SUSTAINABLE AND SCALABLE PRODUCTION OF FOOD AND FEED USING THE AQUATIC CROP PLANTS OF THE LEMNACEAE FAMILY.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1012957
• Project Start Date: Jun 1, 2017
• Project End Date: May 31, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559

Performing Department
Plant Biology

Non Technical Summary
To meet the challenge posed by population increase and environmental degradation due to application of large quantities of agricultural chemicals, this project will seek to create a scalable platform for biomass production using the aquatic plant duckweed. The growth characteristics of this small, floating plant are ideal for vertical farming strategies through modular designs that can tailor productivity per unit area according to need. Systematic standardization of the operating parameters for this prototype will enable its rapid deployment in various application pipelines. Our project's success can unleash this potentially game-changing technology platform for sustainable and continuous biomass production that can create a new sector of crop products, especially in the area of aquaculture. Some key advantages of this approach is that it will not compete with existing crops for arable land while minimizing environmental costs.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	5230	1040	100%

Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
5230 - Feed and feed additives;

Field Of Science
1040 - Molecular biology;

Keywords

duckweed

genotyping

leucine-rich repeat proteins

food security

aquaculture

Goals / Objectives
Agriculture in the 21st Century is facing the challenge of producing sufficient food and bioproducts to provide for more than an estimated 9.6 billion people by 2050. In order to meet this demand, it has been projected that an increase of current crop productivity by about 30% would be needed. At the same time, strategies and technologies to improve crop resilience to more extreme weather conditions resulting from Climate Change and to increase environmental sustainability through reduction in use of Ag chemicals are urgently needed. Plants of the Lemnaceae family, commonly called duckweeds, are aquatic plants found worldwide. These macrophytes are known to be the fastest growing plants in the world and through their rapid growth, can effectively remediate polluted water by assimilation of nitrogen and phosphate into biomass. In the past 5 years, our laboratory at Rutgers University has helped to nucleate a nascent international community of duckweed researchers as well as established a comprehensive germplasm collection, genomic tools and biochemical approaches for the duckweed platform. We are now poised to begin systematic deployment of duckweed as a novel scalable biomass production platform that will complement traditional crops as a sustainable source of nutrition for food and feed. In the next 5 years, we intend to focus on translating and transferring our laboratory results and tools to economically viable commercial ventures that will achieve real societal impacts. The success of our proposed project will relate to multiple NIFA national priority areas, most highly in the area of Aquaculture. Our project will also be relevant to those related to Climate Change issues such as Water Quality and Home Garden and Environment and Global Food Security and Hunger.

