The Second International Conference on Duckweed Research and Applications August 21 – 24, 2013 School of Environmental and Biological Sciences Rutgers, The State University of New Jersey New Brunswick, New Jersey, USA
Program & Abstracts
The Second International Conference on Duckweed Research and Applications
In Dedication to Elias Landolt (1926 – 2013) Dr. Elias Landolt started collecting duckweed in the early 1950s as a young researcher. He likely did not foresee how important this seemingly unimpressive endeavor would turn out to be in the future. Elias not only collected these tiny plants but also systematically classified them, which gave his ever growing collection the rank it still holds today: the gold standard for duckweed research. He made significant contributions to the description of duckweed morphology, geobotany and even physiology. His monographs on duckweed systematics, physiology and biochemistry are considered bibles in the field to this day. In addition to establishing the largest collection of duckweed in the world over the last half century, Elias was well appreciated by his colleagues for his generous sharing of strains as well as providing sage advice upon request. Even in his final days when he was battling serious illness, he continued to assist and train researchers. We fondly remember our visit with Elias during Autumn 2011 when we collected duckweed strains together in Zurich. His enthusiasm for science was infectious and his knowledge about duckweed was truly inspirational. Considering the rising importance of duckweed at this time when sustainable production of biomass for fuel and feed are urgent problems facing humanity, the resources that were established by Elias are more important than ever. The soon-to-be completed genome sequencing of multiple Spirodela strains from the Landolt collection is a good example of the benefits that the scientific community is now beginning to reap from Elias' work. In the coming years, we expect to see an exponential rise in the growth of duckweed research and applications that will leverage the resources enabled by Elias' efforts. We therefore dedicate this meeting to acknowledge the contributions of his life-long passion for duckweed. We hope Elias' inspirational vision and steadfast dedication to the pursuit and sharing of knowledge will serve as a role model for us all in this turbulent time when fundamental changes in the way we manage our planet's resources will determine the sustainability of our world. A figure from The family of Lemnaceae – a monographic study (1986) by Elias Landolt
– Eric Lam and Klaus Appenroth
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The Second International Conference on Duckweed Research and Applications
Organizing Committee Eric Lam Jay J. Cheng Robert Martienssen Todd Michael Tamra Fakhoorian Ryan Integlia
Rutgers, The State University of New Jersey North Carolina State University Cold Spring Harbor Laboratory IBIS Biosciences International Lemna Association em[POWER] Energy Group
Host School of Environmental and Biological Sciences Rutgers, The State University of New Jersey New Brunswick, New Jersey, USA
Sponsors
Administrative Support Ryan Gutierrez, Philomena Chu, Feronda Long & Linda Anthony .............................................. Logistics Dalynn Knigge & Bianca Scardina ................................................................................... Online Registration Phil Wisneski..................................................................................................................................... Web Design Barbara Fitzgerald .................................................................................................................. Business Manager ii
The Second International Conference on Duckweed Research and Applications
Introduction The quest for sustainable production of biomass and renewable fuels with low carbon footprints has become a global priority. They are needed to produce food and nutrition for an estimated 9 billion people on this planet by 2050. Also, the expected decline in fossil fuel production and the need to curb climate change are urgent issues facing humanity in this decade. Creating and adopting new biomass and fuel production platforms that are environmentally and economically sustainable will be essential, but pose a formidable challenge to scientists, engineers and entrepreneurs. In our consideration of alternative sources of renewable biomass that can be "domesticated" for energy production as well as other products, we believe the Lemnaceae family of aquatic plants, commonly called duckweed, holds great potential as a commercially viable feedstock for fuel and feed production driven by sunlight. The chief characteristics that make duckweed ideal for waste-toenergy conversion are their rapid growth rate (up to 120 ton dry mass/hectare/yr) and their ability to grow directly on existing wastewater sites. Importantly, their natural growth characteristics also provide simple harvest strategies. Together with low lignin content and a large surface area to body mass ratio, duckweed provides a promising platform for sustainable biomass production coupled to reclamation of wastewater from different sources. To accelerate development of new businesses and products that are based on these aquatic plants as a novel micro-crop, we are organizing an international workshop with the aim to bring researchers and entrepreneurs together for sharing of information and networking for project development. These efforts will leverage and extend the traditional use of duckweed for plant systematics and biomonitoring of environmental contaminants. Several recent advances in this field should make this conference extremely timely: (1) Quantification of the potential of duckweed for bioethanol and biomass production in pilot scale studies, (2) Completion of the first reference genome of the Greater Duckweed Spirodela polyrhiza, (3) Formation of the International Lemna Association as a nascent grassroots effort to promote the advancements of duckweed- related applications and public awareness, and (4) Recent recognition of duckweed as an additional "feedstock" crop in a proposed tax relief bill by the U.S. government, which signals the emerging acceptance and promotion of the duckweed industry. The International Conference on Duckweed Research and Applications will focus on facilitating close interactions and coordination between duckweed researchers and application specialists from emerging industries. This workshop will be pivotal for charting a new course of development for this novel micro-crop system through creating a new sense of teamwork and focus in the duckweed community. Working together, we can then realize the great promise of these remarkable plants to alleviate urgent problems of water conservation and biomass production facing our planet.
Eric Lam Conference Chair iii
The Second International Conference on Duckweed Research and Applications
Important Contact Information Eric Lam Conference Chair Mobile: (XXX) XXX-XXXX Email:
[email protected]
Ryan Gutierrez Conference Coordinator Mobile: (XXX) XXX-XXXX Email:
[email protected]
Rutgers University Inn and Conference Center Phone: (732) 932-9144 Email:
[email protected]
Wireless Internet Access Network: RUWireless Username: duckweed2013 Password: duckweed
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The Second International Conference on Duckweed Research and Applications
Table of Contents Conference Venues .............................................................................................................................................. 1 Daily Schedule ...................................................................................................................................................... 2 Abstracts for Lectures ......................................................................................................................................... 6 Abstracts for Posters ......................................................................................................................................... 19 List of Participants ............................................................................................................................................. 24
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The Second International Conference on Duckweed Research and Applications
Conference Venues Registration ..................................................... Foran Hall Lobby Lectures ........................................................... Foran Hall, Room 138A Poster Sessions ............................................... Foran Hall Lobby Breakfast, Lunch & Coffee/Snacks ............ Foran Hall, Room 138B Dinner ............................................................. University Inn & Conference Center
Conference Venues
