Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: During the project reporting period, PI Cushman presented several invited lectures about the program including: EOL 407/607 Earth Resources and Energy: The End of Oil Reno, NV, March 31, 2011; EDAWN's Clean Energy Media Tour, Reno, NV. May 10, 2011; Squaw Valley Institute. University of Nevada, Reno, NV, June 17, 2011; and UNR Foundation Board of Directors. University of Nevada, Reno, NV, June 23, 2011. Graduate student Hathwaik presented a poster on the selection and evaluation of algae as biofuel feedstocks at the Nevada Agricultural Experiment Station, Ag Field Day, September 10, 2011. Reno, NV. Postdoctoral scholar Hiibel presented a poster on algal biofuel production using wastewater at the Nevada Agricultural Experiment Station, Ag Field Day, September 10, 2011. Reno, NV. Collaborator Vera Samburova presented a poster at the First International Conference on Algal Biomass, Biofuels and Bioproducts, July 17-20, 2011, St. Louis, MO. Reports on the Dunaliella salina (Ramos et al., 2011) and lipid and fatty acid extraction from algae (Samburova et al., 2012) have now been published or are in revision. PARTICIPANTS: To date, two graduate students (Mark Lemos and Leyla Hernandez-Gomez), one postdoctoral research associate (Dr. Sage Hiibel), and one visiting faculty member on a Fulbright Scholar (Dr. Nita Rukminasari) have been trained as independent scientists and participate in our regular meetings and conference calls. Each is receiving advising and mentoring from the project director about their various research activities and their career development goals. Two undergraduate students (Brian Kelly and Rob Cannon) have also been trained on the project. One lab manager has been trained on the project in a wide range of preparative and analytical techniques related to the specific requirement of the project: Rebecca Albion, Staff Research Associate II, University of Nevada, Reno (UNR). Research on the use of wastewater is being conducted in collaboration with Dr. Eric Marchand in the Department of Civil and Environmental Engineering at UNR. Research on algal paste processing by hydrothermal combustion is being conducted in collaboration with Dr. Kent Hoekman in the Atmospheric Sciences Division at DRI. Dr. Juergen Polle of Brooklyn College, City University of New York collaborates with us on all aspects of Dunaliella genetics and molecular biology. Private sector partners were Alton Reich and Geoffrey Chew, Streamline Automation (http://streamlineautomation.biz/site/). TARGET AUDIENCES: The project results and outcomes were targeted to the scientific community at national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on the development of algae as feedstocks for biofuel production. Outreach projects are also being targeted to the lay public through poster presentations at annual meetings of the Nevada Agricultural Experiment Station (Ag Field Days) at the University of Nevada, Reno, the Nevada Renewable Energy Consortium (NVREC) at the University of Nevada, Las Vegas, NV, and research outcomes are being presented to scientific audiences through oral and poster presentations at regional, national and international meetings. PROJECT MODIFICATIONS: None.