Project Methods
Exploring the use of the highly polymorphic NB-LRR (Nucleotide-Binding site-Leucine Rich Repeat) protein-encoding genes in duckweed as high-resolution genotyping markers. Using a degenerate set of primers that were based on the highly conserved amino acid residues at the NBS domain of NB-LRR proteins, we tested the potential of using this high-copy number and polymorphic gene family as a sensitive genotyping marker for S. polyrhiza strains. Preliminary testing with 3 strains obtained from different continents indicate that this is a promising strategy. We will continue to optimize this approach, such as the use of higher resolution polyacrylamide gels in place of agarose gels for separation of amplified DNA fragments, and will test its robustness using strains of S. polyrhiza from similar locale, such as Germany and New Jersey, as well as for species that have been difficult to distinguish based on plastid-encoded DNA barcodes, such as Lemna minor and Lemna japonica. In addition, we will also characterize the amplified bands by sequencing of the PCR products obtained from several duckweed strains in order to assess their identification and correlation to the NBS-LRR genes. In addition to gel-based methods, we will also synthesize fluorescent-probe-labeled primers to enable high resolution and rapid separation of PCR amplification products using a capillary sequencer. Lastly, we will also test whether we can extend this novel genotyping approach to other plant species by testing multiple characterized ecotypes of A. thaliana. The success of this work may provide a novel genotyping strategy for various plant species to aide molecular breeding applications.Construction of a prototype for the DPM and its systematic standardization. The modular format of our proposed DPM is conceived with the intention of both vertical and horizontal scalability. Leveraging the two-dimensional growth property of duckweeds, vertical farming can greatly increase the expected productivity of duckweed biomass per unit area of space. For example, with only a 1 m2 footprint, we can multiply the production of duckweed biomass per unit area by a factor of 8 by using a stack of 4 modules (each with two tiers) within a total height of 2 meters. At a top growth rate of 22 g dry weight per m2 per day (equivalent to about 75 metric ton per hectare per year) that we have measured for several Spirodela strains in our screen (Lam lab, unpublished data), we should be able to generate 170 g of dried duckweed per m2 per day (equivalent to 620 metric ton/hectare/year). If realized at scale, this will be an unprecedented level of edible biomass productivity that will have significant impact on solving the global food challenge. Our vision is to develop a modular duckweed cultivation platform, designated as a DPM, that can be easily scaled up or down depending on need and available resources. By leveraging the facile connectivity between modules via simple pipes and valves, we should be able to adapt this plant production system to either indoor or outdoor scenarios and to various space configurations in order to maximize the use of available space. The salient features and innovation of this platform are described in the following: Taking advantage of the two-dimensional growth property of duckweeds (i.e. they float on top of the water surface and prefer calm water with minimal agitation), we will build a prototype module during Phase 1 of this project. This module will consist of a tray with 0.5 m2 area (approx. 0.9 m x 0.54 m) and 15 cm in height, and a second tray of identical dimension directly below. The open space between the top and bottom trays can be adjusted by mechanical extenders that connect the two trays, but will initially be set at 10 cm. The total height of this two-tiered module will thus be 50 cm. Growth media or wastewater from a reservoir can be circulated periodically between the top and bottom trays by small pumps and will be facilitated by simple gravity-assisted flow design when possible. To prepare for our optimization study with the DPM, we will carry out a systematic comparison for growth rate and protein content of our 12 Starter strains in 20-20-20 media under non-sterile conditions. The setup of our experiment will be similar in design to that reported in Bergmann et al., with some modifications. Briefly, 75 mL of fresh media will be added to glass baby jars that have been cleaned and autoclaved, and to which one of the Starter strains will be added to cover the surface. At each time point of 7 and 14 days, after growing under long day conditions in a growth room, duckweed will be recovered by filtration and dried overnight at 42oC before weighing. Triplicate analyses will be done for each time point. After weighing, the biomass will be used for protein content assay using a standard protocol in the Lam lab, with soybean as our reference. The top duckweed strains from our "Starter kit" identified by these initial screens will then be used as reference material to begin the DPM optimization studies (described below), focusing on the use of commercial fertilizer for food/feed production.Validation of growth rates and protein content of the two top strains grown in the scaled-up volume and environment of the DPM prototype using synthetic fertilizer. From our current pilot studies in greenhouse studies, our DPM will require approximately 100 L of media (10 cm of liquid in height within the tray) vs. the 75 mL of 20-20-20 fertilizer media that we used for the screen above. Duckweed biomass from each of the two strains will be rapidly bulked up and used to spread on the surface of the respective tray with approximately 50 to 75 % coverage. After a full layer of coverage is achieved in our greenhouse environment - usually within 2 weeks - we will divide the surface area of the DPM by attaching 9 strips of thin plastic dividers [4 cm (W) X 54 cm (L)] of 1-2 mm thick and spaced 9 cm from each other. This will create 10 equal troughs of duckweed on the surface of each tray and allow us to collect duckweed samples from a similar surface area by simple skimming with a net. Samples will be collected at three time points (1, 7, 14 days) to measure total biomass, with triplicates to generate standard deviation values. The biomass collected will be dried and quantified for dry weight and protein content as described above. These results will provide a first comparison of the DPM's performance with synthetic fertilizer in a greenhouse environment vs. that obtained in our growth room, thus setting a first benchmark.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Scientific communities related to duckweed and plant microbiome research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two continuing PhD students, plus one new graduate student who just joined the Lam lab in September 2019, were trained under this research program over the past funding period. All three have been trained in genomics and plant biology techniques, and more specifically in plant-microbe interaction mechanisms. One student, Kenneth Acosta, presented his findings on DABs in an international conference on duckweed research last October held at Rehovot, Israel. His poster on DAB community characterization won the second prize of the poster session in this meeting. How have the results been disseminated to communities of interest?Through peer-reviewed journal publications and lectures at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?We will target to complete our effort to obtain the full roster of 70+ bacterial strains that together correspond to the core taxa we have identified in our published work. These strains will be used to construct a first duckweed synCom and will be used to inoculate gnotobiotic duckweed plants. We will sample the inoculated duckweed plants at various times from 1 to 30 days and metagenome analysis with the 16S rDNA amplicon will be carried out to track the assembly and temporal evolution of the microbiota structure within the duckweed tissue. Results from this work should open the gateway to a systematic characterization of the processes involved in generating a stable duckweed microbiome.