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The Second International Conference on Duckweed Research and Applications
Daily Schedule Wednesday, August 21 2:00 – 7:00 PM
Registration of Attendees (Foran Lobby)
2:00 – 4:30 PM
Reception with Snacks and Refreshments in Foran 138B
4:30 – 6:10 PM
SESSION 1: CONFERENCE OPENING
4:30 PM
Welcoming Address by Bradley Hillman, Senior Associate Director, New Jersey Agricultural Experiment Station, Rutgers University
4:45 PM
Opening Address by Conference Chair: Eric Lam, Rutgers University
5:00 PM
Plenary Lecture: “Introduction to thermochemical technologies for production of fuels and biobased products” (L1) Mark Wright, Iowa State University, Ames, IA, USA
6:00 PM
Welcoming Remarks by Robert Goodman, Executive Dean, School of Environmental and Biological Sciences, Rutgers University
6:10 – 7:00 PM
Poster Session (Foran Lobby)
7:00 – 8:30 PM
Dinner at the University Inn
8:30 – 10:00 PM
Open Discussion moderated by the International Lemna Association Chairs: Tamra Fakhoorian and Ryan Integlia
Thursday, August 22 8:00 AM – 6:00 PM Registration Continues (Foran Lobby) 8:00 – 9:00 AM
Continental Breakfast in Foran 138B
9:00 AM – 12 PM
SESSION 2: DUCKWEED BIODIVERSITY, ECOLOGY AND GENETICS
9:00 AM
“Formation and germination of turion in the duckweed Spirodela polyrhiza” (L2) Chair: Klaus Appenroth, University of Jena, Jena, Germany
9:40 AM
“Use of duckweeds for physiological and genetic researches in the laboratory” (L3) Tokitaka Oyama, Kyoto University, Kyoto, Japan
10:10 AM
Coffee/Snack Break in Foran 138B
10:30 AM
“Using disease resistance genes to identify duckweed plants” (L4) Philomena Chu, Rutgers University, New Brunswick, NJ, USA
Daily Schedule
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The Second International Conference on Duckweed Research and Applications Thursday, August 22 (continued) 11:00 AM
“Creating ‘The Charms of Duckweed,’ an educational website” (L5) John Cross, Alexandria, VA, USA
11:30 AM
“Biodiversity of duckweeds in Hainan” (L6) Jiaming Zhang, Chinese Academy of Tropical Agricultural Sciences, Haikou, China
12:00 – 2:30 PM
Lunch in Foran 138B with concurrent Poster Session (Foran Lobby)
2:30 – 6:00 PM
SESSION 3: DUCKWEED GENOMICS
2:30 PM
“Revolutionizing duckweed research with high throughput sequencing technologies” (L7) Chair: Todd Michael, IBIS Biosciences, Carlsbad, CA, USA
3:10 PM
“The genome of greater duckweed, Spirodela polyrhiza” (L8) Wenqin Wang, Rutgers University, Piscataway, NJ, USA
3:40 PM
“Genomics and transcriptomics of Spirodela polyrhiza” (L9) Doug Bryant, Danforth Plant Science Center, St. Louis, MO, USA
4:10 PM
Coffee/Snack Break in Foran 138B
4:30 PM
“Chromosomal integration of the Spirodela reference genome” (L10) Hieu X. Cao, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany
5:00 PM
“Duckweeds: Genetic study for biofuel production” (L11) Almudena Molla-Morales, Cold Spring Harbor Laboratory, NY, USA
5:30 PM
“Plant small noncoding RNAs and their roles in stress response and biomass accumulation” (L12) Weixiong Zhang, Washington University in St. Louis, MO, USA
6:00 – 8:00 PM
Dinner at the University Inn
8:00 – 10:00 PM
Open Discussion moderated by MamaGrande, Argentina Chairs: Eduardo Mercovich and Sebastián Lagorio
Friday, August 23 8:00 – 9:00 AM
Continental Breakfast in Foran 138B
9:00 – 11:50 AM
SESSION 4: WASTEWATER TREATMENT BY DUCKWEED-MICROBE INTERACTION
9:00 AM
Daily Schedule
“Phenolic compounds degradation in the rhizosphere of giant duckweed” (L13) Chair: Kazuhiro Mori, University of Yamanashi, Kofu, Japan
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The Second International Conference on Duckweed Research and Applications Friday, August 23 (continued) 9:40 AM
“Development of multifunctional vegetation bioprocess by utilizing symbiotic microorganisms in the rhizosphere” (L14) Masaaki Morikawa, Hokkaido University, Sapporo, Japan
10:10 AM
Coffee/Snack/Social Break in Foran 138B
10:50 AM
“Rhizosphere microbial community of duckweed (Lemna minor) in pilot-scale wastewater treatment system” (L15) Yonggui Zhao, Chengdu Institute of Biology, CAS, Chengdu, China
11:20 AM
“Enhanced photosynthesis, biomass production and nutrient uptake of duckweeds by plant growth-promoting bacterium, Sinorhizobium sp. SP4” (L16) Tadashi Toyama, University of Yamanashi, Kofu, Japan
11:50 AM – 2:30 PM Lunch in Foran 138B with concurrent Poster Session (Foran Lobby), followed by Open Discussion on formation of International Duckweed Steering Committee 2:30 – 5:10 PM
SESSION 5: AQUATIC AGRONOMY
2:30 PM
“High starch duckweed cultivation and ethanol fermentation efficiency optimization” (L17) Chair: Hai Zhao, Chengdu Institute of Biology, CAS, Chengdu, China
3:10 PM
“Applications of duckweed: bioproducts” (L18) Jay J. Cheng, North Carolina State University, Raleigh, NC, USA
3:40 PM
Coffee/Snack Break in Foran 138B
4:10 PM
“Nitrogen deficiency enhances accumulation of triacylglycerols in vegetative tissues of Lemna gibba DWC131” (L19) Seung Cho Lee, Cold Spring Harbor Laboratory, NY, USA
4:40 PM
“Pathway to scale-up production of duckweed: sustainability, experience and progress” (L20) Eric Lam, Rutgers University, New Brunswick, NJ, USA
5:10 – 6:00 PM
Poster Session (Foran Lobby)
6:00 – 10:00 PM
Dinner at University Inn with musical entertainment by the Vincent Troyani Jazz Quartet
Daily Schedule
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The Second International Conference on Duckweed Research and Applications Saturday, August 24 8:00 – 9:00 AM
Continental Breakfast in Foran 138B
9:00 – 11:40 AM
SESSION 6: DUCKWEED APPLICATIONS AND SYSTEMS APPROACHES
9:00 AM
“Commercial duckweed start-up case study: GreenSun Products, LLC” (L21) Chair: Tamra Fakhoorian, International Lemna Association, Mayfield, KY, USA
9:40 AM
“MamaGrande, a social company: duckweed-based, cost-effective biorefineries as an integral component of a better socioeconomic sustainable paradigm”(L22) Eduardo Mercovich, MamaGrande, Rosario, Argentina
10:10 AM
Coffee/Snack Break in Foran 138B
10:40 AM
“Duckweed as food: nutrition value, traditional consumption and commercialization challenges” (L23) Benny Shoham, GreenOnyx, Gany-Tikvah, Israel
11:10 AM
“Economic and public health improvements for impoverished communities in Bangladesh using polluted water bodies and duckweed as resources” (L24) Ryan Integlia, em[POWER] Energy Group, Wyckoff, NJ, USA
11:40 AM – 2 PM
2:00 PM
Daily Schedule
Lunch in Foran 138B, followed by Strategic Planning Session, including •
Vote to appoint first members of the International Duckweed Steering Committee
•
Open forum to draft a strategic plan outlining immediate goals and actions for the field
•
Selection of chair, location and timing of next meeting
Meeting Adjourns
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The Second International Conference on Duckweed Research and Applications
Abstracts for Lectures L1 Introduction to thermochemical technologies for production of fuels and biobased products Mark Mba Wright, PhD Iowa State University, Ames, IA, USA Research interest in biomass thermochemical technologies has grown in recent years prompted by environmental concerns and the search for economic alternatives to fossil-based fuels. This interest has led to innovations in the ways that we convert biomass into valuable chemicals and fuels. These innovations could change our fundamental understanding of biomass structures and open avenues to novel biomass-derived products. Mark Mba Wright will cover an overview of thermochemical technologies with a focus on biomass conversion strategies to fuels and biobased products. This presentation will discuss desired feedstock characteristics and describe various technologies under development for converting a wide range of biomass. Specifically, he will cover innovations in gasification, pyrolysis, hydrothermal processing, and solvolysis, and examine how these technologies could utilize duckweed. The presentation will highlight ongoing research by the Center for Sustainable Environmental Technologies (CSET) at Iowa State University (ISU). CSET and ISU have developed several facilities to support thermochemical research including the Biorenewables Research Laboratory and BioCentury Research Farm. About the Speaker Mark Mba Wright received a Doctoral degree in Mechanical Engineering and Masters in Biorenewable Resources and Technologies from ISU. After graduation, Mark accepted a Post-Doctoral research position at Massachusetts Institute of Technology. He is currently an Assistant Professor in Mechanical Engineering at Iowa State University. Mark is an affiliate faculty of the Bioeconomy Institute and the Graduate Program in Sustainable Agriculture. Mark has authored several papers on biofuel technologies as well as book chapters on biomass resources and conversion. His research interests include techno-economic analysis, (bio)energy systems, and life cycle analysis. During his free time, Mark enjoys spending time with his wife and one-year-old son.