Impacts
Method development for the extraction and analysis of lipids (triacylglycerols) and free fatty acid content has been completed. Ultra-high pressure liquid chromatography-tandem mass spectroscopy (UHPLC-MS-MS) was used for the analysis of total fatty acids (FA) and lipid quantification and composition. The two methods were found to be in good agreement. Total lipids were extracted from the algae samples using the Accelerated Solvent Extraction (ASE) process along with a dichloromethane/hexane solvent system and the recoveries for TAGs and FFAs ranged from 80 to 125%. A total of 20 triacylglycerols were identified and quantified in the halophytic microalgae Dunaliella salina strains (Samburova et al., 2012). The most abundant TAGs were C18:3/C16:0/C16:4, C18:3/C18:2/C16:4, C18:3/C18:1/C16:4, C18:3/C18:3/C16:0, C18:3/C18:1/C16:0, and C18:3/C18:3/C18:0. Analysis of FFA compositions demonstrated that the most abundant FFAs were linolenic acid (C18:3), was the largest contributor to the total FFA content (52.3-66.7%), while other FAs were present in relatively lower abundance including palmitic (C16:0, 25.1%), linoleic (C18:2; 11.9%), oleic (C18:1, 9.8%), and hexadecatetraenoic (C16:4, 1.6%) acid in Dunaliella species. Eighteen strains of halophytic Dunaliella were screened and characterized for biomass productivity, free fatty acid, triacylglyceride, carbohydrate, and carotenoid composition and production. Biomass productivity ranged from 0.215 g/L to 0.405 g/L dry cell weight (DCW). Total free fatty acid and triacylglyceride content ranged from 2.97% to 7.42% DCW. Carbohydrates ranged from 4.02% to 18.75% DCW. Production of value-added products including β-carotene ranged from 0.109-1.942 mg/g DCW, xanthophyll ranged from 0.224 to 2.645 mg/g DCW, canthaxanthin ranged from 0.0194 to 0.1847 mg/g DCW, and zeaxanthin ranged from 0.193 to 1.215 mg/g DCW. The Dunaliella salina CCAP 19/18 genome ~4 X assembly was recently updated by JGI revealing that the nuclear genome contains 60% repetitive DNA consisting mainly of simple sequence repeats. Nonetheless, a total of 57,754 contigs totaling 252 MB of 350 Mb currently has now been assembled. EST sequencing of mixed cDNA libraries derived from various environmental stress conditions including a NaCl adaptation series, a salt shock series; an anaerobic stress series, and a high light to low light adaptation and vice versa were performed resulting in 37,391 Sanger ESTs and 2,252,211 Roche 454 ESTs resulting in over 1.5 M cleansed reads and about 21,000 unigenes. In order to identify genes that control oil production, an experiment using a custom microarray containing probes for 21,000 unigenes derived subjecting D. salina cells to 0, 12, 24, 48, and 72 h nutrient deprivation has been completed with the largest change in gene expression patterns occurring after 72 h. Detailed characterization of these gene expression data is underway.
Publications
- Ramos AA, Polle J, Tran D, Cushman JC, Jin ES, Varela JC. (2011) The unicellular green alga Dunaliella salina Teod. As a model for abiotic stress tolerance: Genetic advances and future prospects. Algae. 26:3-20.
- Samburova V, Lemos MS, Hiibel SR, Hoekman SK, Cushman JC, Zielinska B (2012) Analysis of triacylglycerols and free fatty acids in algae using ultra-high-pressure liquid chromatography mass spectrometry. Analytical and Bioanalytical Chemistry. In revision.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: During the project reporting period, PI Cushman presented several invited lectures including: Nevada Renewable Energy Consortium Lecture Series at the University of Nevada, Las Vegas, NV on September 7, 2010 and the 2010 Pacific Rim Summit of the Biotechnology Industry Organization (BIO) in Honolulu, HI on December 12, 2010. In addition, graduate student Hathwaik presented a poster at the 2nd Annual Meeting of the Nevada Renewable Energy Consortium (NVREC) at the University of Nevada, Las Vegas, NV on August 20, 2010. Postdoctoral scholar Hiibel presented three posters at the 2nd Annual Meeting of the Nevada Renewable Energy Consortium (NVREC) at the University of Nevada, Las Vegas, NV on August 20, 2010; at the UNR College of Engineering in Reno, NV on October 14, 2010; and at the Annual Meeting of the American Institute of Chemical Engineers (AIChE), November 7-12, 2010, Salt Lake City, UT. Reports on the Dunaliella salina organelle genomes (Smith et al., 2010), EST collections from salinity shocked cells (Alkayal et al., 2010), and a review on Dunalliella (Ramos et al., 2011) have now been published or submitted. PARTICIPANTS: To date, two graduate students (Mark Lemos and Leyla Hernandez-Gomez) and one postdoctoral research associate (Dr. Sage Hiibel) are being trained as independent scientists and participate in our regular meetings and conference calls. Each is receiving advising