Algal Biology Toolbox Workshop Summary Report San Diego, California May 2016 Summary report from the May 24‒25, 2016, Algal Biology Toolbox Workshop in San Diego, California Workshop and summary report sponsored by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office Preface The U.S. Department of Energy’s (DOE’s) Office of Energy Efficiency and Renewable Energy (EERE) invests in a diverse portfolio of energy technologies to achieve a stronger economy, a cleaner environment, and a more secure energy future for America. This report summarizes the results of a public workshop sponsored by DOE-EERE in San Diego, California, from May 24‒25, 2016. The views and opinions of the workshop attendees, as summarized in this document, do not necessarily reflect those of the U.S. government or any agency thereof, nor do their employees make any warranty, expressed or implied, or assume any liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represent that its use would not infringe upon privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States government or any agency thereof. Preface i ALGAL BIOLOGY TOOLBOX WORKSHOP SUMMARY REPORT Contents Preface .......................................................................................................................................i Introduction ...............................................................................................................................1 Workshop Concept and Process ...........................................................................................2 Summary of Presentations .....................................................................................................3 New Developments in Algal Biology .....................................................................................................3 Molecular Toolbox Development and Gene Expression ................................................................4 Omics Databases ...........................................................................................................................................5 Gene Expression and Silencing ................................................................................................................7 Co-Products ...................................................................................................................................................8 From Flask to Farm ......................................................................................................................................9 Biological Improvements in Cultivation ...............................................................................................11 Open Forum Presentations ......................................................................................................................14 Workshop Participant Responses ........................................................................................16 Focus Question #1: Biology Unknowns ...............................................................................................16 Focus Question #2: Species of Focus .................................................................................................23 Focus Question #3: Future Directions ................................................................................................28 Conclusion ..............................................................................................................................35 Appendix A: Workshop Agenda ..........................................................................................37 Appendix B: Breakout Session Questions ..........................................................................39 Appendix C: Abbreviations and Acronyms .......................................................................40 Appendix D: Related Links ....................................................................................................41 Appendix E: Workshop Participants ..................................................................................42 ii Contents List of Figures Figure 1. Breakdown by sector of Algal Biology Toolbox Workshop attendees ............2 Figure 2. Genetic diversity of algae ......................................................................................3 Figure 3. Focus question #1, priority challenges, breakout group #1 .............................18 Figure 4. Focus question #1, priority challenges, breakout group #2 ............................21 Figure 5. Focus question #1, priority challenges, breakout group #3 ...........................22 Figure 6. Focus question #2, priority strain attributes, breakout group #1 ..................23 Figure 7. Focus question #2, priority strain attributes, breakout group #2 ..................25 Figure 8. Focus question #2, priority strain attributes, breakout group #3 .................27 Figure 9. Focus question #3, priority resources needed, breakout group #1 ...............28 Figure 10. Focus question #3, tools and resources still needed, breakout group #2.................................................................................................................30 Figure 11. Focus question #3, tools and resources still needed, breakout group #3.................................................................................................................32 List of Tables Table 1. Focus Question #1, Priority Challenges, Breakout Group #1 ..............................17 Table 2. Focus Question #1, Priority Challenges, Breakout Group #2 .............................19 Table 3. Focus Question #1, Priority Challenges, Breakout Group #3 ............................22 Table 4. Focus Question #3, Tools and Resources Still Needed, Breakout Group #2 .................................................................................................................31 Table 5. Focus Question #3, Tools and Resources Still Needed, Breakout