Phototrophic pigment production with microalgae Kim J. M. Mulders Thesis committee Promotor Prof. Dr R.H. Wijffels Professor of Bioprocess Engineering Wageningen University Co-promotors Dr D.E. Martens Assistant professor, Bioprocess Engineering Group Wageningen University Dr P.P. Lamers Assistant professor, Bioprocess Engineering Group Wageningen University Other members Prof. Dr H. van Amerongen, Wageningen University Prof. Dr M.J.E.C. van der Maarel, University of Groningen Prof. Dr C. Vilchez Lobato, University of Huelva, Spain Dr S. Verseck, BASF Personal Care and Nutrition GmbH, Düsseldorf, Germany This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Phototrophic pigment production with microalgae Kim J. M. Mulders Thesis submitted in fulfilment of the requirement 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 5 December 2014 at 11 p.m. in the Aula. K. J. M. Mulders Phototrophic pigment production with microalgae, 192 pages. PhD thesis, Wageningen University, Wageningen, NL (2014) With propositions, references and summaries in Dutch and English ISBN 978-94-6257-145-7 Abstract Microalgal pigments are regarded as natural alternatives for food colourants. To facilitate optimization of microalgae-based pigment production, this thesis aimed to obtain key insights in the pigment metabolism of phototrophic microalgae, with the main focus on secondary carotenoids. Different microalgal groups each possess their own set of primary pigments. Besides, a selected group of green algae (Chlorophytes) accumulate secondary pigments (secondary carotenoids) when exposed to oversaturating light conditions. In this thesis it was found for the first time that nutrient-depleted Isochrisis. aff. galbana T-ISO (Haptophytes) accumulates 3-hydroxyechinenone, a precursor of astaxanthin. Besides, it was found that nitrogen-depleted Chromochloris (Chlorella) zofingiensis (Chlorophyes) accumulates astax- anthin, presumably synthesised via echinenone, and ketolutein. Inhibition of production of β- carotene derivatives (e.g. echinenone and astaxanthin) did not lead to increased production of primary carotenoids (e.g. lutein) or ketolutein in this species, suggesting that the regulatory mechanisms controlling the flux towards ketolutein and primary carotenoids are not affected by the decreased levels of β-carotene derivatives. Besides, optimal yields of secondary carotenoids and triacylglycerol (TAG) on light were reached with C. zofingiensis for a range of biomass concentrations at the moment of nitrogen depletion. This indicated that the biomass- specific photon absorption rate did not affect the amounts of energy used for secondary carotenoid and TAG production, for the range of biomass concentrations tested. It was also found that nitrogen-depleted C. zofingiensis resupplied with nitrogen hardly degraded astaxanthin, whereas the other major secondary metabolites were degraded rapidly. This indicated that the overall carotenoid yield on light as well as its content may possibly be improved by applying a repeated batch instead of a series of single batch cultivations, which are traditionally applied. Finally, it was discussed that the highest increases in carotenoid yield on light can be reached by optimizing strain performance (using targeted genetic engineering and/or random mutagenesis), rather than by optimizing the cultivation conditions/operation mode or reactor design. v Photograph of the artwork Pigments From Microalgae: An Ocean Of Colours which is made of filter-dried nutrient-sufficient, nitrogen-depleted and nitrogen-resupplied Chromochloris (Chlorella) zofingiensis cells. The colours comprise mainly the pigments chlorophyll a and b (green), lutein (yellow), canthaxanthin (orange), ketolutein (orange) and astaxanthin (red). vi Contents Abstract v Chapter 1 1 Introduction and thesis outline Chapter 2 9 Phototrophic pigment production with microalgae: biological constraints and opportunities Chapter 3 29 Growth and pigment accumulation in nutrient depleted Isochrysis aff. galbana T-ISO Chapter 4 43 Nitrogen-depleted Chromochloris zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: a carotenoid metabolism study Chapter 5 69 Effect of biomass concentration on secondary carotenoids and triacylglycerol (TAG) accumulation in nitrogen-depleted Chromochloris zofingiensis Chapter 6 89 Dynamics of biomass composition and growth during recovery of nitrogen-starved Chromochloris zofingiensis Chapter 7 109 General Discussion Microalgal carotenoid yields on light: current status and future perspectives References 133 Summary 151 Samenvatting 159 Dankwoord 169 The Author Curriculum