and mentoring from the project director about their various research activities and their career development goals. Four undergraduate students (Kim Rafter, Samantha Kertson, Alexander Lewis, and Brian Lelly) have also been trained on the project. One lab manager has been trained on the project in a wide range of preparative and analytical techniques related to the specific requirement of the project: Rebecca Albion, Staff Research Associate II, University of Nevada, Reno (UNR). Research on the use of wastewater is being conducted in collaboration with Dr. Eric Marchand in the Department of Civil and Environmental Engineering at UNR. Research on algal paste extraction is being conducted in collaboration with Dr. Charles Coronella in the Department of Chemical and Metallurgical Engineering at UNR. Dr. Juergen Polle of Brooklyn College, City University of New York collaborates with us on all aspects of Dunaliella genetics and molecular biology. Private sector partners include Dr. John Bebout, Bebout & Associates (http://bebout-and-associates.com/), Mr. Jeffrey Eppink, Enegis LLC, (http://www.enegis.com/), and Alton Reich, Streamline Automation (http://streamlineautomation.biz/site/). TARGET AUDIENCES: The project results and outcomes were targeted to the scientific community at national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on the development of algae as feedstocks for biofuel production. Outreach projects are also being targeted to the lay public through poster presentations at an annual meeting of the Nevada Renewable Energy Consortium (NVREC) at the University of Nevada, Las Vegas, NV and to scientific audiences through oral and poster presentations at regional, national and international meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Analysis of growth rates, triacylglycerol, free fatty acid content, and insoluble starch content for nineteen (19) strains of halophytic microalgae (Dunaliella salina) has been completed and analysis of results is being finalized for publication. Dunaliella cells contain a relatively low triacylglycerol content averaging only 0.56% on a dry weight basis (range = 0.18-1.91%) with the following fatty acid compositions: linolenic C18:3 > linoleic C18:2 > palmitic C16:0 > oleic C18:3, > palmitoleic C16:1 = hexadecadienoic C16:2 > C16:4. In addition, we have explored the use of centrate, the liquid fraction created when anaerobically digested wastewater sludge is dewatered for disposal purposes, as a low cost, nutrient sources for growing algae. The 18S rRNA gene and adjacent internal transcribed spacer (ITS) regions were used to provide a facile method to unambigouously identify identify and classify the Dunaliella strains. The sequencing of 18s rDNA gene and adjacent ITS region has been completed for eighteen and nineteen strains, respectively. We have also begun the sequencing of ~30 different freshwater green algae strains including Chlorella, Neochloris, and Nannochloropsis species. Transgressive selection trials of wildtype and ethyl methyl sulfonate (EMS) mutagenized strains of Dunaliella salina CCAP 1918 have continued through 53 rounds of selection of wildtype and mutant cells by buoyant density gradient centrifugation. Flow cytometry with Nile Red dye staining has been performed successfully to assess the effects of reiterative selection and associated lipid content changes. Both buoyant density gradient centrifugation and flow cytometry followed by fluorescence activated cell sorting can be effectively used to select for significant increases in lipid and/or starch content from within wildtype or mutagenized algal cell populations without resorting to genetic engineering. Currently, a ~4 X assembly of the genome has been completed by JGI Resulting complete mitochondrial (28.8 kb) and plastid (269 kb) genome sequences have now been published (Smith et al., 2010). The nuclear genome contains 60% repetitive DNA consisting mainly of simple sequence repeats and this is making contig assembly especially challenging with only 191 MB of 350 Mb currently being assembled. EST sequencing of mixed cDNA libraries derived from various environmental stress conditions by Sanger (37,391 ESTs) and Roche 454 (2,252,211 ESTs) sequencing produced over 1.5 M cleansed reads and about 21,000 unigenes. Analysis of a small collection of 2831 ESTs from salt-shocked D. salina cells was also completed (Alkayal et al., 2010). Such information along with additional rounds of paired end reads will be needed to improve the genome assembly and annotation. In order to identify genes that control oil production, an experiment using a custom microarray containing probes for 21,000 unigenes derived subjecting D. salina cells to 0, 12, 24, 48, and 72 h nutrient deprivation has been completed with the largest change in gene expression patterns occurring after 72 h.