Abstracts for Lectures
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The Second International Conference on Duckweed Research and Applications L2 Formation and germination of turion in the duckweed Spirodela polyrhiza Klaus-J. Appenroth University of Jena, Plant Physiology, Dornburger Str. 159, 07743 Jena, Germany Formation of turions, the vegetative perennation organs, plays an important role in the survival of Spirodela polyrhiza. Turion formation was investigated in 32 clones, including the recently sequenced clone 7498, collected from a wide geographical range. The Pearson correlation was tested with (1) duration of growing season (monthly average temperature of ≥ 10oC), (2) relative growth rate of the fronds, (3) longitude and latitude, and (4) several climatic parameters, in all possible single and multiple regressions. All single coefficients of determination were below 0.10. The highest correlation (R2 = 0.61) was found in a multiple linear regression with the following five parameters: average temperatures over the year and during the growing season, duration of the growing season and precipitation over the year and during the growth period. All these parameters were shown to have significant contributions. The average temperature over the year has the highest impact. It is concluded that lower temperatures decrease the survival rate of turions and that adaptation refers to increasing SY (quantified as number of turions formed per frond). The regression equation was used successfully to predict the SY of five newly isolated clones. The different levels of SY in the clones (ranging from SY = 0.22 to 5.9) were detected even after several years of axenic cultivation. It is therefore assumed that these adaptations to the climatic conditions are genetically determined. Moreover, SYs are correlated with the threshold nutrient concentrations at which turion formation is induced: Clones with high SY started turion formation at higher external phosphate concentrations passing a low, clone-dependent threshold. Decrease of the external phosphate concentrations and therefore induction of turion formation is caused by uptake during the growth season. Newly formed turions are dormant and germinate only after breaking dormancy, e.g. by cold afterripening. The light-dependent germination response of turions is mediated by phytochrome and requires the presence of Ca2+ in the medium. L3 Use of duckweeds for physiological and genetic researches in the laboratory Tokitaka Oyama Kyoto University, Graduate School of Science, Kyoto, Japan The duckweeds are tiny and floating flowering plants. The conspicuous traits of them are suitable for various experiments in the laboratory. Plant rhythmic phenomena and their underlying systems are my interest, and duckweed plants are good materials for the basic researches. Lemna gibba G3 and Lemna paucicostata 6746 have been “classic” duckweeds for physiological experiments. There were a number of reports about their flowering time and circadian rhythms, and we begun to use those two species. Through the researches we have developed physiological and genetic tools for the analyses. Gene transfection methods, methods for functional analysis of genes, pure lines, procedures of precise growth record are some of those tools. We also applied them to other duckweed species. I will summarize those tools and the application that are to be useful for experiments in the laboratory.
Abstracts for Lectures
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The Second International Conference on Duckweed Research and Applications L4 Using disease resistance genes to identify duckweed plants Philomena Chu, Nikolai Borisjuk, Angela Wan and Eric Lam Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA The diminutive size and reduced morphology of duckweed pose a significant obstacle for identifying species and, in particular, sub-species strains. Though physically similar, many strains exhibit differing physiological and biochemical characteristics. In order to exploit the large natural diversity of duckweed strains for potential applications, it is imperative to establish a rapid and robust technique to identify genetically unique individuals. We employ a DNA-based method called genotyping to fingerprint duckweed strains. Our prime candidates for genotyping are the nuclear-encoded NBS (nucleotide-binding site)-LRR (leucine-rich repeat) protein-encoding genes, the largest class of disease resistance genes in plants. These genes typically undergo rapid evolution as plants experience strong selective pressures from pathogens in the local environment. Hence, NBS-LRR alleles are highly diverse and can be used to distinguish individuals from different natural populations. Using the recently sequenced Spirodela polyrhiza genome, we designed PCR primers to amplify a region(s) of the NBS-LRR genes in duckweed. Preliminary results suggest that this method can successfully differentiate species, and further work will reveal the potential of this approach to discriminate sub-species strains. L5 Creating ‘The Charms of Duckweed,’ an educational website John W. Cross Alexandria, VA, USA This how The Charms of Duckweed website came to be. In 1989, I was studying the mechanism of action of a plant growth regulator. Duckweed attracted my attention as a model since it grows in a liquid medium and is easy to dose for metabolic studies. So I obtained a culture of Lemna gibba from Janet Slovin at the USDA-ARS in Beltsville, MD and worked with it for several years. I was impressed with the ease of experimentation with Lemna, and with the encouragement of duckweed pioneers Bud Culley at LSU and Elias Landolt at the ETH in Switzerland, I became equally enthusiastic about its potential applications. When I was at the National Science Foundation in 1994 the Internet was in its infancy. One of the other Program Directors gave a lecture on “Home Pages” and how to write them in HTML code. At that time, research interest in duckweeds had dwindled in favor of Arabidopsis, which has unexcelled genetics. I did not dispute this, but considered duckweeds had greater promise for practical applications. So I determined to create a comprehensive duckweed website that would promote their application. Accordingly, The Charms of Duckweed was written to cover everything from molecular biology to wastewater treatment and other applications. It first went on-line in 1998. Since early 2001, it has been hosted by the Missouri Botanical Garden and has received over 300,000 visits. This success has been assisted by many experts who contributed information and beautiful photographs.
Abstracts for Lectures
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The Second International Conference on Duckweed Research and Applications L6 Biodiversity of duckweeds in Hainan Jiaming Zhang, Ming Peng, Zilong Ma and Deguan Tan Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, 571101, China Hainan has a tropical climate, which allows duckweed to grow all year round in the open. It is also one of the largest tilapia growing provinces, with 53,000 hectares of water surface. Wastewater in the fish ponds is currently released at about once every two weeks without treatment, which is 12.8 billion tons of wastewater per year. This amount of wastewater provides great potential for duckweed to show its value in environmental protection and bioenergy production. Due to concerns of possible invasion by alien species to a novel habitat, native duckweed resources were collected and characterized. More than 200 strains have been collected so far. They grew normally in water with high concentrations of ammonium or nitrate and phosphate. Four species from four genera of duckweed were identified morphologically. They were present in multiple community types. Chloroplast rps16 sequencing was used to phylogenetically characterize the isolated strains. They were classified into four species: Lemna aequinoctialis, Spirodela polyrhiza, Wolffia species, and Landoltia punctata. From our data, an intra-species difference in rps16 sequences among the strains analyzed was not found, suggesting low biodiversity in the same species in Hainan. L7 Revolutionizing duckweed research with high throughput sequencing technologies Todd Michael IBIS Biosciences, Carlsbad, CA, USA High throughput sequencing has changed the pace and scope of plant biology. Now species with rich biology and research that were previously intractable to genomic, molecular or genetic studies are experiencing a renaissance. Often referred to as second-generation sequencing, these technologies have enabled the exploration of genome-wide biological features that were previously cost prohibitive or difficult to measure such as plant-microbe interactions, population nucleotide diversity and epigenomics. The true innovation of second-generation sequencing was the generation millions of short 50-150 base pair (bp) reads at the same time, in contrast to first-generation Sanger sequencing where hundreds of longer 500-1,000 bp reads were generated one at a time. This secondgeneration sequencing innovation required new computational tools to make sense of the massive number of low cost short reads, but it allowed researchers to use sequencing as a genomics counting tool where genes, 5-methylcytosine (5mC), transcription factor binding sites, single nucleotide polymorphisms (SNP), histone modifications, and other genomic features could be empirically annotated. More recently, a third-generation of sequencers are emerging that allow real time sequencing of long 5,000-10,000 bp single molecule reads, which enables genome assembly of complex plants and microbes, detection of full length spliced transcripts, phasing of SNPs and the direct detection of modified DNA (5mC). Duckweed is one such organism with a long and rich biological research history that will greatly benefit from the advent of high throughput sequencing technologies. The application of these technologies for the duckweed research community will be discussed.
