Acidophilic green alga Pseudochlorella sp. YKT1 accumulates high amount of lipid droplets under a nitrogen-depleted condition at a low-pH.

Microalgal storage lipids are considered to be a promising source for next-generation biofuel feedstock. However, microalgal biodiesel is not yet economically feasible due to the high cost of production. One of the reasons for this is that the use of a low-cost open pond system is currently limited because of the unavoidable contamination with undesirable organisms. Extremophiles have an advantage in culturing in an open pond system because they grow in extreme environments toxic to other organisms. In this study, we isolated the acidophilic green alga Pseudochlorella sp. YKT1 from sulfuric acid mine drainage in Nagano Prefecture, Japan. The vegetative cells of YKT1 display the morphological characteristics of Trebouxiophyceae and molecular phylogenetic analyses indicated it to be most closely related to Pseudochlorella pringsheimii. The optimal pH and temperature for the growth of YKT1 are pH 3.0-5.0 and a temperature 20-25°C, respectively. Further, YKT1 is able to grow at pH 2.0 and at 32°C, which corresponds to the usual water temperature in the outdoors in summer in many countries. YKT1 accumulates a large amount of storage lipids (∼30% of dry weigh) under a nitrogen-depleted condition at low-pH (pH 3.0). These results show that acidophilic green algae will be useful for industrial applications by acidic open culture systems

Physiolohical features of strain YKT1.

<p>Optimal growth pH (pH_opt), limit of growth pH (pH_limit), optimal growth temperature (T_opt in °C), limit of growth temperature (T_limit in °C) maximum growth rate in exponential phase (GR_max in d⁻¹) and maximum dry weight biomass (Max DW Biomass in g/l).</p><p>Physiolohical features of strain YKT1.</p

Growth of YKT1 under various pH and temperature conditions.

<p>(A) Photograph of the culture (1 day after the onset of culture at the indicated pH) and the growth rate based on increase in the cell number (open bars) and OD₇₅₀ (solid bars) at the indicated pH and 25°C. (B) Micrographs showing the cells cultured at the indicated pH (from pH 1.0 to pH 7.0). The arrowheads indicate dividing cells. (C) Photograph of the culture (1 day after the onset of culture at the indicated temperature) and the growth rate based on the increase in the cell number (open bars) and OD₇₅₀ (solid bars) at the indicated temperature and pH 3.0. (D) Micrographs showing cells cultured at the indicated temperature (from 10 to 35°C). The arrowheads indicate dividing cells. Scale bars: 5 µm (B, D).</p

Accumulation of lipid droplets under the nitrogen-depleted condition (pH 3.0, 25°C).

<p>(A) Growth curves of YKT1 under nitrogen-replete (+N) or nitrogen-depleted (–N) conditions. (B) Photographs of the cultures under +N or –N conditions at 0 and 7 days after the onset of cultivation. (C) Time course of storage lipid accumulation under +N or –N conditions. Lipid droplets were stained with BODIPY (green fluorescence) and cells were observed under fluorescence microscopy. (D) Micrographs of cells that were cultured under the +N or –N conditions for 7 days. Images were obtained by differential interference contrast microscopy (DIC), BODIPY staining (BODIPY), chlorophyll autofluorescence (Chl), and merged images of BODIPY and Chl (Merged) are shown. (E) Storage lipid content (% of dry weight) in YKT1 cultured under +N or –N conditions for 7 days was determined by the Nile Red method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107702#pone.0107702-Chen1" target="_blank">[27]</a>. The bars indicate the standard deviation of three individual experiments. Scale bars: 5 µm (C); 2 µm (D).</p

Phylogenetic position of YKT1.

<p>Phylogenetic trees based on the 18S rDNA (A) and plastid 16S rDNA sequence (B) are shown. The trees were constructed by a maximum-likelihood method (RaxML 7.2.8) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107702#pone.0107702-Stamatakis1" target="_blank">[24]</a>. Maximum likelihood bootstrap values (ML) >50% by RaxML and bayesian posterior probabilities (BI) >0.7 by the Bayesian analysis (MrBayes 3.1.2) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107702#pone.0107702-Ronquist1" target="_blank">[25]</a> are shown above the branches. The accession numbers of the sequences are shown along with the names of the species. The lineage designation follows <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107702#pone.0107702-Darienko1" target="_blank">[31]</a>. The branch length reflects the evolutionary distances indicated by the scale bar.</p

Taming chlorophylls by early eukaryotes underpinned algal interactions and the diversification of the eukaryotes on the oxygenated Earth

Extant eukaryote ecology is primarily sustained by oxygenic photosynthesis, in which chlorophylls play essential roles. The exceptional photosensitivity of chlorophylls allows them to harvest solar energy for photosynthesis, but on the other hand, they also generate cytotoxic reactive oxygen species. A risk of such phototoxicity of the chlorophyll must become particularly prominent upon dynamic cellular interactions that potentially disrupt the mechanisms that are designed to quench photoexcited chlorophylls in the phototrophic cells. Extensive examination of a wide variety of phagotrophic, parasitic, and phototrophic microeukaryotes demonstrates that a catabolic process that converts chlorophylls into nonphotosensitive 13(2),17(3)-cyclopheophorbide enols (CPEs) is phylogenetically ubiquitous among extant eukaryotes. The accumulation of CPEs is identified in phagotrophic algivores belonging to virtually all major eukaryotic assemblages with the exception of Archaeplastida, in which no algivorous species have been reported. In addition, accumulation of CPEs is revealed to be common among phototrophic microeukaryotes (i.e., microalgae) along with dismantling of their secondary chloroplasts. Thus, we infer that CPE-accumulating chlorophyll catabolism (CACC) primarily evolved among algivorous microeukaryotes to detoxify chlorophylls in an early stage of their evolution. Subsequently, it also underpinned photosynthetic endosymbiosis by securing close interactions with photosynthetic machinery containing abundant chlorophylls, which led to the acquisition of secondary chloroplasts. Our results strongly suggest that CACC, which allowed the consumption of oxygenic primary producers, ultimately permitted the successful radiation of the eukaryotes throughout and after the late Proterozoic global oxygenation.</p