Publications
- Smith DR, Lee RW, Cushman JC, Magnuson JK, Tran D, Polle JEW. (2010) The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA. BMC Plant Biology. 10:83.
- Alkayal MF, Albion RL, Tillett RL, Mark S. Lemos, Hernandez-Gomez L, Cushman JC. (2010) Expressed Sequence Tag (EST) profiling in salinity shocked Dunaliella salina reveals high expression of protein synthetic apparatus components. Plant Science. 179: 437-449.
- Ramos AA, Polle J, Tran D, Cushman JC, Jin ES, Varela JC. (2011) The unicellular green alga Dunaliella salina Teod. As a model for abiotic stress tolerance: Genetic advances and future prospects. Algae. Submitted.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: During the project reporting period, collaborator Polle and PI Cushman presented a poster at the JGI User Meeting on March 25-27, 2009 at Walnut Creek, CA. Both graduate students Hernandez-Gomez and Lemos presented posters at the 2009 American Society of Plant Biologist (ASPB) & Phycological Society of America (PSA) on July 18-22th, 2009 in Honolulu, HI and at the NAES Ag Field Day on September 12, 2009 at the Main Station Farm Complex, Reno, NV. Undergraduate student Rafter presented at poster at the Undergraduate Research Opportunities Program Poster Session on August 12, 2009 at the University of Nevada, Reno, NV. Graduate student Hernandez-Gomez also presented a poster at the First Annual UNR Molecular Biosciences Retreat on October 17, 2009 at the University of Nevada, Reno, NV. Dr. Cushman delivered oral presentations to the 2009 Sun Grant Initiative Energy Conference in Washington D.C. on March 12, 2009 and to the Society for General Microbiology Fall Meeting at Heriot-Watt University in Edinburgh, Scotland, UK on September 9, 2009. PARTICIPANTS: To date, two graduate students (Mark Lemos and Leyla Hernandez-Gomez) and one postdoctoral research associate (Dr. Sage Hiibel) are being trained as independent scientists and participate in our regular meetings and conference calls. Each is receiving advising and mentoring from the project director about their various research activities and their career development goals. One lab manager has been trained on the project in a wide range of preparative and analytical techniques related to the specific requirement of the project: Rebecca Albion, Staff Research Associate II, University of Nevada, Reno (UNR). Three undergraduate students (Kim Rafter, Samantha Kertson, and Alexander Lewis) have also been trained on the project. Research on the use of wastewater is being conducted in collaboration with Dr. Eric Marchand in the Department of Civil and Environmental Engineering at UNR. Research on algal paste extraction is being conducted in collaboration with Dr. Charles Coronella in the Department of Chemical and Metallurgical Engineering at UNR. Dr. Juergen Polle of Brooklyn College, City University of New York collaborates with us on all aspects of Dunaliella genetics and molecular biology. Private sector partners include Dr. John Bebout of Bebout & Associates (http://bebout-and-associates.com/) and Mr. Jeffrey Eppink of Enegis LLC, (http://www.enegis.com/). TARGET AUDIENCES: The project results and outcomes were targeted to the scientific community at national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on the development of algae as feedstocks for biofuel production. Outreach projects are also being targeted to the lay public through poster presentations at the annual Agriculture Field Day held by the Nevada Agricultural Experiment Station and to oral presentations to national and international scientific audiences. PROJECT MODIFICATIONS: A 1 year extension request has been made to allow the following two objectives to be completed. Objective 1: Identify mutant strains with enhanced production of oil or starch. Putative mutants from an EMS mutagenesis have been selected. The plan is to conduct genetic crosses with these variants to verify stable phenotypes. Lines will be characterized and monitored for levels for oil and starch. These improved strains will then be tested for production traits in a pilot scale experiment. The purpose is to establish a precedent for using mutagenesis to generate strains with improved biofuels traits. Objective 2: Develop a method to transform D. salina to enable genetic engineering of improved traits. A first generation transformation vector was designed and synthesized (pJ244:26780). This vector was designed for homologous recombination with the chloroplast genome of D. salina, and carries resistance genes for chloramphenicol and BASTA selection. During the next year, a graduate student will continue to modify and test this vector in D. salina and Chlamydomonas reinhardtii. Two important modifications will be tested. First, a GUS marker will be used to determine how much DNA is being transferred into cells by different transformation techniques (variations on microprojectile bombardment and electroporation). Second, new promoters and homologous recombination sites will be introduced to increase marker gene expression levels and stable recombination frequencies, respectively. These changes are now possible in light of the recent sequencing of the complete plastid and mitochondrial genomes of D. salina (Smith et al., 2010). The purpose is to establish a reliable transformation protocol for future genetic engineering of D. salina.