Abstracts for Lectures
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The Second International Conference on Duckweed Research and Applications L8 The genome of greater duckweed, Spirodela polyrhiza Wenqin Wang and Joachim Messing Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ, USA Despite of its small size and simple structure duckweed exhibits attractive traits because of its fast growth, ease of transformation, and compact size. Compared to terrestrial plants, duckweed utilizes turions (rich of starch) as a dormant phase to pass through cold winters in place of seeds. Because of its unique habitat—floating on free water, it needs very little vascular tissue to maintain its stature. In addition, the duckweed community has already taken advantage of duckweed species to clean wastewater and convert their biomass into biofuel. To provide a new reference for the study of this family, we have sequenced, in collaboration with others, all three genomes of one species, greater duckweed or Spirodela polyrhiza, including the nuclear and two organellar genomes. This species has one of the smallest nuclear genomes of monocots at a size of 158 Mb. We have constructed a physical map so that the assembled sequence could be aligned to chromosome-sized supercontigs. The positional information about gene content and repetitive elements could aid our understanding of the chromosomal structure and organization of S. polyrhiza. Assuming our manuscript will be in press by the time of the meeting, the results of this study and a complete list of collaborators will be presented. L9 Genomics and transcriptomics of Spirodela polyrhiza Douglas Bryant1, Philomena Chu2, Ryan Gutierrez2, Nikolai Borisjuk2, Hanzhong Zhang2, Todd Mockler1, Todd Michael3 and Eric Lam2 1Donald
Danforth Plant Science Center, St. Louis, MO, USA; 2Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA; 3IBIS Biosciences, Carlsbad, CA, USA Spirodela polyrhiza is an excellent model for both basic and applied research due to its small genome, global distribution, and ability to transition between protein-rich vegetative leaf-like “fronds” to starch-rich storage organs, or “turions”. Development of genomic and transcriptomic resources for Spirodela is a fundamental first step necessary to facilitate genome-enabled, organismal- and population-level research. To augment the genomic tools available for Spirodela research, we created a high quality reference for an additional Spirodela accession (#9509) utilizing a diverse set of Illumina overlap, paired-end, and mate-pair libraries at ~120x estimated coverage. Transcriptome discovery and functional annotation was performed using de novo and reference-guided methods based on the new Spirodela reference and on extensive transcript sequencing (strand-specific RNA-seq). Using this reference genome we surveyed an additional eight Spirodela accessions at ~40x coverage each to assess intraspecific genomic variations such as single nucleotide polymorphisms (SNPs) and short insertion and deletions (INDELs). We then surveyed three accessions that differed in turion yield under turion-induced (ABA treatment) and control conditions to identify the expression networks and cis-acting elements associated with the high protein to high starch developmental switch. These genomic and transcriptomic resources provide a basis for subsequent molecular, genetic, physiological, and population structure analyses that should help shed light on the adaptation pathways which help shaped the turion yield trait in Spirodela.
Abstracts for Lectures
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The Second International Conference on Duckweed Research and Applications L10 Chromosomal integration of the Spirodela reference genome Hieu X. Cao1, Jörg Fuchs1, Wenqin Wang2, Klaus Appenroth3, Joachim Messing2 and Ingo Schubert1 1Leibniz
Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany; Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ, USA; 3University of Jena, Jena, Germany 2Waksman
The Lemnoideae (duckweeds) are a subfamily of the order of Alismatalis that comprises aquatic plants with a range of different genome sizes, from 0.158 to 1.88 gigabases. Greater Duckweed or Spirodela polyrhiza has one of the smallest monocot genomes with 158 Mb comparable to the dicot Arabidopsis. However, whereas Arabidopsis has only five chromosomes, Spirodela has 20. Given their similar genome sizes, the Spirodela genome has much smaller chromosomes, presenting a challenge to resolve them cytogenetically. Here, we have selected bacterial artificial chromosomes (BACs) with cloned Spirodela DNA that are low in repetitive elements and derived from a physical map as DNA probes to correlate the cytogenetic with the physical map. Based on the cytogenetic analysis, we could identify twenty individual chromosomes by using multicolor fluorescence in situ hybridization (mcFISH) combined with an optimized BAC probe pooling strategy. Correlation of the cytogenetic and physical maps will be beneficial in aligning the contiguous genome sequence information against twenty intact chromosomes. This completion of genome information allows further comprehensive comparative genomic analysis between duckweed, its close relatives, and other monocots of interest. In addition to the molecular cytogenetic map, we established the prerequisites for comparative chromosome painting to study chromosome homoeology and karyotype evolution of duckweed species, which rarely display sexual reproduction and thus are not suitable for conventional genetic mapping. L11 Duckweeds: Genetic study for biofuel production Almudena Mollá-Morales, Alex Cantó Pastor, Evan Ernst, Seung Cho Lee and Robert Martienssen Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA Petroleum availability is one of the main concerns of the era. Energy consumption is increasing and new sources need to be found. Currently, biofuels produced from corn grain and soybean are the two predominant ones. Due to their soil-based nature, they directly compete for land with food production crops, and there is a general concern about their potential of being sustainable. Lemnaceae species, commonly known as duckweeds, present themselves as an ideal organism for biofuel production. The aquatic nature of duckweeds eludes the competition with croplands, their high growth rate yields large amounts of biomass and their simple morphology facilitates their processing. Our goal is to engineer duckweeds to increase their oil levels for biofuel production. In order to do so, we aim to increase the expression of genes related to the production of TAG, silence the genes that have a role in the oxidation of lipid bodies, or redirect the starch metabolism to oil production by silencing the key genes that lead to starch accumulation. We are studying duckweed genetics and metabolism to develop and optimize molecular tools that can be applied to duckweeds and allow us to modify and analyze their oil content and composition.
Abstracts for Lectures
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The Second International Conference on Duckweed Research and Applications L12 Plant small noncoding RNAs and their roles in stress response and biomass accumulation Weixiong Zhang Department of Computer Science and Engineering, Department of Genetics, Washington University in St. Louis, MO, USA Small noncoding RNAs (sncRNAs), particularly microRNAs, have been recognized as key posttranscriptional gene regulators in eukaryotes. In this talk, I will first briefly review the types of sncRNAs in plants and their main modes of action in gene regulation. I will then focus on their functions in regulating genes and pathways related to stress response and biomass accumulation. I will end by a brief discussion of an on-going project on identifying, profiling and characterizing sncRNAs in Spirodela polyrhiza. L13 Phenolic compounds degradation in the rhizosphere of giant duckweed Kazuhiro Mori, Tadashi Toyama and Yasuhiro Tanaka University of Yamanashi, Kofu, Japan Wastewater treatment using aquatic plants has been studied and applied to practical use in the world. This system removes organic compounds (measured as BOD) and inorganic nutrients with low energy input via solar-driven plant uptake of nutrients and microbial degradation in the rhizosphere. However, its applicability to organic compounds recalcitrant to decomposition, including aromatic compounds, is still unknown. In this study, we elucidated the degrading activities of rhizosphere microorganisms of giant duckweed (Spirodela polyrrhiza) against aromatic compounds such as nitrophenols (NPs), chlorophenols (CPs), bisphenol A (BPA), nonylphenol polyethoxylate (NPE), and liner alkylbenzene sulfonate (LAS). Concurrently, the capacity of the plant-microbe system to remove compounds from contaminated water was evaluated. Sequential batch cultivation of material plants showed that the acclimation of the rhizo-microbial communities enhanced the rhizo-degradation capability, and the accelerated duckweed systems removed contaminants in the solution immediately. Almost 1010 CFU/g roots d.w. of microorganisms were observed on the root samples of duckweed, and the microbial flora was highly different from that of bulk cultivated water samples. Cultivation tests controlling the photo condition indicated accelerated microbial degradation due to oxygen transportation by the plant roots. Then, we isolated degrading microorganisms and applied them to bioaugmentation for accelerated duckweed treatment system, resulting in rapid and sustainable removal of contaminants in polluted environmental water through sequential batch treatment. These results indicate that the plant-microbe system plays an important role in the biodegradation and removal of chemical compounds in the water environment and can be a useful tool for polluted water treatment.
