Bio-removal of rare earth elements from hazardous industrial waste of CFL bulbs by the extremophile red alga Galdieria sulphuraria.

In recent decades, a shift has been seen in the use of light-emitting diodes over incandescent lights and compact fluorescent lamps (CFL), which eventually led to an increase in wastes of electrical equipment (WEE), especially fluorescent lamps (FLs) and CFL light bulbs. These widely used CFL lights, and their wastes are good sources of rare earth elements (REEs), which are desirable in almost every modern technology. Increased demand for REEs and their irregular supply have exerted pressure on us to seek alternative sources that may fulfill this demand in an eco-friendly manner. Bio-removal of wastes containing REEs, and their recycling may be a solution to this problem and could balance environmental and economic benefits. To address this problem, the current study focuses on the use of the extremophilic red alga, Galdieria sulphuraria, for bioaccumulation/removal of REEs from hazardous industrial wastes of CFL bulbs and the physiological response of a synchronized culture of G. sulphuraria. A CFL acid extract significantly affected growth, photosynthetic pigments, quantum yield, and cell cycle progression of this alga. A synchronous culture was able to efficiently accumulate REEs from a CFL acid extract and efficiency was increased by including two phytohormones, i.e., 6-Benzylaminopurine (BAP - Cytokinin family) and 1-Naphthaleneacetic acid (NAA - Auxin family).

Changes in glycosyl inositol phosphoceramides during the cell cycle of the red alga Galdieria sulphuraria.

Sphingolipids are significant component of plant-cell plasma membranes, as well as algal membranes, and mediate various biological processes. One of these processes is the change in lipid content during the cell cycle. This change is key to understanding cell viability and proliferation. There are relatively few papers describing highly glycosylated glycosyl inositol phosphorylceramide (GIPC) due to problems associated with the extractability of GIPCs and their analysis, especially in algae. After alkaline hydrolysis of total lipids from the red alga Galdieria sulphuraria, GIPCs were measured by high-resolution tandem mass spectrometry and fragmentation of precursor ions in an Orbitrap mass spectrometer in order to elucidate the structures of molecular species. Fragmentation experiments such as tandem mass spectrometry in the negative ion mode were performed to determine both the ceramide group and polar head structures. Measurement of mass spectra in the negative regime was possible because the phosphate group stabilizes negative molecular ions [M-H]-. ANALYSIS: of GIPCs at various stages of the cell cycle provided information on their abundance. It was found that, depending on the phases of the cell cycle, in particular during division, the uptake of all three components of GIPC, i.e., long-chain amino alcohols, fatty acids, and polar heads, changes. Structural modifications of the polar headgroup significantly increased the number of molecular species. Analysis demonstrated a convex characteristic for molecular species with only one saccharide (hexose or hexuronic acid) as the polar head. For two carbohydrates, the course of Hex-HexA was linear, while for HexA-HexA it was concave. The same was true for GIPC with three and four monosaccharides.

Improving microalgae for biotechnology - From genetics to synthetic biology - Moving forward but not there yet.

Microalgae are a diverse group of photosynthetic organisms that can be exploited for the production of different compounds, ranging from crude biomass and biofuels to high value-added biochemicals and synthetic proteins. Traditionally, algal biotechnology relies on bioprospecting to identify new highly productive strains and more recently, on forward genetics to further enhance productivity. However, it has become clear that further improvements in algal productivity for biotechnology is impossible without combining traditional tools with the arising molecular genetics toolkit. We review recent advantages in developing high throughput screening methods, preparing genome-wide mutant libraries, and establishing genome editing techniques. We discuss how algae can be improved in terms of photosynthetic efficiency, biofuel and high value-added compound production. Finally, we critically evaluate developments over recent years and explore future potential in the field.

Growth under Different Trophic Regimes and Synchronization of the Red Microalga Galdieria sulphuraria.

The extremophilic unicellular red microalga Galdieria sulphuraria (Cyanidiophyceae) is able to grow autotrophically, or mixo- and heterotrophically with 1% glycerol as a carbon source. The alga divides by multiple fission into more than two cells within one cell cycle. The optimal conditions of light, temperature and pH (500 µmol photons m-2 s-1, 40 °C, and pH 3; respectively) for the strain Galdieria sulphuraria (Galdieri) Merola 002 were determined as a basis for synchronization experiments. For synchronization, the specific light/dark cycle, 16/8 h was identified as the precondition for investigating the cell cycle. The alga was successfully synchronized and the cell cycle was evaluated. G. sulphuraria attained two commitment points with midpoints at 10 and 13 h of the cell cycle, leading to two nuclear divisions, followed subsequently by division into four daughter cells. The daughter cells stayed in the mother cell wall until the beginning of the next light phase, when they were released. Accumulation of glycogen throughout the cell cycle was also described. The findings presented here bring a new contribution to our general understanding of the cell cycle in cyanidialean red algae, and specifically of the biotechnologically important species G. sulphuraria.

The biosynthesis of phospholipids is linked to the cell cycle in a model eukaryote.

