Progress 10/01/11 to 09/30/16

Outputs
Target Audience:The target audience that have been reached by our efforts during this reporting period is mainly the scientific community working in Plant Biology, through our publications in peer-reviewed journals as well as presentations in seminars and conferences. In addition, we have collaborated with the commercial sector by assisting several start-ups that seek to develop duckweed-based applications. One is MamaGrande (Rosario, Argentina) that works to create a wastewater to bioplastics pipeline and the other is GreenOnyx (Tel Aviv, Israel) that is creating a compact duckweed production unit for scalable deployment. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Trained the following personnel through the duckweed project: 12 undergraduate students, with 4 currently working in the lab 4 visiting scholars from abroad 3 postdoctoral researchers How have the results been disseminated to communities of interest?6 publications in international journals. 15 invited seminars both nationally and abroad. Produced and maintained the Rutgers Duckweed Stock Cooperative website that disseminated our work and results to the community at-large. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Our project focused on creating the foundation resources to establish the aquatic plant duckweed as a novel micro-crop that will augment traditional agriculture by providing a sustainable platform for feed and fuel production. Over the past five years, my laboratory has helped to build the international community of duckweed researchers and commercial developers. We have demonstrated the feasibility and productivity of cultivating duckweed at existing wastewater sites and we have generated a comprehensive set of genomic resources that will enable the systematic domestication of duckweeds via molecular breeding approaches. 1. Identification of optimal strains of duckweed for biomass production from municipal wastewater. Established a 1,000+ strain stock center for duckweed germplasm Set up methodologies for systematic screening of duckweed strains for growth rate, protein content and starch content Explored methods for identification of high methionine strains of duckweed as optimal animal feed 2. Creating a sustainable wastewater-to-fuel pipeline with the duckweed platform. Work at the Pineland Nursery's fertilizer run-off containment ponds of the Pinelands Nursery in South Jersey provided an assessment of the potential duckweed biomass output that can be produced in an existing wastewater site. We estimated at this pond with an estimated 38,000 sq. ft., 48 tons of fresh duckweed can be produced per year with a 6 month growing season. Worked to optimize approach for economically viable maintenance and production of duckweed stocks for seeding ponds Genomics to enable molecular breeding technologies and genotyping duckweeds: completed a comprehensive barcode collection for all 37 species of duckweed; completed a high resolution and validated genome of Spirodela polyrhiza using a combination of advanced genomic technologies. 3. Outreach activities using the duckweed platform. Helped initiated and organized the past 3 International Conference of Duckweed Research and Applications (ICDRA), working as the lead organizer for the 2nd meeting held at Rutgers-New Brunswick in 2013. Served as a founder for the Duckweed Forum, a community Newsletter that began as a result of the 2nd ICDRA. Maintain and distributed duckweed strains for the community at-large via the establishment of the RDSC (Rutgers Duckweed Stock Cooperative) in 2009. Through the website of the RDSC, updated information relevant to the duckweed community are made accessible to the public. Worked with two start-up companies, MamaGrande (Rosario, Argentina) and GreenOnyx (Tel Aviv, Israel) to assist in their effort to deploy duckweeds for wastewater to bioplastic and for mobile source of nutrition for human consumption.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Comprehensive Definition of Genome Features in Spirodela polyrhiza by High-Depth Physical Mapping and Short-Read DNA Sequencing Strategies. Michael TP , Bryant D , Gutierrez R , Borisjuk N , Chu P , Zhang H , Xia J , Zhou J , Peng H , El Baidouri M , Ten Hallers B , Hastie AR , Liang T , Acosta K , Gilbert S , McEntee C , Jackson SA , Mockler TC , Zhang W , and Lam E . Plant J. 2016 Oct 18. doi: 10.1111/tpj.13400. [Epub ahead of print]

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audience that have been reached by our efforts during this reporting period is mainly the scientific community working in Plant Biology, through our publications in peer-reviewed journals as well as presentations in seminars and conferences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In this past reporting period, we have trained two new undergraduates in the laboratory on plant biology related techniques. In addition, two graduate students are working on our duckweed project as their PhD dissertation research. All these students are being trained in molecular biology, plant physiology, and genomics methods. In addition, these students also learn communication skills through laboratory seminars that they made presentations in on their research projects. How have the results been disseminated to communities of interest?Through publications in refereed journals and presentation in invited seminars internationally. There were also many invited seminars related to this project that have been presented by the PI over the past funding period. What do you plan to do during the next reporting period to accomplish the goals?1) We are currently working on the genome sequence for the smallest of duckweed genera, Wolffia. Working with Todd Michael at IBIS Biosciences in San Diego (CA), we are currently focused on completing the reference genome sequence for W. australiana. The Wolffia are fast growing, with doubling time of less than 30 hours, and has the smallest size at less than 1mm in diameter. Their simple, rootless architecture, with less than 500 cells make it a great plant model for Systems Biology. 2) We will continue to work with MamaGrande Co., to assist them in optimizing the duckweed deployment phase in their Salta wastewater treatment project. We are working closely with their young team of engineers to help design appropriate strategies in systematically identifying the best strain(s) of duckweed to deploy in their wastewater treatment lagoons. The lessons learned from this pilot project will be invaluable to inform future endeavors with similar goals.

Impacts
What was accomplished under these goals? For goal number 1, we have been focused on creating the key resource to associate phenotypes of duckweed to the genotypes in strains of interest that we have identified from our screens. Through work in the past 3 years, we have successfully integrated advanced technologies to produce a high fidelity reference genome for Spirodela polyrhiza, the Greater Duckweed. Our analysis with the validated genome sequence demonstrated that this duckweed has the fewest number of protein coding genes in all flowering plants sequenced to date. Together with a detailed characterization of the small RNAs in this species, our results now set the stage for the deployment of functional genomic approaches to characterize duckweeds. This work is currently being written up for submission to an international journal for consideration to publish. For goals number 2 and 3, we have continued to support and collaborate with MamaGrande Co. in Rosario, Argentina in their effort to build the first wastewater-to-bioplastics pipeline in the world. In Feb., 2015, they have begun their pilot scale project of 150 hectares of wastewater retention ponds in Salta and Tucuman, Argentina. We are assisting them with plant growth optimization strategies as well as supplying them with Argentine duckweed lines that the RDSC maintains and characterized. Lastly, we also helped to organize the 3rd International Duckweed Research and Applications conference that took place in July 2015, at Kyoto, Japan.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: "Natural variance in salt tolerance and induction of starch accumulation in duckweeds" K. Sowjanya Sree, Kai Adelmann, Cyrus Garcia, Eric Lam and Klaus J. Appenroth, Planta, doi: 10.1007/s00425-015-2264-x "Resurgence of duckweed research and applications: report from the 3rd International Duckweed Conference" Klaus?J. Appenroth, K. Sowjanya Sree, Tamra Fakhoorian and Eric Lam, Plant Molecular Biology, doi: 10.1007/s11103-015-0396-9
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: September 11, 2014: The ESALQ-University of Sao Paulo, Piracicaba, Brazil Title: "Duckweed Biotechnology and Genomics: a new path of collaboration with USP"
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: February 13, 2015: Institute of Biosciences, Department of Botany, University of Sao Paulo, Brazil Title: "Fast-forward creation of high-fidelity genome maps: enabling genomics for the sustainable aquatic crop Duckweed (Lemanceae)"
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: March 10, 2015: Institute of Biosciences, Department of Botany, University of Sao Paulo Workshop on Using Systems and Synthetic Biology to Tailor Plant Cell Walls for a Better Future Title: "Building resources to enable the use of duckweeds as a model plant for Systems Biology and Comparative Genomics"
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: April 25, 2015: Penn. State University, University Park, PA ECMS (Environmental Chemistry and Microbiology Student) Symposium, Keynote Speaker. Title: "Producing biomass from wastewater: aquatic agronomy enabled with duckweed as a sustainable crop"
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: August 27, 2015: Foz do Iguacu, Brazil The 44th SBBq (Society of Brazilian Biochemistry) meeting. Title: "Paving the foundation for the novel aquatic agricultural platform of duckweed with genomics
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: July 4, 2015: Kyoto University, Kyoto, Japan The Third International Conference on Duckweed Research and Applications Title: "Fast forward approach to a high-resolution genome sequence map and quantification of intraspecific structural and sequence variations in Spirodela polyrhiza strains"

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Plant Biologists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has helped train a graduate student (Philomena Chu), an undergraduate student (Sarah Pfaff) and a technician (Kenny Acosta), in addition to a Laboratory Researcher (Dr. Ryan Gutierrez). They were all exposed to training in plant physiology, molecular biology, genomics and biochemistry in various components of the project. How have the results been disseminated to communities of interest? A paper on genotyping of duckweeds has been published in Plant Biology last year. What do you plan to do during the next reporting period to accomplish the goals? 1. Continue our collaboration with MamaGrande to help guide their strain selection process in establishing the duckweed wastewater treatment pilot at Salta, Argentina. 2. Complete and publish a more robust draft of the Spirodela polyrhiza reference genome sequence. 3. Continue our effort to isolate, characterize and sequence the genomes of duckweed-associated bacteria strains.

