Current Status of the Department of Energy’s Aquatic Species Program Lipid-Focused Algae Collection

NREL is a national laboratory of the U.S. Department of EnergyOffice of Energy Efficiency and Renewable Energy

Operated by the Alliance for Sustainable Energy, LLC

g p gEric P. Knoshaug, Ryan Sestric, Eric Jarvis, Yat-Chen Chou, Philip T. Pienkos, and Al Darzins

National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO. 80401

Abstract

The Department of Energy’s Aquatic Species Program (ASP) was funded from1978 to 1996 in an effort to develop liquid transportation fuels from microalgae.During this time an extensive algae culture collection was amassed from water

AMPHO1AMPHO27AMPHO28

Strains Listed in either the MicroalgaeCulture Collection  or Addendum

Currently

Endogenous Lipid Vesicles

Algal cells were stained with the neutral lipid specific dye, BODIPY 493/503. The stained cells were excited with480 nm light and the pictures show the juxtaposition of the chlorophyll containing chloroplasts (red) and theputative lipid vesicles (green). When excited with 480 nm light, chloroplasts emit at ≥630 nm while BODIPY stainedvesicles emit at 515 nm. These micrographs were taken of cells in the early stationary phase and have not beenspecifically stressed for lipid production

OOCYS1

g gsamples around the United States. Out of more than 3000 strains, 51 were wellcharacterized in terms of growth and lipid production and these are described in theMicroalgae Culture Collection (http://www.nrel.gov/docs/legosti/old/3079.pdf) andaddendum (http://www.nrel.gov/docs/legosti/old/3079a.pdf). At the close of the ASP,a total of 297 strains of the original 3000, including 37 of the 51 strains listed in theMicroalgae Culture Collection and addendum, were transferred to the Center forMarine Microbial Ecology and Diversity (CMMED) at the University of Hawaii (UH).The complete list of strains transferred is available in the ASP Closeout Report(http://www.nrel.gov/docs/legosti/fy98/24190.pdf). However, this list does notinclude some strains that were listed in the Microalgae Culture Collection yet arepresent in the UH collection. With the resurgence in interest in using microalgae asa feedstock for producing liquid transportation fuels, many inquiries have come upregarding the current status of this important strain collection. Currently, 23 of the51 strains listed in the Microalgae Culture Collection and addendum are still extantand have been re-established at the National Renewable Energy Laboratory. Wepresent here the current status of these strains including a microscopic

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ANKIS1

CHAET6

CHAET59

CHLOR1

CHLOR2

CYCLO1

ELLIP1

yExtant Strains

specifically stressed for lipid production.

CHLOR2

ANKIS1NANNP1

NANNP2

NAVIC7

FRANC1

ELLIP1

NANNO2

OOCYS1present here the current status of these strains including a microscopiccharacterization of potential lipid vesicles using neutral lipid specific dyes.

CHLOR1CHLOR2CHLOR3CYCLO1CYCLO2CYCLO4ELLIP1ENTOM3FRANC1GREEN3ISOCH1MONOR1MONOR2NANNO2NANNO12NANNP1NANNP2

FRANC1

MONOR1

MONOR2

NANNO2

NANNP1

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NAVIC2

NAVIC7

Overview of the DOE Aquatic Species Program Work

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PLEUR1

NANNP2NAVIC1NAVIC2NAVIC6NAVIC7NAVIC8NAVIC24NITZS1NITZS2NITZS13NITZS14NITZS28OOCYS1PHAEO1PHAEO2PLUER1

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NITZS14

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PHAEO1

PHAEO2

PLUER1Long Term Storage via Cryo-Preservation

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Conclusion

An impressive collection of microalgae was assembled under the Department of Energy’s AquaticSpecies Program. Maintenance of microalgal cultures over the long term is challenging requiring

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This list shows the strains that were carefully investigatedduring the Aquatic Species Program and those that weretransferred to the University of Hawaii at the close of thatprogram. The green box shows the strains that still existand are currently available from the University of HawaiiMarine Microbial Culture Center.

Not on list of transferred strains in the close out report

TETRA1

TETRA4

Algal strains that have been re-established at NREL are in theprocess of being cyro-preserved in an effort to reduce the work-loadassociated with maintaining an algae collection and to prevent un-intended loss or genetic drift, a risk with frequent transfer. The cryo-freezer uses liquid nitrogen and cultures are stored at -195oC in thevapor phase. We are now beginning to freeze and evaluate strainsfor survival.

NREL/PO-510-45788May 3-6, 2009 • San Francisco, CA

The information contained in this poster is subject to a government license. Presented at the 31st Symposium on Biotechnology for Fuels and Chemicals.

frequent transfers exposing the cultures to the risks of contamination and genetic drift. The loss ofmany strains from the collection over time highlights the difficulties involved in maintaining algal culturecollections over the long term. New methods and technologies are continually being tried and cyro-preservation, according to the literature, seems a good candidate for some species for long termstorage. Although many of the strains have been lost over time, promising candidates for theproduction of lipids still exist from the original ASP culture collection.

Acknowledgments

The authors acknowledge the University of Hawaii for maintaining the NREL ASP algae collection.

Aquatic Species Program collection sites within the continental United States.

Not on list of transferred strains in the close-out report.Lost prior to transfer to the University of Hawaii.

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