The structural challenges faced by eukaryotic cells through the cell cycle are key for understanding cell viability and proliferation. We tested the hypothesis that the biosynthesis of structural lipids is linked to the cell cycle. If true, this would suggest that the cell's structure is important for progress through and perhaps even control of the cell cycle. Lipidomics (31P NMR and MS), proteomics (Western immunoblotting) and transcriptomics (RT-qPCR) techniques were used to profile the lipid fraction and characterise aspects of its metabolism at seven stages of the cell cycle of the model eukaryote, Desmodesmus quadricauda. We found considerable, transient increases in the abundance of phosphatidylethanolamine during the G1 phase (+35%, ethanolamine phosphate cytidylyltransferase increased 2·5×) and phosphatidylglycerol (+100%, phosphatidylglycerol synthase increased 22×) over the G1/pre-replication phase boundary. The relative abundance of phosphatidylcholine fell by ~35% during the G1. N-Methyl transferases for the conversion of phosphatidylethanolamine into phosphatidylcholine were not found in the de novo transcriptome profile, though a choline phosphate transferase was found, suggesting that the Kennedy pathway is the principal route for the synthesis of PC. The fatty acid profiles of the four most abundant lipids suggested that these lipids were not generally converted between one another. This study shows for the first time that there are considerable changes in the biosynthesis of the three most abundant phospholipid classes in the normal cell cycle of D. quadricauda, by margins large enough to elicit changes to the physical properties of membranes.

Exploring Mycosporine-Like Amino Acids (MAAs) as Safe and Natural Protective Agents against UV-Induced Skin Damage.

Prolonged exposure to harmful ultraviolet radiation (UVR) can induce many chronic or acute skin disorders in humans. To protect themselves, many people have started to apply cosmetic products containing UV-screening chemicals alone or together with physical sunblocks, mainly based on titanium-dioxide (TiO2) or zinc-oxide (ZnO2). However, it has now been shown that the use of chemical and physical sunblocks is not safe for long-term application, so searches for the novel, natural UV-screening compounds derived from plants or bacteria are gaining attention. Certain photosynthetic organisms such as algae and cyanobacteria have evolved to cope with exposure to UVR by producing mycosporine-like amino acids (MAAs). These are promising substitutes for chemical sunscreens containing commercially available sunblock filters. The use of biopolymers such as chitosan for joining MAAs together or with MAA-Np (nanoparticles) conjugates will provide stability to MAAs similar to the mixing of chemical and physical sunscreens. This review critically describes UV-induced skin damage, problems associated with the use of chemical and physical sunscreens, cyanobacteria as a source of MAAs, the abundance of MAAs and their biotechnological applications. We also narrate the effectiveness and application of MAAs and MAA conjugates on skin cell lines.

Response of the Green Alga Chlamydomonas reinhardtii to the DNA Damaging Agent Zeocin.

DNA damage is a ubiquitous threat endangering DNA integrity in all living organisms. Responses to DNA damage include, among others, induction of DNA repair and blocking of cell cycle progression in order to prevent transmission of damaged DNA to daughter cells. Here, we tested the effect of the antibiotic zeocin, inducing double stranded DNA breaks, on the cell cycle of synchronized cultures of the green alga Chlamydomonas reinhardtii. After zeocin application, DNA replication partially occurred but nuclear and cellular divisions were completely blocked. Application of zeocin combined with caffeine, known to alleviate DNA checkpoints, decreased cell viability significantly. This was probably caused by a partial overcoming of the cell cycle progression block in such cells, leading to aberrant cell divisions. The cell cycle block was accompanied by high steady state levels of mitotic cyclin-dependent kinase activity. The data indicate that DNA damage response in C. reinhardtii is connected to the cell cycle block, accompanied by increased and stabilized mitotic cyclin-dependent kinase activity.

Bio-mining of Lanthanides from Red Mud by Green Microalgae.

Red mud is a by-product of alumina production containing lanthanides. Growth of green microalgae on red mud and the intracellular accumulation of lanthanides was tested. The best growing species was Desmodesmus quadricauda (2.71 cell number doublings/day), which accumulated lanthanides to the highest level (27.3 mg/kg/day), if compared with Chlamydomonas reinhardtii and Parachlorella kessleri (2.50, 2.37 cell number doublings and 24.5, 12.5 mg/kg per day, respectively). With increasing concentrations of red mud, the growth rate decreased (2.71, 2.62, 2.43 cell number doublings/day) due to increased shadowing of cells by undissolved red mud particles. The accumulated lanthanide content, however, increased in the most efficient alga Desmodesmus quadricauda within 2 days from zero in red-mud free culture to 12.4, 39.0, 54.5 mg/kg of dry mass at red mud concentrations of 0.03, 0.05 and 0.1%, respectively. Red mud alleviated the metal starvation caused by cultivation in incomplete nutrient medium without added microelements. Moreover, the proportion of lanthanides in algae grown in red mud were about 250, 138, 117% higher than in culture grown in complete nutrient medium at red mud concentrations of 0.03, 0.05, 0.1%. Thus, green algae are prospective vehicles for bio-mining or bio-leaching of lanthanides from red mud.

