Unicellular Red Alga Galdieria Sulphuraria Research Articles

Reversible adsorption of iridium in lyophilized cells of the unicellular red alga Galdieria sulphuraria.

Iridium (Ir) is one of the rarest elements in the Earth's crust and is valuable in industry due to its high corrosion resistance. In this study, we used lyophilized cells of a unicellular red alga, Galdieria sulphuraria for the selective recovery of small amounts of Ir from hydrochloric acid (HCl) solutions. The Ir recovery efficiency of the lyophilized cells was higher than that of activated carbon and comparable to that of an ion-exchange resin in up to 0.2 M acid. Lyophilized G. sulphuraria cells showed different selectivity from the ion-exchange resin, adsorbing Ir and Fe in 0.2 M HCl solution while the ion-exchange resin adsorbed Ir and Cd. The adsorbed Ir could be eluted with more than 90% efficiency using HCl, ethylenediaminetetraacetic acid, and potassium hydroxide solutions, but could not be eluted using a thiourea-HCl solution. After the elution of Ir with a 6 M HCl solution, lyophilized cells could be reused up to five times for Ir recovery with over 60% efficiency. Scanning electron-assisted dielectric microscopy and scanning electron microscopy revealed that Ir accumulated in the cytosol of the lyophilized cells. X-ray absorption fine structure analysis demonstrated the formation of an outer-sphere complex between Ir and the cellular residues, suggesting the adsorption via ion exchange, and explaining the ability to elute the Ir and reuse the cells. Our results provide a scientific basis for inexpensive and environmentally friendly biosorbents as an alternative to ion-exchange resins for the recovery of Ir.

Cell population behavior of the unicellular red alga Galdieria sulphuraria during precious metal biosorption

This study investigates the biosorption mechanism, including cell population behavior, of trace amounts of precious metals (gold, palladium, and platinum) in a unicellular red alga, Galdieria sulphuraria. Single-cell inductively coupled plasma mass spectrometry showed that the number of adsorbing cells and the concentration of adsorbed metal per cell varied depending on solution acidity and metal species. The X-ray absorption fine structure in 5 mM HCl solution indicated that the adsorbed Au formed inner-sphere complexes with S, whereas the adsorbed Pd and Pt formed an inner-sphere complexes with N and/or S. In 500 mM HCl solution, the adsorbed Au and Pd formed inner-sphere complexes only with S, and the Au formed a structure similar to Au2S. At higher acidity, Au and Pd were recovered by interacting with residues that formed more stable complexes, which was accompanied by changes in the behavior of cell populations adsorbing the metals. This is the first study to demonstrate the relationship between changes in the behavior of cell populations and chemical interactions that occur between substrate elements and biomaterial residues during biosorption. The findings of this study provide deeper insights into the biosorption mechanism and a background for the design of an environmentally friendly biosorbent.

Effect of lyophilization on the acid resistance of a unicellular red alga Galdieria sulphuraria during platinum recovery

The widespread use of platinum (Pt) in medical treatments and the industry has led to the increasing presence of low amounts of Pt in various wastewaters, and the sustainable recycling of Pt has garnered attention because of the associated economic and environmental advantages. Herein, we report that the lyophilization of the cells of a unicellular red alga, Galdieria sulphuraria, enabled the recovery of Pt with an efficiency over 90% from a 2 M hydrochloric acid (HCl) solution containing 10 mg/L Pt—comparable to the acid and Pt concentrations found in metal wastewater. Moreover, the lyophilized cells recovered only Pt, with 80% efficiency, from a 2 M HCl solution containing 10 mg/L concentrations of seven metals, and the selectivity of this method for Pt was higher than those of ion-exchange resins and activated carbons. Scanning electron-assisted dielectric microscopy showed that Pt was accumulated inside the cells, especially in chloroplasts. X-ray absorption near-edge structure revealed that Pt(IV) was reduced to Pt(II), forming an inner-sphere complex with S in the thiol residues in the lyophilized cells. The formation of an inner-sphere complex with S appears to increase the acid tolerance while retaining the high selectivity of lyophilized G. sulphuraria cells toward Pt.