Impacts
What was accomplished under these goals? Following our detailed characterization of the duckweed associated microbiome, during the past funding period we have been focused on identifying a set of target bacteria strains that will represent members of the core taxa in order to begin our work to build synthetic communities of duckweed associated bacteria (DABs). Thus far, out of the ~70 strains that we aim to assemble in our first trial, we have been able to procure about 50 from our own collection as well as from other researchers. While we are currently contacting additional laboratories or the ATCC repository for bacteria strains to obtain the missing ones, we have been testing strategies to create a reproducible protocol for generating stocks of each strain of interest that will facilitate the future combinatorial approach to test each strain's efficacy to assemble into a stable microbiome in situ with gnotobiotic duckweed plants. After multiple trials and validation experiments, we now have a working protocol to generate stock culture tubes for each strain of selected bacteria that can be kept at -80C for months without losing their viability. So far, we have now produced these stock cultures for about 25 of the 50 strains of interest that we have in hand. In the new funding period, we aim to complete the rest of the 70 target strains and begin the first synCom experiments for duckweed.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Duckweed hosts a taxonomically similar bacterial assemblage as the terrestrial leaf microbiome Kenneth Acosta, Jenny Xu, Sarah Gilbert, Elizabeth Denison, Thomas Brinkman, Sarah Lebeis and Eric Lam, PLoS One (2020) 15(2): e0228560. https://doi.org/10.1371/journal.pone.0228560. pp.1-24
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Host-specific and tissue-dependent orchestration of microbiome community structure in traditional rice paddy ecosystems Weijuan Huang, Sarah Gilbert, Alexander Poulev, Kenneth Acosta, Sarah Lebeis, Chunlin Long and Eric Lam, Plant & Soil (2020) 452: 379-395. Cover Story
• Type: Journal Articles Status: Published Year Published: 2020 Citation: High saccharification, low lignin, and high sustainability potential make duckweeds adequate as bioenergy feedstocks Debora Pagliuso, Adriana Grandis, Eric Lam and Marcos Silveira Buckeridge, BioEnergy Research,https://doi.org/10.1007/s12155-020-10211-x

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Scientific community related to duckweed and microbiome research Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students were trained under this research program over the past funding period, with one of them - Sarah Gilbert - successfully completed her PhD defense. All of them were trained in genomics and plant biology techniques, and more specifically in the area of plant-microbe interation. Sarah has moved on to a Postdoc fellow position at Jeff Dangl's laboratory in North Carolina after her graduation. How have the results been disseminated to communities of interest?Yes, through publication and lectures at meetings. What do you plan to do during the next reporting period to accomplish the goals?We will continue our work toward creating synthetic bacterial communities with duckweed-associated microbes. These defined mixtures of multiple bacterial strains will be tested with duckweed and the stably assembled communities defined through amplicon sequencing approaches.

