LAAN-C-XX-E026 Application Determination of Storage Lipid Contents by Total Organic Carbon (TOC) Analyzer and Fourier Transform Infrared Spectrophotometer Note (FT-IR) in Non-Destructive Microalgal Cells – A New Method for the Semi-Quantitative Analysis of Long-Chain Ketones (Alkenones) Produced by Marine Haptophyte Algae – No. 43 Yutaka Hanawa (1), Yoshihiro Shiraiwa (1) * 1 μm Energy 1. Introduction Background Recent progress in our understanding of biofuel organic cellular components such as proteins, production has increased the demand for the carbohydrates and lipids in non-disrupted cells. On the development of various technical methods for the other hand, a Total Organic Carbon (TOC) analyzer can quantification of biofuel candidates such as neutral be used for the quantification of total organic and lipids and polar lipids such as fatty acids. The keys inorganic carbons in samples. Therefore, the factors required for increasing biofuel and biorefinery combination of both FT-IR and TOC analysis was production are biological, particularly how to isolate expected theoretically to enable us to get quantities of highly productive microalgal strains and technological individual cellular organic components. However, it is and engineering systems to establish highly impossible to quantify all cellular organic components by energy-efficient high-cell-density-mass cultivation. For FT-IR although it does provide relative amounts of most establishing such systems, the development of major cellular organic components. Therefore, we analytical methods which are useful for easy and rapid sought to develop a new method to get quantification of biofuel compounds is necessary. “semi-quantitative values” of major cellular components. Fourier Transform Infrared Spectrophotometry (FT-IR) is very useful for getting relative quantification data of (1) Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8572, Japan. * Corresponding author 1 Application No. 43 Note Alkenones as a target of analysis and biological alkenone producer In this article, we introduce a newly-developed method considered one of major candidates of fossil fuel for detecting cellular storage lipids which can be used producers in the geological era. One such haptophyte, without disruption of cells. The method focuses the Emiliania huxleyi, a coccolithophore with a calcium storage lipids produced in some marine haptophyte carbonate cell covering (coccoliths), is wide spread and cells, namely long-chain ketones (alkenones) which are produces huge blooms, even in the present-day oceans. very different from triacylglycerols (TAGs). To date, With the potential of huge biomass production, we there has been no useful method to do this, but we have considered these algae as candidates for algal have succeeded in developing a new method for rapid biofuel production. Alkenones are straight carbon chain detection and semi-quantification of alkenones using a compounds with several trans-type carbon-carbon combination of Fourier Transform Infrared double bonds and one keto-group in a C37-C40 Spectrophotometer (FT-IR) and a Total Organic Carbon alkenone molecule. Such alkenones, and alkenes, a (TOC) analyzer. molecule without a keto-group, are expected to be Our research groups have studied physiological, good sources for producing jet fuels as drop-in-fuel biochemical and molecular biological features of without high energy input of added chemical alkenone-producing marine haptophytes, often processes. Analysis of alkenones Alkenones, including various derivatives, have been separation of several lipid classes through separation identified and quantified by gas chromatography (GC) columns are time-consuming and often lead to using well-established methods. GC analysis of increase errors due to many procedures. Biology alkenones is performed routinely in well-equipped laboratories often do not require highly accurate organic geochemistry laboratories where alkenones quantifiable data of each alkenone and derivative are used as a molecular paleothermometer for the molecules, but need to estimate total amounts of purpose of sea surface paleotemperature estimation. those compounds rapidly and easily during mass However, it is quite difficult to set up such analytical cultivation to estimate the production of lipids as one systems in general laboratories such as biology of parameter of biomass and biofuel production. laboratories. We therefore have developed a new semi-quantification On the other hand, biology laboratories are often method of alkenones by FT-IR and TOC using cells of interested in estimating total amount of alkenones, alkenone-producing haptophytes such as a calcareous rather than individual alkenone compounds, since the unicellular alga