A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii

Abstract

SUMMARYInterest in exploiting algae as a biofuel source and the role of inorganic nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can easily be manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements after the cellular ionome would result in optimized cell growth. We determined the trace metal content of several commonly used Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel these measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between different wild-type strains grown in traditional Hutner's supplement. Visual observation did not reveal defects in cell motility or mating efficiency in the new supplement. Ni 2+ -inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased requirement for Ni 2+ because of the introduction of an EDTA buffer system in the revised medium.Other advantages include more facile preparation of trace element stock solutions, a reduction in total chemical use, a more consistent batch-to-batch formulation and long-term stability (tested up to 5 years). Under the new growth regime, we analyzed cells growing under different macro-and micronutrient deficiencies. TAG accumulation in N deficiency is comparable in the new medium. Fe and Zn deficiency also induced TAG accumulation, as suggested by Nile Red staining. This approach can be used to efficiently optimize culture conditions for other algal species to improve growth and to assay cell physiology.

Three Acyltransferases and Nitrogen-responsive Regulator Are Implicated in Nitrogen Starvation-induced Triacylglycerol Accumulation in Chlamydomonas

“…Nutrient-deficient media were prepared as follows. NH 4 Cl was omitted in nitrogen-free TAP; MgSO 4 was omitted in sulfur-free TAP; ZnSO 4 was replaced with ZnCl 2 ; potassium phosphate was replaced with 1 mM KCl in phosphorus-free medium (46); iron-free Hutner's trace element solution was supplemented with 0.25 M FeCl 3 for low iron medium (47) and zinc-free Kropat's trace elements for zinc-deficient TAP medium (21).…”

“…Although biomass from terrestrial plants can be processed and used as a feedstock for generating alcohols and other fuels (8) oil from photoautotrophically grown algae or seeds represents another fuel source, with algae offering distinct advantages over traditional oil crops (9,10). The green alga Chlamydomonas reinhardtii accumulates increased amounts of triacylglycerols (TAGs) 5 under conditions of growth-inhibiting macro-or micronutrient deficiency such as sulfur (11), nitrogen (12)(13)(14)(15)(16)(17)(18)(19)(20), phosphorus (15), zinc, or iron deficiency (21) or other growthinhibiting stressors such as high salt (22) or high light (23). With a sequenced genome (24) and the potential for application of metabolic engineering (25), C. reinhardtii is a powerful reference organism for discovering green algal TAG biosynthetic pathway(s) and the ways in which they are regulated by nutrient deprivation.…”

UV-B Perception and Acclimation in Chlamydomonas reinhardtii

“…Chlamydomonas was grown using Tris acetate phosphate (TAP) media (Gorman and Levine, 1965) with mineral nutrient supplements (Kropat et al, 2011). A working stock of Chlamydomonas was maintained on solid TAP media containing 1.6% Bacto agar (Difco) and was subcultured biweekly.…”

Expression and membrane-targeting of an active plant cytochrome P450 in the chloroplast of the green alga Chlamydomonas reinhardtii

“…Chlamydomonas cultures Strains were grown in acetate-containing (TAP) medium or minimal (HSM) medium as described in (Harris et al, 2009), however a revised trace metal recipe was used as found in (Kropat et al, 2011). Cells were grown in 100 ml cultures, at 25°C, shaking at 110 rpm using illumination at 15 µmol photons m Plasmid design The pASapI vector, described in (Economou et al, 2014), was used for the introduction of the CYP79A1 coding sequence into the chloroplast genome.…”