Different Behaviour between Autotrophic and Heterotrophic Galdieria sulphuraria (Rhodophyta) Cells to Nitrogen Starvation and Restoration. Impact on Pigment and Free Amino Acid Contents

The unicellular red alga Galdieria sulphuraria is a polyextremophilic organism with a metabolic flexibility to grow autotrophically or heterotrophically. Galdieria can also produce and accumulate biotechnologically attractive products such as pigments (phycocyanin) and proteins. In this research we studied the effects of nitrogen starvation and its subsequent restoration on pigment and free amino acid contents both in photoautotrophic and heterotrophic cells. Following the nitrogen starvation, the levels of the primary photosynthetic pigments decreased both in autotrophic and heterotrophic cells, except for the chlorophyll a marginally diminished in heterotrophic cells. Ammonium supply to G. sulphuraria N starved cells caused a significant increase of total chlorophylls both in autotrophic and heterotrophic cells. It was observed how such increase was more rapid and marked in heterotrophic cells than in the autotrophic ones. Under N starvation, phycocyanin contents decreased in both autotrophic and in heterotrophic cells; however, after a time-lapse of 24 hours, they resulted significantly higher in heterotrophic cells. In Galdieria sulphuraria, like in other microalgae, free amino acid contents were profoundly dependent on nitrogen status of the cells but heterotrophic cells maintained much higher levels, especially of glutamate, respect to autotrophic ones. In general, cells grown in the presence and absence of light showed different responses toward N availability; in particular heterotrophic cells seemed to respond quicker to the ammonium restoration compared to autotrophic ones.

Cellular accumulation of cesium in the unicellular red alga Galdieria sulphuraria under mixotrophic conditions

As a consequence of the Fukushima Daiichi Nuclear Power Plant accident which occurred in Japan in 2011, huge amounts of radioactive cesium (Cs) spread across a large area at low concentrations. Bioremediation is known as an effective method for the recovery and concentration of metals at low concentrations. In the present study we showed that G. sulphuraria cells recovered 52 ± 15% of the Cs present in a potassium-deficient medium containing 30 μg L−1 Cs in 10 days. Because of the advantages in biotechnological use, G. sulphuraria is a good candidate for the decontamination of radioactive Cs. In G. sulphuraria cells, Cs was accumulated specifically under mixotrophic condition of the three different culture conditions used (autotorophic, mixotrophic, and heterotrophic condition). Cells in the early logarithmic phase of growth did not accumulate Cs, whereas those in the late logarithmic phase did. Cs accumulated in the cell exists mainly in a soluble form (e.g., salt, chelating complex).

Profiling of lipid and glycogen accumulations under different growth conditions in the sulfothermophilic red alga Galdieria sulphuraria

The unicellular red alga Galdieria sulphuraria grows efficiently and produces a large amount of biomass in acidic conditions at high temperatures. It has great potential to produce biofuels and other beneficial compounds without becoming contaminated with other organisms. In G. sulphuraria, biomass measurements and glycogen and lipid analyses demonstrated that the amounts and compositions of glycogen and lipids differed when cells were grown under autotrophic, mixotrophic, and heterotrophic conditions. Maximum biomass production was obtained in the mixotrophic culture. High amounts of glycogen were obtained in the mixotrophic cultures, while the amounts of neutral lipids were similar between mixotrophic and heterotrophic cultures. The amounts of neutral lipids were highest in red algae, including thermophiles. Glycogen structure and fatty acids compositions largely depended on the growth conditions.

Isolation and characterization of thermostable phycocyanin from Galdieria sulphuraria

Phycocyanin is a highly valuable pigmented protein synthesized by several species of cyanobacteria and red alga. In this study we demonstrate the production of thermostable phycocyanin from the unicellular red alga Galdieria sulphuraria. Phycocyanin was extracted by repeated freeze-thaw cycles and purified in a two-step process using ammonium sulfate fractionation, at 25% and 50% concentrations. Purified phycocyanin exhibited maximum absorbance at 620 nm, and the purity ratio (A620/A280) was found to be greater than 4. The recovery efficiency of phycocyanin from the crude extract was above 80%. In total, approximately 19 milligram pure phycocyanin was obtained from 3 g of wet cell mass of Galdieria sp. Subunits α and β of the protein were separated by SDS-PAGE and analyzed by MALDITOF mass spectrometry for identification, which confirmed that the isolated protein is phycocyanin. The molecular weight of α and β subunits of phycocyanin was found to be 17.6 and 18.4 kDa, respectively.