Impacts
What was accomplished under these goals? Rapid growth and easily digestible walls that are naturally low in lignin make the aquatic plant family Lemnaceae, or duckweed, a promising feedstock for biofuel production. We are carrying out systematic studies on the interaction between duckweed and microbes, specifically the bacterial microbiome, by combining both culture-independent and culture-dependent approaches. We have now characterized the bacterial microbiome of duckweed from various wild-populations as well as re-assembled through inoculation of gnotobiotically grown plants with wastewater. A conserved "core" community structure has been uncovered and we are working toward the creation of synthetic communities of defined combinations of bacteria strains to help us dissect the functions and roles of these bacteria.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Sowinski EE, Gilbert S, Lam E and Carpita NC. 2019. Linkage structure of cell-wall polysaccharides from three duckweed species. Carbohydrate Polymers 223:115119.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Researchers and application specialists in the growing field involving duckweed as a source of sustainable biomass for food and feed. Plant systematicists and breeders interested in genotyping technologies. Aquaculture and aquaponics researchers and application specialists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduate students (Katherine Scott and Megan Wang) and three graduate students (Kenneth Acosta, Sarah Gilbert, and Shawn Sorrels) have been contributing to the duckweed project in the Lam lab over the past period. They have been trained in various aspect of our basic research as well as applied studies related to duckweed biology and technology development. How have the results been disseminated to communities of interest?Through peer reviewed publications What do you plan to do during the next reporting period to accomplish the goals?We will continue our studies related to the following two topics: 1) Continuing our microbiome studies by focusing on functionally characterizing the role of several duckweed associated microbes from our collection. Some of the key studies that we intend to complete will be to focus on determining how colonization by one or more of the microbes may facilitate more robust growth of duckweeds under environmental stresses such as heat and/or high light. 2) We plan to continue our optimization of the DPM module through comparison of biomass production rates under varying light conditions as well as through continual adjustment of nutrient levels. Furthermore, product development with duckweed biomass that are produced from the DPM pipeline will be a new area that we need to move forward in order to help monetize the value of this crop through market creation. In addition to collaborating with Prof. Ron Huang at Food Science in Rutgers to develop protein extracts from duckweed biomass, we will also explore the fermentation of high starch duckweeds into beverages (e.g. beer, cider) that may find niches in the marketplace.

Impacts
What was accomplished under these goals? In the past reporting period, we have accomplished the follow objectives: 1) We have continue our basic research to characterize the microbiome of duckweed in order to enable our sustainable farming of these aquatic marcrophytes. We have published the first systematic work with 47 sequenced bacteria strains that we have isolated as endophytes of duckweed and characterized their ability to influence their plant hosts through their production of the phytohormone auxin. 2) In collaboration with Prof. Marcos Buckeridge's lab at the University of Sao Paulo, Brazil, we have completed a systematic comparison of cell wall associated carbohydrates of several duckweed species, focusing on the possible role of apiose as a potential biomarker for growth potential. 3) Continuing our development of a scalable Duckweed Production Module (DPM), we have successfully integrated a six-tier prototype with a 300 Gal. fish tank and demonstrated continuous production of duckweed biomass using aquaculture water, without addition of any fertilizer, for more than 5 months. In parallel, we have completed several systematic comparisons with different fertilizer compositions using two smaller DPM prototypes with 3-tier configuration. Tracking nutrient levels over 3-month periods using varying levels of starting fertilizer concentrations, we have collected quantitation data that should allow us to correlate biomass production rate to nutritent content in the growth medium. These results will be used to guide us in developing the precision farming protocol for the farming of duckweeds.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: "Bacterial Production of Indole Related Compounds Reveals Their Role in Association Between Duckweeds and Endophytes" Gilbert S, Xu J, Acosta K, Poulev A, Lebeis S, and Lam, E. Front Chem. 6:265. doi: 10.3389/fchem.2018.00265. eCollection 2018.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: "Correlation of Apiose Levels and Growth Rates in Duckweeds" Pagliuso D, Grandis A, Igarashi ES, Lam E, and Buckeridge MS. Front Chem. 6:291. doi: 0.3389/fchem.2018.00291.

Progress 06/01/17 to 09/30/17

Outputs
Target Audience: Nothing Reported 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? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will focus on carrying out the work as outlined in our recently accepted project proposal to NIFA.

Impacts
What was accomplished under these goals? Between June and September of the past reporting period, we have constructed the first prototypes for our Duckweed Production Module (DPM) and are beginning to test its functional characteristics in producing two different species of duckweed (Spirodela polyrhiza and Lemna minor). In parallel, fish tanks obtained from the NJ Aquaculture Innovation Center (AIC) are being integrated with some of the DPMs in order to test its functionality with aquaculture.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Michael, T. P., Bryant, D., Gutierrez, R., Borisjuk, N., Chu, P., Zhang, H., Xia, J., Zhou, J., Peng, H., El Baidouri, M., ten Hallers, B., Hastie, A. R., Liang, T., Acosta, K., Gilbert, S., McEntee, C., Jackson, S. A., Mockler, T. C., Zhang, W. and Lam, E. (2017), Comprehensive definition of genome features in Spirodela polyrhiza by high-depth physical mapping and short-read DNA sequencing strategies. Plant J, 89: 617635. doi:10.1111/tpj.13400