Abstracts for Lectures
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The Second International Conference on Duckweed Research and Applications L14 Development of multifunctional vegetation bioprocess by utilizing symbiotic microorganisms in the rhizosphere Masaaki Morikawa1, Masayuki Sugawara1, Kyoko Miwa1, Ayaka Makino2, Hideyuki Tamaki2, Yoichi Kamagata2, Tadashi Toyama3, Yasuhiro Tanaka3, Kazuhiro Mori3, Daisuke Inoue4, Kazunari Sei4, Masashi Kuroda5 and Michihiko Ike5 1Hokkaido
University, Sapporo, Japan; 2National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Japan; 3University of Yamanashi, Kofu, Japan; 4Kitasato University, Japan; 5Osaka University, Japan Objective: Reduction of fossil fuel use in industries is important not only for business but also for environmental conservation especially avoidance of global warming. Wastewater treatment is an energy consuming process in manufacturing factories. Our final goal is to develop basic technologies for effective wastewater treatment processing using aquatic plants including duckweeds. The key idea is to explore and make the best use of unknown beneficial bacteria that are in symbiotic interaction with aquatic plants. Results: A phenol degrading Acinetobacter sp. P23 could colonize the surface of Lemna minor and enhance its growth rate two-fold. This effect was not due to the rescue of L. minor from growth inhibition by phenol. P23 did not produce indole-3-acetatic acid and ACC deaminase but showed phosphorus solubilizing activity. Efficiency of water purification, in terms of phosphorus and nitrogen removal activities in wastewater and river water, was also increased accordingly in the Lemna/P23 system. When we tested phenol degradation activity, Lemna/P23 continuously degraded phenol for more than one week. We noticed that the chlorophyll contents of L. minor were increased upon colonization by P23. Then, we asked if this effect of P23 is limited to the family Lemnaceae. It was found that P23 was capable of colonizing the roots of a hydroponic vegetable, Lactica sativa (lettuce), and increased its chlorophyll content under nutrient poor conditions. Conclusion: We successfully attached Acinetobacter sp. P23 on the surface of L. minor and demonstrated the efficacy of Lemna/P23 both in wastewater treatment and biomass production technologies. This project has been supported by Japan Science and Technology Agency (JSTALCA). L15 Rhizosphere microbial community of duckweed (Lemna minor) in pilot-scale wastewater treatment system Yonggui Zhao and Hai Zhao Duckweed Research Group, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China In duckweed wastewater treatment systems, rhizosphere microbes of duckweed play an important role in removal of COD, TN, TP from wastewater, and also might exert a beneficial or deleterious influence on duckweed growth. Unfortunately, little was known about rhizosphere microbial communities of duckweed, especially in pilot-scale system. In this study, rhizosphere microbial communities of duckweed (Lemna minor) in pilot-scale systems operating under different HRTs
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The Second International Conference on Duckweed Research and Applications (hydraulic retention time) were investigated by 454 pyrosequencing. A total of 16,616 bacterial partial 16S rRNA gene sequences from four samples (four HRTs: 3, 6, 10 and 15 days) were acquired. All reads were affiliated with 23 phyla. Proteobacteria (60-80%) was the most abundant phylum in all analyzed samples. Other dominant phyla were Bacteroidetes, Cyanobacteria, Actinobacteria, Verrucomicrobia, Actinobacteria and so on. At the genus level, the predominant genera found were Rhodobacter, Pseudomonas, Flavobacterium, Perlucidibaca, Nitrosomonas, Rhizobacteria. Interestingly, as the HRT increased, the nutrient concentrations and removal amount of sewage in the treatment system decreased. The abundance of some genera, associated with the removal of COD and ammonia nitrogen—such as Nitrosomonas and Pseudomonas—decreased too, whereas the abundance of Rhizobacteria, which contributes to nitrogen fixation, increased significantly. This study reveals a high level of microbial diversity and a significant difference in the microbial community in the rhizosphere of duckweed in pilot-scale systems operating under different HRTs. L16 Enhanced photosynthesis, biomass production and nutrient uptake of duckweeds by plant growth-promoting bacterium, Sinorhizobium sp. SP4 Tadashi Toyama1, Yasuhiro Tanaka1, Kazuhiro Mori1 and Masaaki Morikawa2 1University
of Yamanashi, Kofu, Japan; 2Hokkaido University, Sapporo, Japan
The objectives of this study were to isolate plant growth-promoting bacteria (PGPB) from Spirodela polyrrhiza roots and to examine their effects on photosynthesis, biomass production and nutrient uptake of S. polyrrhiza. The study is an answer to the question, “Can PGPB improve nutrient removal and starch-biomass production from wastewater by duckweed?” More than 50 bacterial strains were isolated from S. polyrrhiza roots. Sinorhizobium sp. SP4 showed the highest growth-promoting effect on S. polyrrhiza in vitro assays. After co-culture of S. polyrrhiza with SP4 in Hoagland solution for 3 days, the rates of biomass (dry weight) growth, nitrate uptake, phosphate uptake and chlorophylls a/b content of SP4-inoculated S. polyrrhiza were up to 1.8, 2.2, 2.2 and 2.5 times higher, respectively, than those of non-inoculated S. polyrrhiza. The biomass production and nutrient removal from secondary effluent of sewage treatment plant was assessed using a laboratory-scale sequencing batch reactor. The rates of biomass production, nitrate removal, phosphate removal and starch production (biomass × starch content) by SP4-inoculated S. polyrrhiza were more than 2 times higher than those by non-inoculated S. polyrrhiza. In addition, SP4 showed the growth-promoting effect on 3 other duckweed (Lemnaceae) species: Lemna minor, Lemna aoukikusa and Wolffia arrhiza. The results suggest that the use of PGPB (e.g., SP4) with duckweed will improve both nutrient removal and starchbiomass production from wastewater. L17 High starch duckweed cultivation and ethanol fermentation efficiency optimization Hai Zhao Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China Liquid biofuels, such as bioethanol, converted from biomass are considered a promising alternative for traditional fossil fuels. However, existing bioethanol production modes have some inherent problems, including the adverse impacts on food security, environment and insufficient agricultural
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The Second International Conference on Duckweed Research and Applications land. As a new bioethanol production feedstock, duckweed has a longer yearly production period than most of other plants. It can thrive on eutrophic wastewater and accumulate high starch percentage. In our study, we found that the starch percentage of duckweed accumulated from the original 3.0% to 45.8% (dry matter) in 5 days when grown in distilled water. Activity of key enzymes involved in starch biosynthesis were up-regulated and enzymes involved in starch degradation were downregulated significantly. Comparative transcriptome analysis results showed that nutrient starvation suppressed most metabolic pathways in duckweed and redirected carbon metabolic flux by fixing more CO2 into the starch synthesis pathway, resulting in higher starch accumulation. We use biochemical, physiological and molecular evidence to reveal the mechanism of starch accumulation induced by nutrient starvation. Moreover, we established a large-scale cultivation technology and cultivated high-starch duckweed using wastewater in the fields. In our testing grounds, duckweed growing in wastewater can archive a high starch percentage of 52.9%. At Tien Lake, in the Yunnan province of China, the starch percentage of duckweed increased to 40% by treating with Tien lake water or living wastewater for about a week, and the annual production of this duckweed was 3 tons, corresponding to an annual production of 800 kg starch. To improve ethanol production, pectinase pretreatment was used to release much more glucose from duckweed mash. Results showed that maximum glucose yield of pectinase-pretreated mash was 218.6 mg/g dry matter, corresponding to 2.4 times that of the control. Scanning electron microscopy analysis showed that pectinase pretreatment appeared to change the ultrastructure of duckweed. Basing on this duckweed mash, we achieved an ethanol concentration of 30.8 g/L. The fermentation efficiency was 90.04%, which is the highest ethanol concentration reported to date using duckweed as the feedstock. L18 Applications of duckweed: bioproducts Jay J. Cheng North Carolina State University, Raleigh, NC, USA Duckweed (Lemnaceae) is a fast-growing, free floating aquatic plant. It has high contents of proteins and carbohydrates. Duckweed has been used for wastewater treatment to remove nutrients from the wastewater. The harvested duckweed can be utilized for the production of biofuels, animal and poultry feed, and even nutrition supplements. Duckweed yields 39.1–105.9 tons per hectare per year, which is much higher than the yields of most other crops. Duckweed starch content can be as high as 45.8% dry weight and up to 94.7% of the starch can be converted to ethanol using the existing technologies for corn starch conversion. Many duckweed strains have a high protein content of 3040% with well balanced amino acids including eight basic ones. The feeding trials of duckweed to hens and goats indicates that the egg quality from the hens is much better than the controls, and the growth of the goats is the same as the controls. Duckweed also has a capability of enriching minerals such as iron, zinc, and copper in its biomass, which shows a great potential to make duckweed-based green nutraceutical products.
