Relationship between starch and lipid accumulation induced by nutrient depletion and replenishment in the microalga Parachlorella kessleri
Introduction
During photosynthetic processes, over short periods of time (about 5 days), some microalgae accumulate significant quantities of lipids (60% of DW) (Li et al., 2013) and carbohydrates (55% of DW) (Brányiková et al., 2011, Yao et al., 2012). These can be commercially processed into biofuels, particularly biodiesel (Yang et al., 2011) and bioethanol (Fernandes et al., 2012). However, microalgal lipids are valuable not only from the viewpoint of renewable energy, but they can also be used for the production of biochemicals, nutraceuticals, cosmetics or food-additives. Several studies have demonstrated that it is possible to control cell metabolism to yield a high content of energy-rich compounds; either starch (Brányiková et al., 2011, Dragone et al., 2011) and/or lipids (Chen et al., 2011, Lee, 2011). Although the mechanism of induction of lipid accumulation can be different from that of starch, there are several common approaches to induce both starch and lipid overproduction (Brányiková et al., 2011, Li et al., 2013). Lipid content can be increased by nitrogen or phosphate limitation (Hsieh and Wu, 2009, Rodolfi et al., 2009), high salt concentrations (Takagi et al., 2006), high iron concentrations (Liu et al., 2008) or growth under heterotrophic/mixotrophic culture conditions (Heredia-Arroyo et al., 2010, Shen et al., 2010).
Accumulation of starch can be induced by nitrogen depletion (Dragone et al., 2011), sulfur depletion, high light intensity (Brányiková et al., 2011) or a high CO2 concentration (Izumo et al., 2007). It was also shown that algal strains appropriate for overproducing starch are not usually suitable for overproducing lipids and vice versa (Li et al., 2013, Li et al., 2010). The microalga Parachlorella kessleri, strain CCALA 255, is characterized by a high growth rate, tolerance to high temperatures, resistance to shear stress, poor adhesion to bioreactor surfaces and a low tendency to form aggregates; this was previously tested in a large-scale thin-layer bioreactor to simulate the industrial production of microalgal lipid-rich biomass (Li et al., 2013). These are positive characteristics for its use in large-scale production bioreactors, with a potential for biofuel production. Under optimal conditions, the strain is characterized by energy storage in the form of starch rather than lipid (Li et al., 2013). If untreated, the cultures propagate rapidly, producing large amounts of biomass in a relatively short period of time. The cells contained negligible lipid storage (1–10% of DW) but it was possible to induce hyper-production of storage lipids in P. kessleri biomass using various methods (Li et al., 2013, Přibyl et al., 2012).
Under favorable growth conditions, algae synthesize fatty acids principally for esterification into glycerol-based polar lipids, the major constituents of intracellular membranes. However, under unfavorable environmental or stress conditions, many algae alter their lipid biosynthetic pathways towards the formation and accumulation of neutral lipids, mainly in the form of triacylglycerol (TAG) (Breuer et al., 2012, Li et al., 2011). These storage neutral lipids (especially TAGs) are the preferred lipids for most applications, since they can be overproduced up to very high cellular levels. TAGs have potential especially for biodiesel production, since they can be readily converted to biodiesel through existing oil refining processes (Hu et al., 2008).
It is known that alterations in nutrients can modify both growth and secondary metabolism of microalgae (Behrens and Kyle, 1996, Hsieh and Wu, 2009). Furthermore, microalgal growth depends not only on an adequate supply of essential macronutrients (carbon, nitrogen, phosphorus) and major ions (Mg2+, Ca2+, Cl−, and ) but also on a number of micronutrient metals such as iron, manganese, zinc, cobalt, copper, and molybdenum (Dragone et al., 2011, Sunda et al., 2005). Since reduction in nutrient supply is a simple and inexpensive methodology, medium dilution (5 and 10 times) was used to increase the lipid content in P. kessleri.
Regulatory mechanisms that control the accumulation of starch and lipid in response to changes in growth conditions, and possible interactions between storage and consumption of starch and lipid are unclear (Li et al., 2010, Rawsthorne, 2002, Siaut et al., 2011). According to Siaut et al. (2011) improving microalgal strain performance requires a sound understanding of the mechanisms and regulation of carbon fixation, carbon allocation between biosynthetic pathways and induction under adverse growth conditions. Therefore, the aim of this work was to describe photosynthetic carbon partitioning between starch and neutral lipid in P. kessleri, i.e. the temporal relationship between accumulation/consumption of starch and lipid in response to nutrient depletion and subsequent replenishment. Variations in starch and lipid concentrations are compared with the concentrations of pigments and values of other cellular growth parameters, in order to elucidate how photosynthetic carbon partitioning between starch and lipid is affected by growth conditions that are known to induce neutral lipid production.
Section snippets
Strains and growth conditions
The green microalga P. kessleri (Krienitz et al., 2004), strain CCALA 255, was provided by the Culture Collection of Autotrophic Organisms (CCALA) in Třeboň, Czech Republic (http://ccala.butbn.cas.cz/index.php). In the collection, the strain was maintained on agar slants under an irradiance of about 23 μmol m−2 s−1, 12/12 h (light/dark) regime and at a temperature of 12–15 °C.
Experimental cultures were prepared by transfer of algal inoculum from an agar slant into liquid mineral medium and
Effect of mineral medium depletion and replenishment on chlorophyll content
The green1 color of the microalgal suspension, seen at the beginning of the experiment (Fig. 1A), was yellowish after 7.5 days of growth in 0.1 medium and yellow-green 1 day later in 0.2 medium (Fig. 1B, vessels 0.1 and 0.2), while cultures grown in complete medium became dark-green during continuous growth (Fig. 1B, vessel 1). These color changes, from dark green to yellow-green, observed in
Conclusions
P. kessleri synthesized starch as a primary storage form of carbon and energy. Starch content decreased more slowly under nutrient limiting conditions than in control cultures because energy-requiring growth and reproductive processes were slowed. The hypothesis that starch is converted into storage lipids was not supported and storage lipids were shown to be synthesized mostly de novo. Cells recovered growth and division in replenished medium, utilizing both lipids and starch as sources of
Acknowledgements
This study was supported by grant CREST of Japan Science and Technology Agency, by Grant No. LH12145 of Ministry of Education, Youth and Sports of the Czech Republic, by Grant for International Collaboration Academy of Sciences of the Czech Republic No. M200201205 and by the Technology Agency of the Czech Republic, project No. TE01020080.
References (35)
- et al.
Nutrient limitation as a strategy for increasing starch accumulation in microalgae
Appl. Energy
(2011) - et al.
Cultivation of microalgae for oil production with a cultivation strategy of urea limitation
Bioresour. Technol.
(2009) - et al.
Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review
Bioresour. Technol.
(2011) - et al.
Physicochemical properties of starch in Chlorella change depending on the CO2 concentration during growth: comparison of structure and properties of pyrenoid and stroma starch
Plant Sci.
(2007) Algal biodiesel economy and competition among bio-fuels
Bioresour. Technol.
(2011)- et al.
The microalga Parachlorella kessleri – a novel highly-efficient lipid producer
Biotechnol. Bioeng.
(2013) - et al.
Photosynthetic carbon partitioning and lipid production in the oleaginous microalga Pseudochlorococcum sp. (Chlorophyceae) under nitrogen-limited conditions
Bioresour. Technol.
(2011) - et al.
Effect of iron on growth and lipid accumulation in Chlorella vulgaris
Bioresour. Technol.
(2008) Absorption of light by chlorophyll solutions
J. Biol. Chem.
(1941)- et al.
Sequential accumulation of starch and lipid induced by sulfur deficiency in Chlorella and Parachlorella species
Bioresour. Technol.
(2013)
Carbon flux and fatty acid synthesis in plants
Prog. Lipid Res.
Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells
J. Biosci. Bioeng.
Life-cycle analysis on biodiesel production from microalgae: water footprint and nutrients balance
Bioresour. Technol.
Enhancing starch production of a marine green microalga Tetraselmis subcordiformis through nutrient limitation
Bioresour. Technol.
Multiple pathways for triacylglycerol biosynthesis in Streptomyces coelicolor
Appl. Environ. Microbiol.
Microalgae as a source of fatty acids
J. Food Lipids
Microalgae-novel highly efficient starch producers
Biotechnol. Bioeng.
Cited by (118)
Separation of lipids and proteins from clarified microalgae lysate: The effect of lipid-protein interaction on the cross-flow and shear-enhanced microfiltration performances
2024, Separation and Purification TechnologyMicroalgae biomass as an alternative source of biocompounds: New insights and future perspectives of extraction methodologies
2023, Food Research InternationalStudy of carbon fixation and carbon partitioning of evolved Chlorella sp.'s strain under different carbon dioxide conditions
2023, Biocatalysis and Agricultural Biotechnology