High throughput imaging to the diatom Cyclotella cryptica demonstrates substantial cell-to-cell variability in the rate and extent of triacylglycerol accumulation Jesse C. Traller, Mark Hildebrand ⁎ Marine Biology Research Division, Scripps Institution of Oceanography, UCSD, La Jolla, CA, United States abstract article info Article history: Received 19 October 2012 Received in revised form 15 March 2013 Accepted 22 March 2013 Available online xxxx Keywords: Diatom Imaging flow cytometry Triacylglycerol Nutrient starvation Cell-to-cell variability BODIPY In microalgal cultures, most analyses of cellular processes are done in bulk, on the entire population of cells. Information gained from this is representative of the mean; however, it obscures the richness of cell-to-cell variation, which is a well-documented phenomenon. Using imaging flow cytometry, we evaluate changes in triacylglycerol (TAG) content and chlorophyll resulting from silicon or nitrogen deprivation in the diatom Cyclotella cryptica. This approach allows detailed interrogation of large numbers of individual cells and reveals cell-to-cell variation. This study demonstrates several previously undescribed phenomena related to TAG accumulation in microalgae. First, the rate of TAG accumulation varies over time, with a faster rate occurring at the latter stage of the process, resulting in hyperaccumulation in which the majority of the cell volume is comprised of lipid droplets. In C. cryptica and other diatoms this hyperaccumulation occurs strictly under autotrophic conditions. Second, there are distinct responses to silicon or nitrogen limitation, and variation within a given type of limitation treatment. Under most conditions there is a large spread in the population when measuring either chlorophyll or TAG. Heterogeneity within the total population indicates that caution should be taken in interpreting bulk measurements for a variety of variables (TAG, transcript, protein, metabolites, etc.) related to cellular responses. However, a potential means to couple subpopulation-level responses with bulk analysis approaches is described, which could take advantage of the nuances observed during the TAG accumulation process. © 2013 Elsevier B.V. All rights reserved. 1. Introduction The development of microalgal biofuels is gaining momentum, as economic and environmental factors drive the need for renewable and sustainable sources of liquid transportation fuels. Many algal spe- cies synthesize abundant amounts of neutral lipids suitable for con- version to high energy density liquid fuels, producing 30–40% dry weight in lipids, with exceptional species reaching levels of 50–70% [1–4]. A number of analyses indicate that unicellular algae produce more biomass and oil than plants by a large margin [5]. Microalgae can also be grown on non-arable land in non-potable, saline water, reducing competition for resources for human needs. Currently lack- ing, however, is the infrastructure to efficiently grow algae on a large scale, and at the cellular level, an understanding of the basic mecha- nisms underlying controllable and reproducible lipid accumulation. In terms of the economics of production, the research focus has large- ly been to increase the amount of fuel precursor molecule; however, it may not be appreciated that the rate at which the molecule accu- mulates will also have a strong impact on the cost. An ideal production organism will produce abundant neutral lipid or triacylglycerol (TAG) yet still rapidly grow to high biomass. In the natural environment, diatoms (Bacillariophyceae) often outcompete other classes of algae for nutrients and growth and are among the most productive and environmentally-flexible eukaryotic microalgae on the planet [6]. Extensive screening in the Aquatic Species Program (ASP) identified diatoms as some of the best candidate organisms for lipid-based biofuels production [3]. The centric diatom Cyclotella cryptica was defined as a potential model organism with high lipid accumulation ability [7,8], and with consistent productivity levels of 20 g·m -2 ·d -1 in outdoor ponds [9]. One distinguishing feature of diatoms is their cell wall, which is a composite of silica and organic material [10–12]. Most diatoms have an absolute requirement for silicon (Si) in order to grow [13], and re- quire it to the same extent as nitrogen [14]. Depletion of silicon in di- atom cultures leads to cessation of cell division and consequentially accumulation of organic carbon in the form of TAG in some species [4,8,15]. Silicon is not directly involved in many aspects of cellular metabolism [16] and intracellular stores of Si are low [13], which is in contrast to nitrogen, a commonly used limiting nutrient for TAG ac- cumulation in microalgae [3,17,18]. Nitrogen metabolism is tightly Algal Research xxx (2013) xxx–xxx ⁎ Corresponding author at: 9500 Gilman Dr., La Jolla, CA 92093-0202, United States. Tel.: +1 828 822 0167. E-mail address: mhildebrand@ucsd.edu (M. Hildebrand). ALGAL-00049; No of Pages 9 2211-9264/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.algal.2013.03.003 Contents lists available at SciVerse ScienceDirect Algal Research journal homepage: www.elsevier.com/locate/algal Please cite this article as: J.C. Traller, M. Hildebrand, High throughput imaging to the diatom Cyclotella cryptica demonstrates substantial cell-to- cell variability in the rate ..., (2013), http://dx.doi.org/10.1016/j.algal.2013.03.003