Group #3 ................................................................................................................33 Contents iii ALGAL BIOLOGY TOOLBOX WORKSHOP SUMMARY REPORT Introduction On May 24–25, 2016, DOE-EERE’s Bioenergy Technologies Office (BETO) hosted the “Sharpening Our Tools: Algal Biology Toolbox Workshop” in San Diego, California. BETO works to accelerate the development of a sustainable, cost-competitive, advanced biofuel industry that can strengthen U.S. energy security, environmental quality, and economic vitality, through research, development, and demonstration projects in partnership with industry, academia, and national laboratory partners. BETO’s Advanced Algal Systems Program (also called the Algae Program) has a long-term applied research and development (R&D) strategy to increase the yields and lower the costs of algal biofuels. The team works with partners to develop new technologies, to integrate technologies at commercially relevant scales, and to conduct crosscutting analyses to bet- ter understand the potential and challenges of the algal biofuels industry. Research has indicated that this industry is capable of producing billions of gallons of renewable diesel, gasoline, and jet fuels annually. R&D activities are integrated with BETO’s longstanding effort to accelerate the commercialization of lignocellulosic biofuels. Several goals and objectives support the long-term strategy of the Advanced Algal Systems Program, which aims to develop and demonstrate technologies that produce sustainable algal biofuel intermediate feedstocks that can perform reliably in conversion processes to yield renewable diesel, jet, and gasoline. BETO has an aggressive productivity target of 2,500 gallons of biofuel intermediate per acre annual average by 2018, and 5,000 gallons by 2022. The goal is to achieve a $3 per gasoline gallon equivalent price point and a minimum of 50% reduction in greenhouse gas emissions compared to gasoline. BETO is incorporating a near-term focus on high-value nutritional and chemical products to enable the technology development necessary for commercialization of cost-competitive algal biofuels by 2030. BETO’s Advanced Algal Systems Program regularly hosts algal biofuels strategy workshops to help break down critical technical barriers and promote sustainable and affordable algal biofuels.1 At previous events, stakeholder input established three main points of understanding on which to build future discussions: (1) algal bioproducts should be able to act as building blocks for the future development of algal biofuels, (2) economic viability of the target product should be taken into consideration, and (3) products should be environmentally favorable.2 This workshop was the first algal biofuels strategy session to focus specifically on improvements in algal biology—a key research focus required to advance the economic viability of algae-based biofuels. The objective of this event was to discuss R&D needed to achieve affordable, scalable, and sustainable algae-based biofuels. Presentations from algae experts overviewed the tools and resources available to grow, monitor, valorize, and genetically enhance algae for biofuel and bioproduct production. Attendees participated in breakout sessions to provide input about the challenges in algal biology, the metrics of success in algae strain development, and the resources needed to overcome challenges and achieve success. Fifty-eight stakeholders attended the event, repre- senting industry, national laboratories, universities, government agencies, and research institutions (Figure 1). 1 “Algal Biofuels Strategy Workshops,” U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, accessed June 1, 2016, www.energy.gov/eere/bioenergy/algal-biofuels-strategy-workshops. 2 Andrea Bailey, G. Jeremy Leong, and Nichole Fitzgerald, Bioproducts to Enable Biofuels Workshop Summary Report, U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, Bioenergy Technologies Office, December 2016, http://energy.gov/sites/prod/ files/2015/12/f27/bioproducts_to_enable_biofuels_workshop_report.pdf. 1 Introduction Figure 1. Breakdown by sector of Algal Biology Toolbox Workshop attendees 2% 5% 15% 31% University Industry National laboratory Government agency Research institute Other 22% 25% Workshop Concept and Process The purpose of the Algal Biology Toolbox Workshop was to collect input from lead experts in the field of algal biology regarding (1) the current state of algal biological tools, including our understanding of algal biology and biochemistry, available molecular toolboxes, omics databases, and other resources; (2) challenges to developing and applying a full suite of biological tools to improve algae performance and system robustness; and (3) strategies to advance progress toward commercial algal biofuels. BETO emphasized a focus on near-term opportunities to advance commercialization of algal biofuels and enable the production of algae-based bioproducts. Attendees were asked to provide input regarding the following questions: • What are challenges in algal biology, specific to genetic tools, which pose significant hurdles to progress over the next 5 years? • What does success look like; what attributes would the ideal commercial algae strain(s) possess? • What tools and resources exist to overcome the challenges to achieve the ideal strain(s)? - What tools and resources are still needed? Responses from stakeholders affiliated with industry, national laboratories, universities, and research institutions will help BETO shape its near-term research funding strategy. The workshop began with an opening presentation from BETO to provide attendees with necessary context about BETO’s mission and the goals of the workshop. Also on the first day of the workshop, several experts in the field of algal biology gave presentations on recent developments in their R&D efforts. On the second day of the event, BETO facilitated