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The Second International Conference on Duckweed Research and Applications L19 Nitrogen deficiency enhances accumulation of triacylglycerols in vegetative tissues of Lemna gibba DWC131 Seung Cho Lee, Keith Rivera, Evan Ernst, Darryl Pappin and Robert A. Martienssen Cold Spring Harbor Laboratory, 1 Bungtown Rd, Cold Spring Harbor, NY 11724, USA The smallest flowering plants, duckweeds are considered biofuel feed stocks because of their fast growth rates and clonal propagation. The whole genome of Lemna gibba DWC131 has been sequenced, and gene annotation is currently available. Nitrogen deficiency is one of the environmental stresses that can enhance biosynthesis and storage of starch and triacylglycerol (TAG) in vegetative tissues of plant species such as Arabidopsis thaliana and Chlamydomonas reinhardtii. Biodiesel can be produced by transesterification with plant oils which contain the high TAG contents. After they are synthesized in the endoplasmic reticulum or chloroplast, TAG molecules are transported and accumulated in lipid droplets with a mono-layer membrane in the cytosol and inside of the chloroplast. Based on transmission electron microscopy, we showed that L. gibba plants grown on nitrate-lacking media increased the numbers of lipid droplets mostly in the chloroplast, as compared to the plants on normal growth media. Enhanced TAG accumulation under 6 d nitrogen deficiency was confirmed by thin layer chromatography. Further TAG profiling was performed using liquid chromatography-mass spectrometry to identify and quantify different species of TAGs in L. gibba tissues. As a result, saturated and monounsaturated fatty acids such as palmitic acid (16:0) and palmitoleic acid (16:10) were enriched in TAGs extracted from nitrogen deficient L. gibba tissues. To monitor changes in gene expression leading to TAG accumulation during nitrogen deficiency, we obtained RNA-seq data from the control and nitrogen-deficient plants after 24 h. L20 Pathway to scale-up production of duckweed: sustainability, experience and progress Eric Lam1, Michelle Low2, Philomena Chu1, Jessica Kretch1, Ryan Integlia3, David Byrnes1 and Nikolai Borisjuk1 1Department
of Plant Biology and Pathology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA; 2School of Chemical and Metallurgical Engineering, University of the Witwatersrand, Johannesburg, South Africa; 3em[POWER] Energy Group A crucial component for realizing the commercial production of duckweed-based bioproducts is a reliable and economical mid-scale (in the kilogram range) cultivation method that can provide the necessary "seeding stock" for commercial scale, open-air ponds at the metric-ton scale. In the past 3 years, our lab has: (1) Designed, constructed and successfully tested a low-cost, floatation barrier system that can effectively stabilize seeded duckweed population on open ponds; (2) Carried out a careful cost comparison as well as measured growth rates for multiple strains/species of duckweed for various commercially-available or home-made hydroponic media in order to define the most economical and reliable media for duckweed cultivation under greenhouse conditions; and (3) Initiated scale-up production of selected duckweed strains in a greenhouse setting in order to begin a systematic cost analysis for the creation and maintenance of such a "seeding stock" facility. This work will help to create the necessary foundation that should pave the way for a reliable pipeline of translating knowledge generated in laboratory screens and selections to the field. In addition, it
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The Second International Conference on Duckweed Research and Applications should also provide the necessary duckweed biomass for experimental optimization of downstream processing methods. We will share our experience and expectations during this presentation. L21 Commercial duckweed start-up case study: GreenSun Products, LLC Tamra Fakhoorian International Lemna Association GreenSun Products, LLC was founded in 2010 by Tamra Fakhoorian of Mayfield, KY as an online retailer of solar ovens and green household cleaning products for sustainable lifestyles. Ms. Fakhoorian’s long-time interest in duckweed led to a vision for commercial, sustainable duckweed production. This vision began to take shape in 2012 when along with Ryan Integlia, Tamra cofounded the International Lemna Association. In February of this year, Tamra made the move to restructure GreenSun as one of the first privately held commercial duckweed producers/processors in the US. She began operations with a one-acre pilot pond and has since expanded to several more. GreenSun currently produces two strains of duckweed, S. polyrhiza and L. turionifera for boutique animal feeds. While still in the start-up phase, Tamra has much to share with entrepreneurs in terms of designing a business plan around duckweed niche markets and pointers for pond siting, seeding, and production. L22 MamaGrande, a social company: duckweed-based, cost-effective biorefineries as an integral component of a better socioeconomic sustainable paradigm Sebastián Lagorio, Federico Seineldin, and Eduardo Mercovich MamaGrande, Rosario, Argentina MamaGrande is a social company created to provide energy and biomaterial solutions while regenerating ecosystemic services for our challenged and fast changing world. Our deep aim is to create a cyclic industry that gives products and services with a positive balance in all social, natural and economic areas. We developed a biorefinery process that converts wastewater into clean water + ethanol or biodegradable plastic monomer + animal food + inclusive jobs. Using technological recombinant innovation, we integrated current Lemnaceae knowledge and techniques with a new biological product. With social recombinant innovation, we created a government-communityuniversity alliance to develop the biorefinery pilot and developed a business model dedicated to maximize social and natural impact while maintaining a positive economic balance: an assisted model for small towns (up to ~12 to 15,000 inhabitants), and a dedicated service for feedlots, the food industry and big cities (summing more that 4,000,000 inhabitants with only the most probable ones to expand in the next 2 years in Argentina). We are currently working in our first pilot plant in Totoras, an 11,000-inhabitant town with a ~750 m3/day wastewater flux in 4.16 ha of wastewater lagoons in the province of Santa Fe, Argentina. Estimated results for this pilot in the next year are almost 30,000 l of ethanol, 16 T of animal food, +270,000 l of cleaned water and 2 new direct jobs. Our current technical challenges are how to maximize system-wide productivity and scale up Lemnaceae cultivation and harvesting for starch.
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The Second International Conference on Duckweed Research and Applications L23 Duckweed as food: nutrition value, traditional consumption, and commercialization challenges Benny Shoham and Tsipi Shoham GreenOnyx, Gany-Tikvah, Israel [Abstract will be available online.] L24 Economic and public health improvements for impoverished communities in Bangladesh using polluted water bodies and duckweed as resources Ryan Integlia and Tamra Fakhoorian em[POWER] Energy Group, International Lemna Association In Bangladesh, a significant portion of the population does not have access to the safe drinking water and public health amenities. One of the reasons behind these issues is that the economy of the country does not facilitate the administration of basic amenities to the impoverished communities residing near wastewater bodies or grey water bodies. However, Bangladesh has an abundant resource of surface water bodies. These surface water bodies work as collecting points for runoff from nearby agricultural, mixed waste dumping ground and wastewater from local communities. The adjacent communities use these water bodies for bathing, washing and as drinking water. Due to poor water quality of these water bodies, the residents of the local communities suffer from various water borne diseases. These water bodies also work as the breeding ground of various disease carrying mosquitoes. We have identified a potential public health enhancement by using these water bodies as a resource where duckweed and fish can be grown together. After harvesting, the duckweed will be used for biofuel production and fish will be sold to the local market. Duckweed also helps to improve water quality of the water bodies by absorbing various pollutants. We are also working toward culturing local fish species which prey on the mosquito larvae and help to minimize mosquito borne diseases. The duckweed itself can potentially serve to minimize the accessible surface area available to mosquitoes and thus potentially aiding to limit the mosquito population. The entire project is designed to improve the economic and public health condition of the community members. To do so we propose using a labor-intensive industrial process so that a part of the operating cost will be distributed to the local community members as employees engaged in the harvesting, processing and distribution. Thereby, we create jobs from the inception of the industry. We also plan to measure water quality of the water bodies, conduct experiments to measure effect on mosquito population and record the health related issues of the community members, with our partner the Gambangla Unyanna Committee (GUC), to find the effects of this project on the local economy, and public health conditions of the community members.