Emiliania huxleyi and non-calcareous alga total amount of lipids produced is important as one of Tisochrysis lutea which are strong candidates for biofuel parameters of biomass and biofuel production during producers (see flow chart in Fig. 1). The FT-IR and TOC mass cultivation. Additionally, the quantification of analyzer combination method is advantageous for use each alkenone is difficult, even in well-equipped with non-disrupted algal cells for semi-quantifying laboratories since alkenone composition is different storage lipids “alkenones” without extraction and among algal species. The processes of extraction and separation processes of compounds. Haptophyte algae cell suspension in culture Harvested haptophyte algae cells Living cells (in non-disrupted condition) Extraction by using methanol and dichloromethane TOC analyzer FT-IR Extraction by using hexane and ethyl acetate Determination of the ratio GC analysis GC analysis (relative values) of alkenone, Organic carbon content of cells protein, carbohydrate, and lipid content (100% TOC) (Excluded other components) Quantification of each alkenone Quantification of other (Approx. 100% TOC) molecule lipid molecules (C37:2 : C37:3 : C38:2 : C38:3 : C39:2 and (Excluded alkenone) others) Determination of semi-quantitative Determination of quantitative values of alkenone content values of alkenone content Flow chart of the procedures for determining alkenone contents using GC-FID and the combination of FT-IR and TOC analyzer. 2 2. Characteristics of alkenones as a target of FT-IR and TOC analyses Alkenones are long-chain unsaturated ketones of geosciences to estimate sea surface paleotemperature which length of molecules are C37-C39 in major and when and where alkenone-containing marine sediments known to be produced by only five species of marine were produced by haptophytes [7]. This trend can be seen haptophyte algae [1]. One alkenone molecule contains in algal cells fresh prepared in culture in addition to two to four trans-type carbon-carbon double (C=C) marine sediments in which alkenones have been bonds, whereas a fatty acid molecule contains cis-type preserved during geological time scale. double bonds, and one keto-group (Fig. 2). The Several cellular functions of alkenones have been molecules of alkenones and those derivatives were proposed. First, alkenones may regulate the formation found in oceanic sediments 30 years ago but no of membrane vesicles and the physicochemical information how those molecules were produced and properties of membranes within membranes [8]. preserved in the sediments. Later, marine Second, alkenone accumulation may contribute haptophytes were identified as alkenone-producers toward buoyancy regulation by influencing the and alkenones have been used as a biomarker of specific gravity of cells [9]. Third, alkenones may serve alkenone-producing haptophytes. as storage compounds because of their carbon A few genera of haptophytes, such as Emiliania and abundance and energy in short hydrated carbon Gephyrocapsa (family Noëlaerhabdaceae, formerly chains [10], [11], [12]. The storage function of alkenones is Gephyrocapsaceae), and Tisochrysis, Isochrysis and also supported by a recent finding showing that the Chrysotila (family Isochrysidaceae), have been lipid bodies of the alkenone-producing haptophyte reported to produce alkenones [2], [3]. Although the Tisochrysis lutea mostly contain alkenones (about 75 %, phylogenetic relationships between Isochrysidaceae w/w), with only small proportions of alkenes (1 %, and Noëlaerhabdaceae have not been fully elucidated, w/w) and other lipids such as sterols (24 %, w/w); thus 18S rDNA sequence analyses confirmed that Emiliania, these lipid bodies were named "alkenone bodies" [13]. [4] Gephyrocapsa, and Isochrysis are monophyletic . At Recently, alkenones are proposed to be potential present, it is still unknown whether other sources of renewable fuels since the pyrolysis of dry alkenone-producers exist and how alkenone cells of alkenone-producing microalgae generates production is regulated physiologically. The hydrocarbons that are very similar to the components identification of other alkenone producers through a of crude oils [14], [15], [16]. The interest in application of broad screening of algae strains could reveal very alkenones as potential sources of biofuels was further interesting information relevant to not only cell increased by the support of experimental evidences biology but also geology and paleontology. which showed the alkenone-producing haptophyte E. Alkenones, in fact, are excellent biological markers huxleyi contains more lipids and hydrocarbons than and geological paleothermometers, whose presence other microalgae such as the cyanobacterium in sediments