Scenedesmus Obliquus
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The dynamics of oil accumulation in Scenedesmus obliquus Guido Breuer Thesis committee Promotor Prof. Dr R.H. Wijffels Professor of Bioprocess Engineering Wageningen University Co-promotors Dr D.E. Martens Assistant professor, Bioprocess Engineering Wageningen University Dr P.P. Lamers Assistant professor, Bioprocess Engineering Wageningen University Other members Prof. Dr V. Fogliano, Wageningen University Dr J.C. Weissman, Exxonmobil, Annandale, USA Prof. Dr B. Teusink, VU University Amsterdam Dr L.M. Trindade, Wageningen University This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). The dynamics of oil accumulation in Scenedesmus obliquus Guido Breuer Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 6 March 2015 at 1:30 p.m. in the Aula. G. Breuer The dynamics of oil accumulation in Scenedesmus obliquus, 268 pages. PhD thesis, Wageningen University, Wageningen, NL (2015) With references, with summaries in Dutch and English ISBN 978-94-6257-234-8 Contents Chapter 1 Introduction 9 Chapter 2 Analysis of Fatty Acid Content and Composition in 19 Microalgae Chapter 3 The impact of nitrogen starvation on the dynamics of 37 triacylglycerol accumulation in nine microalgae strains Chapter 4 Effect of light intensity, pH, and temperature on 67 triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus Chapter 5 Superior triacylglycerol (TAG) accumulation in starchless 89 mutants of Scenedesmus obliquus: Evaluation of TAG yield and productivity in controlled photobioreactors Chapter 6 Photosynthetic efficiency and carbon partitioning in 109 nitrogen-starved Scenedesmus obliquus Chapter 7 Opportunities to improve the areal oil productivity of 143 microalgae Chapter 8 General discussion 193 References 221 Summary 239 Samenvatting 247 Dankwoord 257 About the author 263 List of publications 265 Overview of completed training activities 266 Chapter 1 Introduction Chapter 1 Introduction 9 Chapter 1 1.1 Microalgae as a sustainable substitute for plant products Global demands for food and fuels are rapidly increasing as a result of an increasing world population and wealth of developing countries (Godfray et al., 2010). Furthermore, renewable and sustainable alternatives for fossil-oil-derived fuels and chemicals are required because of depleting fossil resources, and concerns for climate change. Currently, these alternatives are mainly derived from agricultural crops. Their products can be used directly for food, fuel or chemical production (e.g. production of biodiesel from vegetable oil) or are converted by heterotrophic microbial fermentation to fuels and chemicals (e.g. fermentation of sugars, starch or lignocellulose to for example ethanol-fuel). The production of food and renewable-fuel commodities currently thus relies heavily on the cultivation of agricultural crops. The areal productivity of commonly used crops is reaching a ceiling and the availability of arable land, as well as of fresh water and nutrients, is limited (Godfray et al., 2010). Novel sustainable and renewable sources for both food-commodities and fuels are therefore highly desired (Draaisma et al., 2013). Microalgae-derived products are often considered as a promising substitute for these crop-derived commodities (Draaisma et al., 2013; Wijffels & Barbosa, 2010). Microalgae are essentially unicellular plants (Fig. 1.1A) and can produce the same products as terrestrial crops. Microalgae can produce large quantities of protein, vegetable oil, and carbohydrates (Draaisma et al., 2013). Cultivation of microalgae has several advantages over the use of traditional agricultural crops. Microalgae can achieve much higher photosynthetic efficiencies. As a result, much higher areal productivities can be accomplished (Chisti, 2007; Moody et al., 2014; Wijffels & Barbosa, 2010). Furthermore, microalgae are cultivated in photobioreactors (Fig. 1.1B) and do therefore not require arable land. Potentially, microalgae can even be cultivated off-shore. Some microalgae species can grow efficiently on seawater, which can largely reduce freshwater requirements (Wijffels & Barbosa, 2010). Finally, fertilizer requirements could be reduced because no nutrients are lost to the environment in a photobioreactor (Chisti, 2013). Despite these advantages of microalgae, the technology for production of microalgae needs to be further developed before large scale production is feasible. Today, the cost for production and the energy consumption involved in construction and operation of photobioreactors are too high (Acién et al., 2012; Brentner et al., 2011; Davis et al., 2011; Norsker et al., 2011). Current estimates claim that a several fold reduction in 10 Introduction cost-price needs to be realized before the production of bulk products using microalgae can become feasible (Klok et al., 2014). Such improvements could for example be made by increasing the microalgal productivity. Fig. 1.1. Microscopic photograph of the microalga Scenedesmus obliquus (A). The scale bar indicates 50µm. Photograph of a lab-scale photobioreactor that was used for the work performed for this thesis (B). 