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The Second International Conference on Duckweed Research and Applications
Abstracts for Posters P1 Small scale biodigestion of duckweed for methane production Zachary Dowell, Ryan Integlia, Tamra Fakhoorian, Amanda Luther, Nasir Uddin and Kevin Kung em[POWER] Energy Group; Biogas Institute; International Lemna Association; Rutgers, The State University of New Jersey; Massachusetts Institute of Technology Duckweed has been used in developing nations for water remediation and fish culture. In most cases, the duckweed is dumped alongside water bodies after maturation and left to decompose through natural processes, which contributes to greenhouse emissions through methane gas release. Alternatively, anaerobic digestion (AD) of the duckweed could be applied for biogas production to recover energy from this biomass. Research suggests that co-digestion of vegetative matter with manure can increase biogas yield from these materials. Overloading a bioreactor with duckweed can destabilize the reactor due to a shift in the microbial community towards an increase in acidogenic bacteria populations over methanogens, leading to reduced biogas production and possible failure of the bioreactor. The objective is for duckweed alone to be fed to the digester at the proper controlled loading rate after being “seeded” with manure. Co-digestion of duckweed and manure at a 1:1 ratio is feasible for maintaining a stable microbial community for biogas production and waste degradation within the bioreactor. While manure may be available in some developing countries, there may not be a steady and sufficient supply to support long term co-digestion at this rate. The intent of this research is to test co-digestion of duckweed with manure at different ratios by starting with a 1:1 ratio and incrementally reducing the amount of manure added. Other types of biomass that may serve as a substitute for manure may also be considered as appropriate. The goal is to determine the minimum amount of manure needed for co-digestion with duckweed to maintain a healthy microbial ecosystem with steady biogas production. P2 Rapid prototyping using duckweed feedstock based bio-resin Alexander Pluke, Ryan Integlia, Tamra Fakhoorian, Ryan Hunt and Nasir Uddin em[POWER] Energy Group; The Plastic Economy; International Lemna Association; Algix A method is proposed to transform duckweed, known for its ability to remediate wastewater, into a rapid prototyping sustainable bio-feedstock to produce bio-plastic products that meet a niche demand for the rapid prototyping industry. Duckweed can be either wild harvested, farmed or grown using local wastewater sources, then processed and extruded into bio-plastic resin filament. This filament will be examined for use in 3D printers, which will produce tailored products on demand to meet local needs. The compatibility of the attributes of the duckweed based resin and the properties of the rapid prototyping system will also be analyzed. In general, this process takes what was formerly considered a nuisance plant, which is now considered a feedstock, and creates a useful resource that will sustainably grow new high-value markets of on-demand production.
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The Second International Conference on Duckweed Research and Applications P3 Improving production of bioethanol from duckweed Yanling Jin, Qian Chen, Yang Fang and Hai Zhao Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences (CAS), Chengdu 610041, China Landoltia punctata, a widely distributed duckweed strain with the ability to accumulate starch, was used as a novel feedstock for bioethanol production by Saccharomyces cerevisiae. To improve ethanol production, pectinase pretreatment was used to release much more glucose from L. punctata mash, and the pretreatment conditions (enzyme loading, temperature and pretreatment time) for the duckweed were optimized by using a surface response design. The results showed that maximum glucose yield was 218.64 ± 3.10 mg/g dry matter, which is a 142% increase compared to the untreated mash, with a pectinase dose of 26.54 pectin transeliminase unit/g mash at 45 °C for 300 min. Pectinase pretreatment apparently changed the ultrastructure of L. punctata, as evidenced by scanning electron microscopy analysis. Further fermentation experiments were performed, and 30.8 ± 0.8 g/L of ethanol concentration, 90.04% of fermentation efficiency and 2.20 g/L/h of productivity rate were achieved. This is the highest ethanol concentration reported to date using duckweed as the feedstock. P4 Characterization of cellular circadian rhythms in Lemna gibba by using an in vivo bioluminescence monitoring system Masaaki Okada, Tomoaki Muranaka, Toshihiro Yoshihara and Tokitaka Oyama Department of Botany, Graduate School of Science, Kyoto University Most organisms have the circadian clock as a timekeeping mechanism to anticipate daily environmental changes. In plants the circadian rhythm is involved in many physiological phenomena, including development, growth and flowering. Studies using Arabidopsis have identified a number of clock-related genes and transcriptional-translational feedback loops of these genes form the core of the circadian clock in cells. However, characteristics of individual “cellular circadian clocks” remain to be elucidated. We established the in vivo imaging system that enabled monitoring of bioluminescence reporter activities of individual cells using Lemna plants. We used AtCCA1::luciferase as a morning-expressed reporter gene and this construct was transfected into cells by a particle bombardment method. Using this monitoring system, we attempted to assay the effects of growth conditions, such as illumination conditions, and nutrients on the cellular circadian rhythms. We also tried co-transfection assays for gene manipulation of circadian clock systems.
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The Second International Conference on Duckweed Research and Applications P5 Highly-specific gene silencing by artificial microRNAs in Lemnaceae Alex Cantó Pastor1, Almudena Molla-Morales1, Evan Ernst1, Jixian Zhai2, Blake Meyers2 and Robert Martienssen1,3 1Cold
Spring Harbor Laboratories, NY, USA; 2University of Delaware, DE, USA; 3Howard Hughes Medical Institute - Gordon and Betty Moore Foundation, NY, USA Endogenous micro (mi)RNAs trigger cleavage and/or translational repression of mRNA targets. miRNA precursor sequences are highly variable, yet, mature sequences display high target sequence complementarity, resulting in specific post-transcriptional gene silencing (PTGS). Artificial (a)miRNAs can be designed by taking the endogenous precursor and replacing the mature miRNA sequence (21nt) to target any gene of interest. We predicted a series of endogenous Lemna gibba G3 miRNA precursors, by utilizing our genomic scaffolds and small RNA sequencing libraries. We further tested several the predicted precursors in vivo, looking for correct processing and accumulation of their mature form. Using these endogenous precursors, we have successfully developed a novel amiRNA platform that targets any gene of interest. This amiRNA based PTGS, shall provide a valuable approach for high-throughput and highly specific gene silencing in various Lemnaceae species. P6 Preliminary lab-scale studies on the use of duckweed for the treatment of sewage and starch production in Totoras, Santa Fe (Argentina) L. Mariano Paz1, Mariano Bello2, Diego Wassner3, Federico Seineldín4, Sebastián Lagorio4, Eduardo Mercovich4 and Elena Mongelli1 1Cátedra
de Biomoléculas, Facultad de Agronomía (UBA), Av. San Martín 4453 (1417), Buenos Aires, Argentina; 2Jardín Botánico, Facultad de Agronomía (UBA), Av. San Martín 4453 (1417), Buenos Aires, Argentina; 3Cátedra de Cultivos Industriales, Facultad de Agronomía (UBA), Av. San Martín 4453 (1417), Buenos Aires, Argentina; 4MamaGrande, Av. Francia 889 (2002), Rosario, Argentina Duckweed is a plant that plays an important role in wastewater treatment. It is known for its excellent growth and protein content. On the other hand, its starch content can be increased by manipulating growing conditions, which makes it a possible feedstock for bioethanol production. In order to know the performance of this plant in the treatment of sewage from Totoras, Santa Fe (Argentina) and its starch production, some lab-scale studies were carried out. A local duckweed strain of Spirodela polyrrhiza was initially grown in gutters with effluent from the Totoras sewage lagoon. This plant was used to inoculate containers (0.04 m3) with different dilutions of effluent (100, 50, 25 and 10 % V/V) in order to evaluate the biomass growth and starch production. The effluent used in the experiments was extracted during the autumn, one of the moments of the year with high phytoplankton content. Due to photosynthesis activity, the initial effluent pH was 9.95. The containers were cultivated outdoors during 21 days, with daily external temperature and pH measurements. The average temperature and pH were 22.3 ºC and 9.85,