Structurally reduced monosaccharide transporters in an evolutionarily conserved red alga.

The unicellular red alga Galdieria sulphuraria is a facultative heterotrophic member of the Cyanidiaceae, a group of evolutionary highly conserved extremophilic red algae. Uptake of various sugars and polyols is accomplished by a large number of distinct plasma membrane transporters. We have cloned three transporters [GsSPT1 (G. sulphuraria sugar and polyol transporter 1), GsSPT2 and GsSPT4], followed their transcriptional regulation and assayed their transport capacities in the heterologous yeast system. SPT1 is a conserved type of sugar/H(+) symporter with 12 predicted transmembrane-spanning domains, whereas SPT2 and SPT4 represent monosaccharide transporters, characterized by only nine hydrophobic domains. Surprisingly, all three proteins are functional plasma membrane transporters, as demonstrated by genetic complementation of a sugar uptake-deficient yeast mutant. Substrate specificities were broad and largely redundant, except for glucose, which was only taken up by SPT1. Comparison of SPT1 and truncated SPT1(Delta1-3) indicated that the N-terminus of the protein is not required for sugar transport or substrate recognition. However, its deletion affected substrate affinity as well as maximal transport velocity and released the pH dependency of sugar uptake. In line with these results, uptake by SPT2 and SPT4 was active but not pH-dependent, making a H(+) symport mechanism unlikely for the truncated proteins. We postulate SPT2 and SPT4 as functional plasma membrane transporters in G. sulphuraria. Most likely, they originated from genes encoding active monosaccharide/H(+) symporters with 12 transmembrane-spanning domains.

Redox Regulation of Chloroplast Enzymes in Galdieria sulphuraria in View of Eukaryotic Evolution

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.

Accumulation of phycocyanin in heterotrophic and mixotrophic cultures of the acidophilic red alga Galdieria sulphuraria

The relationship between light intensity, nitrogen availability and pigmentation was investigated in mixotrophic and heterotrophic cultures of the unicellular red alga Galdieria sulphuraria 074G, a potential host for production of the blue pigment, phycocyanin (PC). During the exponential growth phase of batch cultures, G. sulphuraria 074G contained 2–4 mg phycocyanin per g dry weight. In carbon-limited and nitrogen-sufficient batch cultures grown in darkness, this value increased to 8–12 mg g −1 dry weight during the stationary phase, whereas the phycocyanin content in nitrogen-deficient cells decreased to values below 1 mg g −1 dry weight during stationary phase. Light intensities between 0 and 100 μmol photons m −2 s −1 had no influence on phycocyanin accumulation in mixotrophic cultures grown on glucose or fructose, while light stimulated phycocyanin synthesis in cultures grown on glycerol, in which the phycocyanin content in stationary phase was increased from 10 mg g −1 dry weight in darkness to 20 mg g −1 dry weight at a light intensity of 80 μmol photons m −2 s −1. At higher light intensities, less phycocyanin accumulated than at lower intensities, irrespective of the carbon substrate used. In carbon-limited continuous flow cultures grown on glucose or glycerol at a dilution rate of 0.63 day −1, corresponding to 50% of the maximum specific growth rate, the highest steady-state phycocyanin content of 15–28 mg g −1 dry weight was found at 65 μmol photons m −2 s −1. In contrast to the apparent glucose repression of light-induced PC synthesis observed in batch cultures, no glucose repression of the light stimulation was observed in continuous flow cultures because the glucose concentration in the culture supernatant always remained at limiting levels. Despite the fact that G. sulphuraria 074G contains less phycocyanin than some other microalgae and cyanobacteria, the ability of G. sulphuraria 074G to grow and synthesize phycocyanin in heterotrophic or mixotrophic cultures makes it an interesting alternative to the cyanobacterium, Spirulina platensis presently used for synthesis of phycocyanin.

