of project management, progress for the 2012-2013 timeframe will be discussed in terms of feedstocks, biobased products/bioenergy and sustainability. Project Management – The working group, which consists of representatives of Cooper, Yulex, ARS and Arizona State University, meet regularly to discuss both technical and management issues regarding the project. Two face-to-face complete team meetings were held during the period to discuss technical progress. Feedstocks – Guayule genome sequencing – The team is in the process of acquiring the guayule transriptome. 2x 454 GS FLX+ runs 700+bp reads (Approximately 2 million ESTs) Tools for molecular breeding - The team is in the process of creating a diversity panel from the broadest sources available. This will be used to connect phenotype (plant size, architecture, rubber and resin content) to genotype (genome size, ploidy, genotyping-by-sequencing (GBS)). ~100 guayule accessions from several sources have been planted in a greenhouse and will serve as the foundation for ploidy analysis and the SNP discovery projects. Guayule direct seeding - Based on viability and germination results, a specific line was selected for the direct seeding experiments. The fields at MAC were prepared in March and cover crops were planted in April. The seeding study was begun in May using variables of irrigation type, cover crop type, seed type (primed vs pelleted) and also used transplants as controls. Guayule irrigation - A 15-station SDI system was installed in a 100-m by 120-m field at the Maricopa Agricultural Center (MAC) in 2012. The system was designed to provide separate control of water and nutrients to 15 plots. Guayule seedlings were transplanted in October in the surface irrigation field and later in the same month in the SDI field. Installation of neutron access tubes is complete. Data collection includes soil physical and chemical properties, soil water content, irrigation water flow rate and pressure and irrigation water chemistry. Plants will be sampled and biomass, height, volume, and leaf area measured, as well as rubber and resin content. Guayule harvesting – A new smaller scale harvester was purchased and will be tested in 2013. If successful, higher throughput harvesting equipment will be purchased for scale up trials, along with additional hauling equipment. Biobased products and bioenergy Extraction/stabilization/drying of guayule - A trial of an alternative milling process to improve cell rupturing and increase rubber extraction was successful. A process was developed to produce tire grade rubber from latex which meets specifications set by Cooper. Enough material was prepared to do small scale compounding on all tire components in the benchmark CS4 tire, assuming no processing issues. Extremely cold weather in Arizona in late 2012-early 2013 has damaged the guayule crop and this will prevent additional rubber shipments to Cooper for the remainder of 2013. Guayule compounding – A specification has been defined for tire grade guayule from latex which provides sufficient latitude in both rubber manufacture and formulation development as to make guayule substitution for components in the CS4 feasible. Guayule based formulas for two components of the CS4 have been defined and others are being developed. Polymer-protein interactions – These are being studied so as to make guayule strain crystallize like conventional NR. Addition of amino acids and proteins to guayule latex generally improves oxidative stability. One amino acid acts as both a plasticizer and an accelerator in guayule rubber latex. In general, however, addition of amino acids/ proteins do not affect film green strength although one exception was identified. Energy from guayule bagasse - New options for gasification cogeneration technology are being explored with two companies. An agreement for biofuels testing with a biofuels technology company is being finalized. Sustainability Amy Landis of Arizona State has replaced Anthony Halog on this portion of the project because Dr. Halog has moved out of the country. Dr. Landis will use ISO 14040 series to develop the LCA analysis. A scoping meeting with all stakeholders was held to define LCA goals and complete project scope.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: SECURING THE FUTURE OF NATURAL RUBBER  AN AMERICAN TIRE AND BIO-ENERGY PLATFORM FROM GUAYULE H.A. Colvin, L. P. Christoffersen, C. M. McMahan, and Amy Landis