1.2 TAG production by microalgae Although microalgae could be a source for a large variety of commodities, the work presented in this thesis focusses on the production of triacylglycerol (TAG). TAGs are a lipid consisting of a glycerol backbone containing three fatty acid groups (Box 1). TAGs are the main component of vegetable oil and the composition of microalgal derived TAGs is similar to that of currently used vegetable oils derived from oil-crops (Draaisma et al., 2013). These TAGs are therefore suitable as a resource for both food and biofuel applications (Chisti, 2007; Draaisma et al., 2013; Wijffels & Barbosa, 2010). In potential, TAG production using microalgae can be more sustainable and productive than TAG production using agricultural oil-crops. However, with current technology TAG production is not yet cost-effective and still requires more energy input than is produced in the form of TAG. An increase in the TAG productivity can help to decrease the cost- price and specific energy consumption of the production process. To achieve these high 11 Chapter 1 TAG productivities, a quantitative understanding of the processes involved in microalgal TAG production is essential. Box 1. Lipids in microalgae. Lipid is an ambiguous term and lipids often refers to hydrophobic organic molecules or to the fraction of the biomass that is soluble in a specific organic solvent (e.g. hexane or chloroform). Because a large variety of lipid classes are produced by microalgae, and the relative abundance of these lipids can vary to a large extent, it is important to distinguish between these lipids. From a chemical point of view, these lipids could be classified as fatty-acyl lipids and non-fatty-acyl lipids. Table 1.1. Lipids in microalgae. The values represent typical values found for most microalgae and can be used as a rule of thumb. For specific species, exceptions exist that exceed these values (e.g. large quantities of hydrocarbons in Botryococcus braunii or β- carotene in Dunaliella salina). Component Main function Abundance Reference (% DW) (Breuer et triacylglycerol storage or as al., 2012; 0-50 (TAG) energy sink Hu et al., 2008) cell and lipids - phospholipids organelle mono-, and (Breuer et membranes 5-10 glycerolipids diacylglycerols al., 2012) thylakoid galactolipids membranes fatty acyl fatty carotenoid- secondary & fatty-acyl- primary (Mulders et esters pigments al., 2014a; 0-5 (photoprotective Mulders et carotenoids & al., 2014b) photosynthesis) (Mulders et primary al., 2014a; chlorophylls pigments 1-3 acyl acyl lipids Mulders et - (photosynthesis) al., 2014b) fatty various, - e.g. waxes, including Other sterols, 5-20 Estimated1 non structural and hydrocarbons signalling 1Estimated from total lipids (15-25%)(Griffiths & Harrison, 2009; Pruvost et al., 2009; Renaud et al., 1999) minus the other lipid classes. Based on nitrogen replete conditions. Microalgae produce many different types of lipids (Table 1.1). These lipids serve various cellular functions. Triacylglycerol (TAG) is present as droplets in the cell and serves to store carbon and energy. Cell, organelle, and thylakoid membranes mainly consist of diacylglycerols and these have a structural role. Pigments such as chlorophylls and carotenoids have a role in photosynthesis for light harvesting or photo-protection. Other lipids present in microalgae include waxes, sterols, and hydrocarbons. Among other roles, these lipids have a function in cell signalling, or contribute to the structure of, for 12 Introduction example, membranes (Table 1.1). Fatty-acyl lipids are lipids that contain fatty acid groups. An important example of these fatty-acyl lipids is TAG. TAG consists of a glycerol backbone with three fatty acid groups (Fig. 1.2). Of all microalgal lipids, TAGs receive most attention because these TAGs are similar to vegetable oil and can be used as an edible oil. In addition, especially TAGs, but also other fatty-acyl lipids, are of interest because these can be trans-esterified using methanol to yield fatty acid methyl esters (FAMEs). These FAMEs can be used as a biodiesel. Fig. 1.2. Chemical