Impacts
What was accomplished under these goals? We have essentially completed objectives 1 and 2 of the project and have identified a number of duckweed strains that can grow well in municipal wastewater and those that can produce high starch or protein. For objective 2 however, we are currently not pursuing the LCA analysis since we do not have the resources to carry out a scale-up production line for the wastewater-to-fuel technology ourselves. Instead, we are partnering with a start-up company, MamaGrande, in Argentina to eventually implement this. We are at present screening the 20 Argentine strains of duckweed in our collection to generate the growth rate and starch/protein content data for MamaGrande. In return, we will learn about their scale-up production platform and wastewater treatment economics in their new, 150 hectare wastewater treatment project in Salta, Argentina. As a social company that works very closely with the local municipalities to integrate education and environmental conservation in their business plan, we believe our work with MamaGrande will also address directly our Objective 3 in the project. To facilitate the creation of a new aquatic cropping system with duckweed, we have focused on developing two areas of high technology that will pave the way toward breeding elite duckweed strains with desirable traits. The first is a facile genotyping technique that will allow rapid and economical identification of duckweeds. We have now completed the first database for two molecular barcodes with data from all 37 known species of duckweed. We show that this resource can now be deployed to identify 30 out of the 37 species within 3 days via a generic PCR-based protocol. This work has recently been published and should help duckweed researchers and application specialists in more accurate typing of duckweed isolates worldwide. Currently, we are developing more refined genotyping technologies by testing nuclear loci that are known to be polymorphic in plants. To help advance genomic approaches in duckweed, a highly accurate genome sequence map is essential. Toward this end, we are finalizing our work in de novo genome sequencing and assembly for a strain of Greater Duckweed, S. polyrhiza (9509). Using a novel integration approach with genome draft sequence of another strain of S. polyrhiza (7498), we have produced a high fidelity reference genome for this species of duckweed and have validated its genome assembly as well as the predicted transcript models. This work, which should be submitted for publication in this quarter, should help set the stage for genomics-guide breeding of duckweeds in the near future. Lastly, as we worked toward scale-up production of duckweed under greenhouse and field conditions, it became apparent that association of particular bacterial strains may be an important factor for more robust growth of these aquatic plants. This empirical observation, together with the realization that many of the duckweed strains that we are maintaining in the RDSC carry tightly-associated bacteria, sparked our interest to begin systematic analysis of the microbiome associated with duckweeds. We have now set up a collaboration with Dr. Sarah Lebeis, the head of an Arabidopsis microbiome lab, at the University of Tennessee to begin a study on duckweed-associated-bacteria. This project has been awarded support in August by the JGI-DOE EMSL funding mechanism and we hope to help create the foundation to begin a systematic analysis of these microbes in the coming year.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: "Assessment, validation and deployment strategy of a two-barcode protocol for facile genotyping of duckweed species" Nikolai Borisjuk, Philomena Chu, Ryan Gutierrez, Honzhong Zhang, Kenny Acosta, Nikolai Friesen, Sowjanya Sree, Cyris Garcia, Klaus-J. Appenroth and Eric Lam, Plant Biol (Stuttg), August 12. doi: 10.1111/plb.12229.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Commercial developers interested in bioproducts; college students interested in sustainable agriculture; national funding agencies such as the National Science Foundation Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has trained 4 undergraduate students, two graduate students and one post-graduate workers in the past year. How have the results been disseminated to communities of interest? Yes. 1) published one paper in a refereed journal. 2) organized an international conference that was attended by over 50 participants. 3) visited several commercial developers to initiate discussions of collaborations. 4) presented several lectures on duckweed technology in both national and international venues. What do you plan to do during the next reporting period to accomplish the goals? 1) Continue to develop social media products to promote awareness and appreciation of the duckweed technology platform. 2) Take major steps to solidify collaborations with commercial developers to facilitate their deployment of this technology. 3) Publishing the first series of genome papers related to duckweed in order to move the field forward scientifically.