breakout discussions that aimed to identify challenges in algal biology and strategies for advanc- ing the field. Breakout session participants provided their input regarding available molecular toolboxes, omics databases, and other algal biology and biochemistry resources. Participants also discussed potential strategies to overcome challenges and to measure the field’s progress toward commercial algal biofuels. See appendix A for a detailed agenda. Workshop Concept and Process 2 ALGAL BIOLOGY TOOLBOX WORKSHOP SUMMARY REPORT Summary of Presentations Experts in the field of algal biology shared their recent successes with event attendees to further the algal biology community’s understanding of the current state of the art. The speakers were selected with a goal of ensuring that a range of expertise was represented within the presentations. This section of the workshop summary report provides highlights of each speaker’s presentation with a focus on current challenges in algal biology, definitions of success in strain improvement, and the tools and resources needed in order to address near-term challenges to viable com- mercial algal biofuels and bioproducts. New Developments in Algal Biology BETO has hosted several algal biofuels strategy workshops to discuss R&D needed to achieve affordable, scal- able, and sustainable algae-based biofuels. This particular event focused more specifically on algal biology, a field critical to the advancement of algal biofuels. As a starting point, an introductory presentation by Dr. Amanda Barry provided a thorough overview of recent developments in biology and biochemistry, as they relate to algae. New Discoveries in Biology and Biochemistry Amanda Barry, Ph.D. U.S. Department of Energy, Bioenergy Technologies Office, Advanced Algal Systems Program Dr. Barry, Scientist and Technology Development Manager in the BETO Advanced Algal Systems Program, opened the 2-day Algal Biology Toolbox Workshop with an overview of new discoveries in biology and biochem- istry. Her presentation began with a brief overview of the genetic tools for strain optimization, which include directed evolution, heterologous and native gene expression, genome editing, and breeding. Next, she provided an overview of omics approaches and bioinformatics approaches, which include genomics, transcriptomics, proteomics, metabolomics and lipidomics, and phenomics. Dr. Barry explained that the incredible genetic diversity of algae makes it difficult to create tools and methods that are universally applicable, necessitating an extensive suite of genetic tools and omics approaches. Figure 2 below illustrates algae’s genetic diversity and, therefore, its diversity of opportunity in commercial development. Figure 2. Genetic diversity of algae3 Plantae Mesostigmatophyceae Red algae Plants Mamialleles Galdieria Green algae (UTC clade) Chromalveolates Auxenochlorella, Glaucophytes Clorella, Picochlorum Cryptomonads Cyanobacteria Excavates Haptophytes Euglenophytes Dinoflagellates (peridinin) Other excavates Dinoflagellates (fucoxanthin-Karenia) Apicomplexa Stramenopiles Nannochloropsis Chlorarachniophytes Opisthokonta Amoebozoa (animals & fungi) Diatoms Other rhizaria Rhizaria Unikonts 3 Summary of Presentations As an introduction to the current state of research, Dr. Barry stated that gene transfer system development, an important milestone, had been established in many algal strains, but not all. She also differentiated between the statuses of development in different strains. In cyanobacteria, for example, transgenic approaches have enabled the production and secretion of cellulose, sucrose, ethanol, isobutanol, ethylene, and free fatty acids. In eukaryotic strains of microalgae, targeted metabolic engineering to increase the production of lipids has begun in green algae, diatoms, and brown algae. Dr. Barry also reviewed results from two major public-private partnerships supported by DOE with the goals of advancing algae science and commercializing algal biofuel. One of these partnerships, the National Alliance for Advanced Biofuels and Bioproducts, was successful in developing technologies with the potential to reduce the cost of algae-based biocrude from $240 to $7.50 per gallon. The alliance, led by the Donald Danforth Plant Science Center in St. Louis, Missouri, had over 30 partners, including national laboratories, universities, and companies. The collaborative effort helped to establish a sustainable algal biofuels and bioproducts industry. A final report published in 20144 summarized important technical results such as new strain development, improved cultivation, low-energy harvesting technology, and high-yield extraction and conversion technology. Another public-private partnership, the Consortium for Algal Biofuel Commercialization, recently published its final report.5 DOE established this group with the goal of enabling commercial viability of algae-based biofuels with a focus on key aspects of algal biofuel production, such as development of genetic tools, crop protection, and nutrient utilization and recycling. The consortium, led by the University of California, San Diego, also developed education programs that have trained more than 200 research scientists and laboratory technicians and have intro- duced more than 50,000 students worldwide to the benefits of sustainable alternative fuels. Molecular Toolbox Development and Gene Expression Molecular toolbox development has been identified as a crucial effort in advancing the field of algal biol- ogy. Professor Matt Posewitz with the Colorado School of Mines focuses research efforts at his laboratory on Nannochloropsis, Chlorella, and cyanobacteria strains. Professor Mark Hildebrand, who runs a research laboratory at the University of California, San Diego, focuses his research efforts on diatoms. Molecular Tools for Improving Biofuel Phenotypes in Algae and Cyanobacteria Matthew Posewitz, Ph.D. Colorado School of Mines Colorado