Impacts
We have determined the growth rates, lipid content, and insoluble starch content for nineteen (19) strains of halophytic microalgae. We have also completed the analysis of 11 strains of freshwater algae. Analysis of 12 brackish water strains is in progress. Freshwater algae strains can serve as potential feedstocks for biofuel production using wastewater. In order to test the transformability of Dunaliella salina targeting either the plastome or nuclear genome, we have completed the sequencing of the organellar genomes, wherease the nuclear genome is still in progress due to an unexpectedly high content of repetitive DNA resulting in a large genome size. Total genomic DNA and RNA from Dunaliella salina CCAP 19/18 was submitted to the Joint Genome Institute (JGI) for genomic sequencing and annotation. Currently, a 1.5 X assembly of the genome has been completed by JGI resulting in complete mitochondrial (28.8 kb) and plastid (269 kb) genome sequences (Smith et al., 2010) and a nuclear genome of approximately 300 Mb with 60% repetitive DNA content. EST sequencing of mixed cDNA libraries derived from various environmental stress conditions by Sanger (37,391 ESTs) and Roche 454 (2,252,211 ESTs) sequencing produced over 1.5 M cleansed reads and about 21,000 unigenes. This information will be instrumental in improving the accuracy of genome annotation and mRNA expression analysis to test the effects of nutrient deprivation or other stress conditions on lipid production. In order to identify genes that control oil production, hydrogen production, salt tolerance, and freezing tolerance we are in the process of completing the design of a custom microarray containing probes for 21,000 unigenes derived from the above transcriptome sequencing. An experiment subjecting D. salina cells to nutrient deprivation over a time course of up to 72 h has been completed and will be analyzed once the microarray design and fabrication is completed. Transgressive selection trials of wildtype and ethyl methyl sulfonate (EMS) mutagenized strains of Dunaliella salina CCAP 1918 have continued. After 20 rounds of reiterative transgressive selection using continuous Percoll buoyant density gradient centrifugation, we have been able to increase lipid content by 39% and starch content by 227%. Genetic analysis of these strains is now in progress as are continued rounds of selection. .
Publications
- Smith DR, Lee RW, Cushman JC, Magnuson JK, Tran D, Polle JEW. (2010) The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA. Genome Biology. Submitted.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The long-term goal is to develop a biofuels crop for the great basin based on a salt-water loving algae (Dunaliella salina). PARTICIPANTS: This project includes graduate training for Leyla Hernandez and Mark Lemos. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Two major advances have been made during the past year. First, outdoor ponds for growing algae have been constructed to permit the evaluation of harvesting protocols and oil yields. Second, in developing a genetic transformation system for algal strains of interest, we have identified a BASTA resistance marker as our selection marker of choice. Although a stable line of transgenic Dunaliella salina has not yet been obtained in our lab, transient expression experiments have verified that a standard BASTA resistance gene can be used to provide herbicide resistance. Attempts to increase the frequency of stable transgene integrations are underway.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The long-term goal is to develop a biofuels crop for the great basin based on a salt-water loving algae (Dunalliela salina). This project has just been initiated with the first aim focused on identifying the best algae strains to use for laboratory and field experiments.
PARTICIPANTS: Graduate Student Leyla Hernandez under the supervision of Jeff Harper and John Cushman.
Impacts
Several algae strains have been identified that produce high levels of oil.
Publications
- No publications reported this period
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