Establishment of a bioenergy-focused microalgal culture collection Lee G. Elliott a, c , Corinne Feehan c , Lieve M.L. Laurens c , Philip T. Pienkos c , Al Darzins c, 1 , Matthew C. Posewitz b, ⁎ a Department of Civil and Environmental Engineering, Colorado School of Mines, 1500 Illinois St., Golden, CO 80401, United States b Department of Chemistry and Geochemistry, Colorado School of Mines, 1500 Illinois St., Golden, CO 80401, United States c National Bioenergy Center, National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, CO 80401, United States abstract article info Article history: Received 20 October 2011 Received in revised form 10 March 2012 Accepted 4 May 2012 Available online 12 June 2012 Keywords: Microalgae Bioprospecting FACS Cryopreservation Lipids Biofuels A promising renewable energy scenario involves growing photosynthetic microalgae as a biofuel feedstock that can be converted into fungible, energy-dense fuels. Microalgae transform the energy in sunlight into a variety of reduced-carbon storage products, including triacylglycerols, which can be readily transformed into diesel fuel surrogates. To develop an economically viable algal biofuel industry, it is important to maxi- mize the production and accumulation of these targeted bioenergy carriers in selected strains. In an effort to identify promising feedstock isolates we developed, evaluated and optimized contemporary high-throughput cell-sorting techniques to establish a collection of microalgae isolated from highly diverse ecosystems near geographic areas that are potential sites for large-scale algal cultivation in the Southwest United States. These efforts resulted in a culture collection containing 360 distinct microalgal strains. We report on the es- tablishment of this collection and some preliminary qualitative screening studies to identify important biofuel phenotypes including neutral lipid accumulation and growth rates. As part of this undertaking we determined suitable cultivation media and evaluated cryopreservation techniques critical for the long-term storage of the microorganisms in this collection. This technique allows for the rapid isolation of extensive strain biodiversity that can be leveraged for the selection of promising bioenergy feedstock strains, as well as for providing fun- damental advances in our understanding of fundamental algal biology. © 2012 Elsevier B.V. All rights reserved. 1. Introduction As concerns over national energy security and global warming in- crease, the need to develop alternative and renewable sources of en- ergy is more important than ever. One promising scenario involves using microalgae as biochemical factories to capture and convert the energy in sunlight into organic hydrocarbons. Through photosynthe- sis, inorganic carbon is reduced and incorporated into biomass. Among the most reduced, energy-rich hydrocarbons in algal biomass are triacylglycerol storage lipids (TAGs) composed of 3 fatty acid moi- eties bound to a glycerol backbone [1–5]. These and other algal cellu- lar components can be converted into a variety of biofuels [6]. A recent technology and engineering assessment of the algal biofuel industry [7] concluded that, in addition to coupling biofuel production to waste water treatment and nutrient recovery to lower costs, a dou- bling of current reported algal productivities needs to be achieved to reach economic feasibility. To circumvent productivity limits, tools are being developed to metabolically engineer microalgae to optimize bio- energy carrier accumulation, for example by manipulating lipid and car- bohydrate metabolism [8–11]. These endeavors are primarily focused on well-studied model organisms (e.g. Chlamydomonas reinhardtii and Phaeodactylum tricornutum), but the process can be greatly facilitated by identifying and developing superior microalgal strains that already possess certain genetic traits better adapted for biofuel production rel- ative to model laboratory organisms [12,13]. There are approximately 40,000 different algal species identified to date, with some conservative and more generous estimates of actual diversity ranging from one to two orders of magnitude more extant species that have yet to be described [14]. Relatively little of this bio- logical and genetic diversity has been examined in detail; however, a recent surge of interest in algal-based biotechnology is helping to rap- idly expand the list of described species. The biological diversity in nature is attributed to the natural selec- tion of traits that improve species fitness. Through evolution, algae have acquired the ability to survive prolonged exposure to conditions of low nutrients, turbulence, temperature extremes, wide ranges of salinity and pH, fluctuating light levels, niche competition from other organisms, predatory grazing, toxins, and periodic droughts. This level of environmental adaptability is a reflection of complicated metabolic strategies used in response to environmental stressors that can act to hinder as well as advance the utility of algae as a feedstock. Understand- ing the mechanisms behind these responses will significantly help to develop the full potential of the algal biotechnology industry. The idea that novel microalgal strains with desirable biofuel phe- notypes exist in nature was extensively explored during the 1980's Algal Research 1 (2012) 102–113 ⁎ Corresponding author. Tel.: + 1 303 384 2425. E-mail address: mposewit@mines.edu (M.C. Posewitz). 1 Current address: Gas Technology Institute, 1700 S. Mount Prospect Road, Des Plaines, IL 60018, United States. 2211-9264/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.algal.2012.05.002 Contents lists available at SciVerse ScienceDirect Algal Research journal homepage: www.elsevier.com/locate/algal