Impacts
What was accomplished under these goals? During the past funding period, we have actively pursued the following goals, with the aim to drive the duckweed technology platform toward successful commercialization: 1) While the potential of duckweed for wastewater remediation and biomass production is now well established in the lab, we realize thst the importance of public education at this stage will be critical in order to promote awareness as well as acceptance of this new technology and create the market for it. In this regard, we have accomplished 3 activities in the past year: a) We created an award-winning video of duckweed to biofuel and begin to promote this technology through public media. b) We established more collaboration and interactions between the growing number of stakeholders that are interested in duckweed worldwide, we hosted an international conference on Duckweed Research and Applications at Rutgers in August 2013. c) We have initiated steps to create collaboration with a start-up company MamaGrande in Rosario, Argentina, who has now begun to implement this technology for wastewater to bioethanol conversion. A visit to MamaGrande by the Lam group was completed in November 2013. 2) In order to better understand the science that will be necessary for the future development of duckweed as a new crop model for aquatic agriculture, we have now completed a reference genome for one strain of Spirodela polyrhiza. In addition, we have also resequenced 8 other strains of S. polyrhiza with increasing capacity for turion formation. This work should provide critical clues for us to begin understanding the mechanism of this developmental switch that is critical for climate adaptation of duckweed at the molecular level. In addition, since starch accumulation is dramatically increased in turions, we expect also to uncover the key regulators that can increase starch production as well.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Telling duckweed apart: genotyping technologies for the Lemnaceae (2013) Klaus-J. Appenroth, Nikolai Borisjuk and Eric Lam, Chinese Journal of Applied & Environmental Biology 19, 1-10.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: 1. Mentored two graduate (PhD track) students and 3 undergraduate (BS track) students on research projects related to duckweed utilization. 2. Co-organized the First International Conference on Duckweed Research and Applications. This meeting took place in Chengdu, China from October 7 to 10, 2011. 3. Participated in the New Jersey Governor's School of Engineering and Technology (July 2, 2012 to July 27, 2012) by providing lectures and hands-on experience to selected high school students on the use of duckweed for wastewater remediation and biofuel production. 4. Maintain and expanded the Rutgers Duckweed Stock Cooperative to more than 800 accessions/strains. Supplied duckweed strains to more than 15 users nationwide. 5. Presented a talk "Using duckweed for wastewater to fuel" to the Rutgers NJAES Board of Managers Research Committee and Extension Committee on Feb. 23, 2012. PARTICIPANTS: 1. Nikolai Borisjuk, Laboratory Researcher III. 2. Philomena Chu, Graduate Student 3. Jorge Montalvo, Graduate Student 4. Kenny Acosta, Undergraduate Student 5. Cyrus Garcia, Undergraduate Student 6. Angela Wan, Undergraduate Student 7. Klaus Appenrof, Jena Germany, Collaborator 8. Hai Zhao, Chengdu China, Collaborator TARGET AUDIENCES: 1. Classroom lectures to students at Rutgers. 2. Outreach seminars to the students in 4-H. 3. Seminars to NJAES Board of Managers at EcoComplex (Burlington, NJ). 4. Posters and Lectures in Universities and Conferences overseas: Chengdu (China), Beijing (China), Sao Paulo (Brazil), Recife (Brazil), Ohio (USA), Rutgers IGERT Summer Symposium (USA). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
There are two major thrust areas of our duckweed platform that we focused on developing in the past reporting year: 1) Demonstrating the feasibility of planting a desirable strain of duckweed at a wastewater test site. Using our barrier system that we designed and constructed, we were able to establish at the Pinelands Nursery fertilizer run-off pond the method to remove most of the indigenous duckweed from within the barrier by a pump-and-filter method. Subsequently, we successfully planted our selected strain of duckweed within the barrier and showed that it can be effectively propagated and retained within this system. This work pave the way for future commercial deployment of selected duckweed strains. 2) To provide the necessary information for intellectual property protection of the duckweed platform that arose from use of our large duckweed collection, we have focused on developing 2 key resources: a reference genome sequence for duckweed and a rapid genotyping method that is both economical and sensitive. Working with the largest commercial genome center in the world, the Beijing Genome Institute (BGI), we are in the process of completing the genome sequence for 9 strains of Spirodela (Greater Duckweed). In addition, we have generated encouraging results from experiments leading to conserved genes in plants for rapid and accurate genotyping analyses. These studies should pave the way for technologies and resources for protecting intellectual properties relating to duckweed strains identified for optimal production of target products.

Publications

• Zhao, H., Appenroth, K., Landesman, L., Salmean, A.A., and Lam, E. (2012) Duckweed rising at Chengdu: summary of the 1st International Conference on Duckweed Application and Research. Plant Mol. Biol. 78:627-632.
• Appenroth, K., and Lam, E. (2012) Wasserlinsen als Nutzpflanzen. Biol. Unserer Zeit 3:181-187.