Short Communication Early detection of protozoan grazers in algal biofuel cultures John G. Day ⇑ , Naomi J. Thomas, Undine E.M. Achilles-Day, Raymond J.G. Leakey Scottish Association for Marine Science, Scottish Marine Institute, Oban, Argyll PA37 1QA, UK article info Article history: Received 27 January 2012 Received in revised form 5 March 2012 Accepted 5 March 2012 Available online 14 March 2012 Keywords: Algal biofuels Ciliates FlowCAM Grazers Lipids abstract Future micro-algal biofuels will most likely be derived from open-pond production systems. These are by definition open to ‘‘invasion’’ by grazers, which could devastate micro-algal mass-cultures. There is an urgent requirement for methodologies capable of early detection and control of grazers in dense algal cul- tures. In this study a model system employing the marine alga Nannochloropsis oculata was challenged by grazers including ciliates, amoebae and a heterotrophic dinoflagellate. A FlowCAM flow-cytometer was used to detect all grazers investigated (size range <20–>80 lm in length) in the presence of algae. Detec- tion limits were <10 cells ml 1 for both ‘‘large’’ and ‘‘small’’ model grazers, Euplotes vannus (80  45 lm) and an unidentified holotrichous ciliate (18  8 lm) respectively. Furthermore, the system can distin- guish the presence of ciliates in N. oculata cultures with biotechnologically relevant cell densities; i.e. >1.4  10 8 cells ml 1 (>0.5 g l 1 dry wt.). Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Microalgae have significant advantages over ‘‘higher’’ plants for the production of future biofuels,; these include very high growth rates, capacity to utilize a large fraction of the solar energy (in the- ory 10% of the total solar energy can be fixed into biomass) and capacity of many taxa to grow under conditions that are not suit- able for terrestrial biomass growth, specifically with regards to water quality and salinity (Carlsson et al., 2007; Day et al., 2012). Furthermore, algal cultivation could be directly coupled with CO 2 elimination/sequestration from industrial flue gases derived from fossil fuels. It has been suggested that, at 2007 oil prices, a biomass yield in the region of 100 tonnes ha 1 yr 1 would be required for an open pond system to be economically viable (Carlsson et al., 2007). However, as with higher plants, grazing has the potential to com- promise productivity and final yields so it is vital that any proto- zoan detection system is capable of identifying ciliates in dense algal cultures capable of this level of productivity. The yields re- quired for the oleaginous alga N. oculata, which is a strong candi- date for future algal-derived biofuels, are likely to be >1.0  10 8 cells ml 1 and any detection system applicable to commercial production systems must be capable of recognising potential graz- ers in these extremely dense algal cultures. Grazing by ciliates, amoeba, rotifers and other zooplankton taxa has a significant influence on natural aquatic ecosystems and they are critical to the functioning of the marine food web (Sherr and Sherr, 2002). Although grazing is a widespread problem in the algal biotechnology field, to date relatively little has been published on this topic. There have been a small number of reports on loss of al- gae associated with insect larvae grazing in Spirulina ponds (Venkatamaran and Kanya, 1981; Belay, 1997), and on rotifers and other metazoan zooplankton (Becker, 1994), as well as amoeba and ciliates (Post et al., 1983) grazing in large-scale Dunaliella cul- tures. Moreno-Garrido and Canavate (2001) also observed that grazing ciliates can visually clarify dense outdoor mass cultures of Dunaliella salina within 2 days. Such catastrophic losses are com- mercially unacceptable and strategies to prevent and/or control grazers need to be developed for each application. A prerequisite for any control measure is the early detection of grazers before they have adversely effected productivity of the algal crop. In this study we assessed the applicability of the FlowCAM, a flow-cytom- eter that employs continuous digital imaging to measure the num- ber, size and shape of microscopic particles in a fluid medium, to provide early warning of ciliate contamination of N. oculata cultures. 2. Methods 2.1. Cultures and growth conditions Nannochloropsis oculata CCAP 849/1 was cultured in static flasks (100 ml medium in 250 ml flasks) in f/2 medium (see www.ccap.a- c.uk/media/pdfrecipes.htm). Cultures were maintained at 15 °C un- der a mixture of cool-white and warm-white fluorescent tubes at 30 lmol photon m 2 s 1 PAR and a 12–12 h light–dark regime. 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.03.015 ⇑ Corresponding author. Tel.: +44 1631 559349; fax: +44 1631 559001. E-mail address: jgd@sams.ac.uk (J.G. Day). Bioresource Technology 114 (2012) 715–719 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech