Elsevier

Bioresource Technology

Volume 144, September 2013, Pages 268-274
Bioresource Technology

Relationship between starch and lipid accumulation induced by nutrient depletion and replenishment in the microalga Parachlorella kessleri

https://doi.org/10.1016/j.biortech.2013.06.096Get rights and content

Highlights

  • Parachlorella cells, starved by medium depletion, ceased growth and division.

  • During starvation, chlorophyll was degraded and starch content decreased.

  • During starvation, storage lipids, but not other cellular lipids, were overproduced.

  • Starch was not used for storage lipid synthesis, whereas cellular lipids were.

  • Algae that were transferred to complete medium recovered growth and cell division.

Abstract

Photosynthetic carbon partitioning into starch and neutral lipids, as well as the influence of nutrient depletion and replenishment on growth, pigments and storage compounds, were studied in the microalga, Parachlorella kessleri. Starch was utilized as a primary carbon and energy storage compound, but nutrient depletion drove the microalgae to channel fixed carbon into lipids as secondary storage compounds. Nutrient depletion inhibited both cellular division and growth and caused degradation of chlorophyll. Starch content decreased from an initial value of 25, to around 10% of dry weight (DW), while storage lipids increased from almost 0 to about 29% of DW. After transfer of cells into replenished mineral medium, growth, reproductive processes and chlorophyll content recovered within 2 days, while the content of both starch and lipids decreased markedly to 3 or less % of DW; this suggested that they were being used as a source of energy and carbon.

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 SO42-) 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)

  • S. Rawsthorne

    Carbon flux and fatty acid synthesis in plants

    Prog. Lipid Res.

    (2002)
  • M. Takagi et al.

    Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells

    J. Biosci. Bioeng.

    (2006)
  • J. Yang et al.

    Life-cycle analysis on biodiesel production from microalgae: water footprint and nutrients balance

    Bioresour. Technol.

    (2011)
  • C.H. Yao et al.

    Enhancing starch production of a marine green microalga Tetraselmis subcordiformis through nutrient limitation

    Bioresour. Technol.

    (2012)
  • A. Arabolaza et al.

    Multiple pathways for triacylglycerol biosynthesis in Streptomyces coelicolor

    Appl. Environ. Microbiol.

    (2008)
  • P.W. Behrens et al.

    Microalgae as a source of fatty acids

    J. Food Lipids

    (1996)
  • I. Brányiková et al.

    Microalgae-novel highly efficient starch producers

    Biotechnol. Bioeng.

    (2011)
  • Cited by (118)

    View all citing articles on Scopus
    View full text