Comparative Genomics of Two Closely Related Unicellular Thermo-Acidophilic Red Algae, Galdieria sulphuraria and Cyanidioschyzon merolae, Reveals the Molecular Basis of the Metabolic Flexibility of Galdieria sulphuraria and Significant Differences in Carbohydrate Metabolism of Both Algae

Unicellular algae serve as models for the study and discovery of metabolic pathways, for the functional dissection of cell biological processes such as organellar division and cell motility, and for the identification of novel genes and gene functions. The recent completion of several algal genome sequences and expressed sequence tag collections and the establishment of nuclear and organellar transformation methods has opened the way for functional genomics approaches using algal model systems. The thermo-acidophilic unicellular red alga Galdieria sulphuraria represents a particularly interesting species for a genomics approach owing to its extraordinary metabolic versatility such as heterotrophic and mixotrophic growth on more than 50 different carbon sources and its adaptation to hot acidic environments. However, the ab initio prediction of genes required for unknown metabolic pathways from genome sequences is not trivial. A compelling strategy for gene identification is the comparison of similarly sized genomes of related organisms with different physiologies. Using this approach, candidate genes were identified that are critical to the metabolic versatility of Galdieria. Expressed sequence tags and high-throughput genomic sequence reads covering >70% of the G. sulphuraria genome were compared to the genome of the unicellular, obligate photoautotrophic red alga Cyanidioschyzon merolae. More than 30% of the Galdieria sequences did not relate to any of the Cyanidioschyzon genes. A closer inspection of these sequences revealed a large number of membrane transporters and enzymes of carbohydrate metabolism that are unique to Galdieria. Based on these data, it is proposed that genes involved in the uptake of reduced carbon compounds and enzymes involved in their metabolism are crucial to the metabolic flexibility of G. sulphuraria.

Purification and characterization of UDP‐D‐galactose 4‐epimerase from the red alga Galdieria sulphuraria

UDP‐D‐galactose 4‐epimerase of the unicellular red alga Galdieria sulphuraria has been purified to apparent electrophoretic homogeneity by chromatography on DEAE‐Fractogel, hydroxylapatite and by affinity chromatography on Dyematrex Orange. The holoenzyme is a homodimer with an apparent molecular mass of 83 and 76 kDa as determined by gelfiltration and by sucrose gradient centrifugation, respectively. The size of the subunits was 42 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 4‐epimerase from G. sulphuraria does not require external NAD for activity, unlike the enzyme from some other organisms, and inhibition by NADH was not observed. The apparent Km for UDP‐D‐galactose was 64 μM. The pH optimum was at 8 and the apparent equilibrium constant for UDP‐Glc/UDP‐Gal was 3.5. The enzyme in crude as well as in purified samples was unusually stable and was not inactivated even on incubation at 46°C for several hours.

Organization of plastid-encoded ATPase genes and flanking regions including homologues of infB and tsf in the thermophilic red alga Galdieria sulphuraria.

We have cloned and sequenced the plastid ATPase operons (atp1 and atp2) and flanking regions from the unicellular red alga Galdieria sulphuraria (Cyanidium caldarium). Six genes (5 atpI, H, G, F, D and A 3) are linked in atp1 encoding ATPase subunits a, c, b, b, delta and alpha, respectively. The atpF gene does not contain an intron and overlaps atpD by 1 bp. As in the genome of chloroplasts from land plants, the cluster is located downstream of rps2, but between this gene and atp1 we found the gene for the prokaryotic translation elongation factor TS. Downstream of atpA, we detected two open reading frames, one encoding a putative transport protein. The genes atpB and atpE, encoding ATPase subunits beta and epsilon, respectively, are linked in atp2, separated by a 2 bp spacer. Upstream of atpB, an uninterrupted orf167 was detected which is homologous to an intron-containing open reading frame in land plant chloroplasts. This orf167 is preceded on the opposite DNA strand by a homologue to initiation factor 2 in prokaryotes. The arrangement of atp1 and atp2 is the same as observed in the multicellular red alga Antithamnion sp., indicating a conserved genome arrangement in the red algal plastid genome. Differences compared to green chloroplast genomes suggest a large phylogenetic distance between red algae and green plants, while similarities in arrangement and sequence to chromophytic ATPase operons support a red algal origin of chlorophyll a/c-containing plastids or alternatively point to a common prokaryotic endosymbiont.