School of Mines collaborates on research regarding generalized photoautotrophic biofuel schematics. During his presentation, Professor Posewitz reported results from several efforts. One study used carbon partition- ing to optimize fatty acid levels, starch accumulation, and photosynthetic performance. Another project involved prospecting for better organisms and better proteins from new ecosystems with various environments. In order to isolate genes that will survive high light and temperature conditions, researchers selected strains found in hot and salty locations. Another effort focused on developing Nannochloropsis gaditana into a model marine, biocommodity production strain. This species is a promising lipid producer, and the strain’s cell walls are known for their durability and robustness. Continued work on this species is focusing on improving harvesting efficiency. As part of this effort, 3 Beverley R. Green, “Chloroplast Genomes of Photosynthetic Eukaryotes,” The Plant Journal 66 (2011): 34‒44, doi:10.1111/j.1365-313X.2011.04541.x. 4 National Alliance for Advanced Biofuels and Bioproducts, National Alliance for Advanced Biofuels and Bioproducts Synopsis Final Report, http://www.energy.gov/eere/bioenergy/downloads/national-alliance-advanced-biofuels-and-bioproducts-synopsis-naabb-final. 5 Consortium for Algal Biofuel Commercialization, Consortium for Algal Biofuel Commercialization Final Report, Dr. Stephen Mayfield (principal investigator), DE-EE0003373, December 4, 2015, http://www.osti.gov/scitech/ biblio/1234700-consortium-algal-biofuel-commercialization-cab-comm-final-report. Summary of Presentations 4 ALGAL BIOLOGY TOOLBOX WORKSHOP SUMMARY REPORT the Colorado School of Mines laboratory is developing transformation techniques for many algal strains. Professor Posewitz sees several opportunities for future research, including efforts to decrease light harvesting antennae, improve carbon assimilation, tailor carbon products, harvest cells from water, and generate secreted products. Genetic Manipulation Approaches for Metabolic Engineering of Microalgae Mark Hildebrand, Ph.D. University of California, San Diego – Scripps Institution of Oceanography Dr. Hildebrand began his presentation with a statistic about the power of genetic tools: Since the 1930s, corn yields have increased by 600%. Similarly, he explained, genetic alteration is required in order to impart a stable pheno- typic environment that allows for cost-effective production of microalgal strains. With respect to algae, choosing which genes to manipulate is still a challenge. It is clear, he stated, that lipid content and growth rate are the two traits that are major drivers for economical biofuel production. At this time, genetic engineering is accepted as the preferred approach to genetic manipulation because it is easier to incorporate multiple traits. Limited or undeveloped methodology for preferred species, the importance of using supporting insight from fields of study such as transcriptomics, and the possibility of missing valuable genes all contribute to the complexity of genetic engineering. Given these complexities, mutagenesis and selection approach- es are also valuable in improving performance of microalgal strains for cost-effective production. Dr. Hildebrand’s laboratory team used a technique called mutagenesis, which involved the engineering of gene mutations and resulted in significantly higher lipid accumulation in 36% of cells screened with no detrimental growth effects. Dr. Hildebrand noted that genetic manipulation is more readily achievable for some types of microalgae than for others. Two important factors are (1) transformation ability, which is driven by identification of appropriate pro- moters, and (2) proficiency, which indicates the ability to acquire new genetic material. Another practical aspect of genetic manipulation is genome sequence determination, which Dr. Hildebrand described as a requirement for in- formed manipulation. The genome sequence enables identification of potential target genes to manipulate, provides a means to derive metabolic maps of the organism, and provides native expression control elements to control and alter the expression of introduced genes. Lastly, tool development is also a critical aspect of genetic manipulation. Regarding diatoms, the focus of Dr. Hildebrand’s work, tools developed include selectable gene markers (such as fluorescent tagging), codon optimization, and transformation approaches. The tools developed for one particular diatom species have generally worked in other genera. In conclusion, Dr. Hildebrand emphasized that genetically engineered organisms are not necessarily genetically modified. Organisms are classified as genetically modified only if deoxyribonucleic acid (DNA) from another genus is introduced. Recombination and crossover can be generated by the native host and are not classified as genetic modification because, in theory, the organism could have done this type of evolution on its own. Dr. Hildebrand identified several priorities for future research, including further refinement of manipulation tools, detailed characterization of carbon flux and metabolism in diverse classes of microalgae, expanded understanding of the factors that control growth rate and biomass accumulation, and application of these approaches to actual production conditions. Omics Databases Omics databases are a valuable contribution to the field of algal biology given that they enhance the efficiency of research efforts by sharing previously established research. In this context, omics approaches cover the following: • Genomics: Sequenced genomes are essential for determining the physiological potential of production strains and for strain improvement. Genome size in algae can vary substantially. • Transcriptomics: Analysis of gene expression will illuminate environmental response and enable gene annotation of other species. 5 Summary of Presentations