Vitae 176 List of publications 177 Overview of completed training activities 178 vii n Chapter 1 Introduction and thesis outline High lights o The consumer demand for naturally produced pigments is rising. o Microalgae possess a large variety of pigments and grow fast on few resourses. o For commercialization, optimizing the pigment production process is desirable. o This requires insight in the algal pigment metabolism, as described in this thesis. IN THE PICTURE IN BEELD To know very precisely which pigments and Om heel precies te weten welke pigenten en how much of each pigment is present in the hoeveel van ieder pigment aanwezig is in de orange and green coloured microalgae, the oranje en groen uitziende microalgen moeten chlorophylls and carotenoids first need to be de carotenoïden en chlorofylen eerste uit de extracted. This occurs in glass tubes using a cel gehaald worden. Dit gebeurt in reageer- mixture of chloroform and methanol. After buizen met behulp van chloroform en metha- shaking and centrifugating the cells, the nol. Na flink schudden en centrifugeren ein- pigments end up in the chloroform (bottom) digen de pigmenten in de (onderste) chloro- phase, separated from the cell debris. form fase, gescheiden van de rest van de cel. 1 Chapter 1 1.1 Microalgae as a source of natural pigments Pigments are molecules that absorb part of the visual light spectrum. The part that is not absorbed is reflected and caught by the human eye, and hence determines their colour. Pigments are used in a wide range of products, including food and cosmetics. Although many pigments can be produced synthetically (e.g. from petroleum, organic acids and inorganic chemicals) for a lower price than by natural production, the consumer demand for naturally produced pigments is rising (BCC Research 2008, Farré et al. 2010, Kobylewski and Jacobson 2010). Pigments of natural origin can be obtained from insects (e.g. cochineals and aphids), vegetables, fruits, petals of flowers, or from microorganisms such as microalgae (Eriksen 2008; Farré et al. 2010; Mendez et al. 2004; Piccaglia 1998; Vilchez 2011). Microalgae are a biotechnologically interesting source of pigments because they possess a large variety of such molecules, including chlorophylls (green), carotenoids (red, orange and yellow) and phycobiliproteins (red and blue). Part of these pigments can be produced in concentrations exceeding those found in higher plants by one or more orders of magnitude (Fig. 1.1). Fig. 1.1 Carotenoid contents (in mg/g dry weight) of raw carotenoid-rich fruits and vegetables, petals of marigold flowers and some microalgae (Del Campo et al. 2007; Lamers et al. 2008; NEVO 2001; Piccaglia et al. 1998) Besides, in contrast to plants, very high areal productivities can be achieved with microalgae, which can be obtained without the use of arable land (Del Campo et al. 2001, 2007; Fernández-Sevilla et al 2010; Lamers et al. 2008). In addition, as many algal species are salt tolerant (Ben-Amotz et al. 1982), seawater can often be used for their cultivation. Using seawater is more sustainable than using freshwater and may in addition be cheaper. Furthermore, microalgae can simultaneously produce other high-value compounds, such as long-chain polyunsaturated fatty acids, vitamin E and C, and bulk compounds such as starch, 2 Introduction proteins and lipids (Abe et al. 1999; Borowitzka 2013). The lipid triacylglycerol (TAG) is currently of particular interest, because it is a promising alternative source for vegetable oils and next generation biofuels (Chisti 2013; Draaisma et al. 2013; Hu et al. 2008). Despite all these advantages of using microalgae for pigment production, and despite the potential to produce a large variety of pigments, microalgae are currently exploited commercially for only three pigments, namely β-carotene (Dunaliella salina), astaxanthin (Haematococcus pluvialis) and phycocyanin (Arthrospira platensis). The carotenoids β-carotene and astaxanthin are claimed to have beneficial health effects, due to their anti-oxidative activity (Spolaore et al. 2006; Harari et al. 2013), which makes them particularly valuable for application in nutraceuticals. Phycocyanobilin, on the other hand, is used mainly in food applications. The reason that only those three pigments are currently exploited commercially is that the production costs of microalgal pigments are generally too high. In the cases of the aforementioned pigments that are already being commercialized, however, the market values and obtained yields are sufficiently high to lead to economically feasible microalgae-based production. However, to