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The Second International Conference on Duckweed Research and Applications respectively. The biomass (dry weight) rose 23.5% when the effluent percentage increased from 25 to 50%, and 30.5% when the percentage was increased from 50 to 100%, without significant biomass differences between the 10 and 25% experiments. Total sugars analysis indicated that the highest production in these conditions was 39.7 % W/W using 50% effluent dilution. P7 Influence of heavy metals on accumulation of starch in Lemna minor Sowjanya Sree Kandregula1, Klaus-J. Appenroth2 1Amity
Institute of Microbial Technology, Amity University Uttar Pradesh, India; 2Institute of Plant Physiology, University of Jena, Germany Lemna minor (clone 9441) is one of the duckweeds growing in the lentic ecosystems with a wide geographical distribution. Tolerance to heavy metals and their uptake by these aquatic plants has been well documented, and this feature allows them to grow also in many of the polluted ponds and lakes. In the present study we investigated the influence of a dozen of heavy metals on the accumulation of starch in the fronds of L. minor. The starch content, Pulse Amplitude Modulated fluorescence and the chlorophyll content of the plants treated with EC50 concentrations (50% effective concentration for the inhibition of growth) of heavy metals and the control plants were measured. Results indicated that at the EC50 concentration of the heavy metal photosynthesis was often not yet strongly inhibited and that the photosynthates were therefore accumulated as starch. For example, in the presence of EC50 of Zn2+, the L. minor fronds showed a minor 9.5% reduction in photosynthetic rate while the accumulation of starch was recorded. On the other hand, EC50 of Tl+ had a heavy inhibitory effect of 40.7% on the photosynthetic rate and the starch content was lower than in the control plants. In conclusion, the photosynthate is accumulated in the fronds in the form of starch because of the inhibition of growth by the heavy metal. Thus, photosynthates cannot be consumed as fuel for growth. This will be of interest when we are looking for a high quantity of starch from less biomass which might be the case in the application of duckweed in biofuel production. P8 Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation Xiang Tao, Yang Fang and Hai Zhao Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan 610041, China Duckweed can thrive on anthropogenic wastewater and produce tremendous biomass. Due to its relatively high starch and low lignin percentage, duckweed is a good candidate for bioethanol fermentation. Previous studies have observed that water devoid of nutrients is good for starch accumulation, but its molecular mechanism remains unrevealed. This study globally analyzed the response to nutrient starvation in order to investigate starch accumulation in duckweed (Landoltia punctata). L. punctata was transferred from nutrient-rich solution to distilled water and sampled at different time points. Physiological measurements demonstrated
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The Second International Conference on Duckweed Research and Applications that the activity of ADP-glucose pyrophosphorylase, the key enzyme of starch synthesis, as well as the starch percentage in duckweed, increased continuously under nutrient starvation. Samples collected at 0 h, 2 h and 24 h time points respectively were used for comparative gene expression analysis using RNA-Seq. A comprehensive transcriptome, comprising of 74,797 contigs, was constructed by a de novo assembly of the RNA-Seq reads. Gene expression profiling results showed that the expression of some transcripts encoding key enzymes involved in starch biosynthesis was up-regulated, while the expression of transcripts encoding enzymes involved in starch consumption were down-regulated; the expression of some photosynthesis-related transcripts were down-regulated during the first 24 h, and the expression of some transporter transcripts were up-regulated within the first 2 h. Very interestingly, most transcripts encoding key enzymes involved in flavonoid biosynthesis were highly expressed regardless of starvation, while transcripts encoding laccase, the last rate-limiting enzyme of lignification, exhibited very low expression abundance in all three samples. Our study provides a comprehensive expression profiling of L. punctata under nutrient starvation, which indicates that nutrient starvation down-regulates the global metabolic status and redirects the metabolic flux of fixed CO2 into the starch synthesis branch, resulting in starch accumulation in L. punctata.
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List of Participants Name
Institution/Company
City
Country
Email
Kenneth Acosta
Rutgers University
New Brunswick, NJ
USA
[email protected]
Klaus Appenroth
University of Jena
Jena
Germany
[email protected]
L2
Doug Bryant
Danforth Plant Science Center
St. Louis, MO
USA
[email protected]
L9
Alex Cantó Pastor
Cold Spring Harbor Laboratory
Cold Spring Harbor, NY
USA
[email protected]
P5
Hieu Cao
Leibniz Institute of Plant Genetics and Crop Research
Gatersleben
Germany
[email protected]
L10
Chia-Hui Chen
Rutgers University
New Brunswick, NJ
USA
[email protected]
Jay Cheng
North Carolina State University
Raleigh, NC
USA
[email protected]
L18
Philomena Chu
Rutgers University
New Brunswick, NJ
USA
[email protected]
L4
John Cross
The Charms of Duckweed
Alexandria, VA
USA
[email protected]
L5
Weihua Cui
China University of Geosciences
Beijing
China
[email protected]
Marvin Edelman
Weizmann Institute of Science
Rehovot
Israel
marvin.edelman@ weizmann.ac.il
Evan Ernst
Cold Spring Harbor Laboratory
Cold Spring Harbor, NY
USA
[email protected]
Tamra Fakhoorian
International Lemna Association
Mayfield, KY
USA
[email protected]
Yang Fang
Chengdu Institute of Biology, Chinese Academy of Sciences
Chengdu
China
[email protected]
Paul Fourounjian
Rutgers University
Piscataway, NJ
USA
[email protected]
Adrianus Cornelis Gauw
Van Hall Larenstein
Leeuwarden
Netherlands
[email protected]
Ryan Gutierrez
Rutgers University
New Brunswick, NJ
USA
[email protected]
Bradley Hilborn
Nagase America
New York, NY
USA
[email protected]
Ryan Integlia
em[POWER] Energy Group
Wyckoff, NJ
USA
[email protected]
L24, P1-2
Yanling Jin
Chengdu Institute of Biology, Chinese Academy of Sciences
Chengdu
China
[email protected]
P3
Sowjanya Sree Kandregula
Amity University Uttar Pradesh
Noida
India
[email protected]
P7
Bert Knol
Algaecom
Eelde
Netherlands
[email protected]
Sebastián Lagorio
MamaGrande
Rosario
Argentina
[email protected]
P6
Eric Lam
Rutgers University
New Brunswick, NJ
USA
[email protected]
L20
Seung Cho Lee
Cold Spring Harbor Laboratory
Cold Spring Harbor, NY
USA
[email protected]
L19
Zilong Ma
Chinese Academy of Tropical Agricultural Sciences
Haikou
China
[email protected]
Robert Martienssen
Cold Spring Harbor Laboratory
Cold Spring Harbor, NY
USA
[email protected]
Autar Mattoo
United States Department of Agriculture
Beltsville, MD
USA
[email protected]
Eduardo Mercovich
MamaGrande
Rosario
Argentina
[email protected]
List of Participants
Abstract
L21, P1-2
L22, P7
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Name
Institution/Company
City
Country
Email
Abstract
Todd Michael
IBIS Biosciences
Carlsbad, CA
USA
[email protected]
L7
Almudena Molla Morales
Cold Spring Harbor Laboratory
Cold Spring Harbor, NY
USA
[email protected]
L11
Kazuhiro Mori
University of Yamanashi
Kofu
Japan
[email protected]
L13
Masaaki Morikawa
Hokkaido University
Sapporo
Japan
[email protected]
L14
Naomi Nakayama
ENS de Lyon
Lyon
France
[email protected]
Fumiki Nomoto
Nagase
Tokyo
Japan
[email protected]
Masaaki Okada
Kyoto University
Kyoto
Japan
m_okada@ cosmos.bot.kyoto-u.ac.jp
P4
Tokitaka Oyama
Kyoto University
Kyoto
Japan
oyama@ cosmos.bot.kyoto-u.ac.jp
L3
Ming Peng
Chinese Academy of Tropical Agricultural Sciences
Haikou
China
[email protected]
Jin Saimaru
Nagese
Tokyo
Japan
[email protected]
Ron Salpeter
Hinoman Ltd
Dekel
Israel
[email protected]
Benny Shoham
GreenOnyx, Ltd.
Tel Aviv
Israel
[email protected]
Tsipi Shoham
GreenOnyx, Ltd.
Tel Aviv
Israel
[email protected]
Ko Sukmin
Jeju National University
Jeju
Korea
[email protected]
Deguan Tan
Chinese Academy of Tropical Agricultural Sciences
Haikou
China
[email protected]
Xiang Tao
Chengdu Institute of Biology, Chinese Academy of Sciences
Chengdu
China
[email protected]
Nathan Tivendale
University of Minnesota
Saint Paul, MN
USA
[email protected]
Tadashi Toyama
University of Yamanashi
Kofu
Japan
[email protected]
L16
Wenqin Wang
Rutgers University
Piscataway, NJ
USA
[email protected]
L8
Tyler Wibbelt
Rutgers University
New Brunswick, NJ
USA
[email protected]
Mark Wright
Iowa State University
Ames, IA
USA
[email protected]
L1
Jiaming Zhang
Chinese Academy of Tropical Agricultural Sciences
Haikou
China
[email protected]
L6
Weixiong Zhang
Washington University in St. Louis
St. Louis, MO
USA
[email protected]
L12
Hai Zhao
Chengdu Institute of Biology, Chinese Academy of Sciences
Chengdu
China
[email protected]
L17
Yonggui Zhao
Chengdu Institute of Biology, Chinese Academy of Sciences
Chengdu
China
[email protected]
L15
L23
P8
List of Participants
25