The microalga Parachlorella kessleri--a novel highly efficient lipid producer.

The alga Parachlorella kessleri, strain CCALA 255, grown under optimal conditions, is characterized by storage of energy in the form of starch rather than lipids. If grown in the complete medium, the cultures grew rapidly, producing large amounts of biomass in a relatively short time. The cells, however, contained negligible lipid reserves (1-10% of DW). Treatments inducing hyperproduction of storage lipids in P. kessleri biomass were described. The cultures were grown in the absence or fivefold decreased concentration of either nitrogen or phosphorus or sulfur. Limitation by all elements using fivefold or 10-fold diluted mineral medium was also tested. Limitation with any macroelement (nitrogen, sulfur, or phosphorus) led to an increase in the amount of lipids; nitrogen limitation was the most effective. Diluted nutrient media (5- or 10-fold) were identified as the best method to stimulate lipid overproduction (60% of DW). The strategy for lipid overproduction consists of the fast growth of P. kessleri culture grown in the complete medium to produce sufficient biomass (DW more than 10 g/L) followed by the dilution of nutrient medium to stop growth and cell division by limitation of all elements, leading to induction of lipid production and accumulation up to 60% DW. Cultivation conditions necessary for maximizing lipid content in P. kessleri biomass generated in a scale-up solar open thin-layer photobioreactor were described.

DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda.

DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase.

Chlamydomonas reinhardtii: duration of its cell cycle and phases at growth rates affected by temperature.

Synchronized cultures of the green alga Chlamydomonas reinhardtii were grown photoautotrophically under a wide range of environmental conditions including temperature (15-37 °C), different mean light intensities (132, 150, 264 µmol m⁻² s⁻¹), different illumination regimes (continuous illumination or alternation of light/dark periods of different durations), and culture methods (batch or continuous culture regimes). These variable experimental approaches were chosen in order to assess the role of temperature in the timing of cell division, the length of the cell cycle and its pre- and post-commitment phases. Analysis of the effect of temperature, from 15 to 37 °C, on synchronized cultures showed that the length of the cell cycle varied markedly from times as short as 14 h to as long as 36 h. We have shown that the length of the cell cycle was proportional to growth rate under any given combination of growth conditions. These findings were supported by the determination of the temperature coefficient (Q10), whose values were above the level expected for temperature-compensated processes. The data presented here show that cell cycle duration in C. reinhardtii is a function of growth rate and is not controlled by a temperature independent endogenous timer or oscillator, including a circadian one.

Glutathione peroxidase activity in the selenium-treated alga Scenedesmus quadricauda.

The function of selenium in an organism is mediated mostly by selenoproteins including glutathione peroxidase. Glutathione peroxidase is a potent anti-oxidative enzyme, scavenging a variety of peroxides. The green alga Scenedesmus quadricauda was used to investigate the relationship between the toxicity of selenium and the glutathione peroxidase activity. Selenium resistant strains SeIV and SeVI were synchronized and grown in high concentrations of Se (selenite or selenate). As a measure of selenium toxicity the EC(50) values were determined. During growth of the untreated wild type, glutathione peroxidase activity increased slightly and then declined gradually until the end of the cell cycle. A similar pattern was observed in untreated resistant strains and when resistant strains were grown in the presence of selenium in the oxidation state to which they were resistant. In the wild type cultivated with 50 mg Se L(-1) (selenite or selenate), activity increased to a high level and slowly declined until the end of the cell cycle. Similarly, activity increased in strains SeIV and SeVI when grown in the presence of selenium in the oxidation state to which they were not resistant. We followed the effect of selenium on the ultrastructure of S. quadricauda. After exposure to selenite, the chloroplast membranes of wild type were reorganized into thick bundles of thylakoids and the stroma became granulose. When selenate was added, the chloroplast of wild type had a fingerprint-like appearance, the stroma became less dense and starch production increased. In selenium resistant strains, when treated with the selenium form to which they were resistant, the chloroplast was affected, but not to such an extent as in the wild type. The activity of glutathione peroxidase in Scenedesmus was affected by selenium in an oxidation state-dependent manner. The most apparent effects of selenium on the ultrastructure involved impairment of the chloroplast and the overproduction of starch.

Chlamydomonas reinhardtii: duration of its cell cycle and phases at growth rates affected by light intensity.

In the cultures of the alga Chlamydomonas reinhardtii, division rhythms of any length from 12 to 75 h were found at a range of different growth rates that were set by the intensity of light as the sole source of energy. The responses to light intensity differed in terms of altered duration of the phase from the beginning of the cell cycle to the commitment to divide, and of the phase after commitment to cell division. The duration of the pre-commitment phase was determined by the time required to attain critical cell size and sufficient energy reserves (starch), and thus was inversely proportional to growth rate. If growth was stopped by interposing a period of darkness, the pre-commitment phase was prolonged corresponding to the duration of the dark interval. The duration of the post-commitment phase, during which the processes leading to cell division occurred, was constant and independent of growth rate (light intensity) in the cells of the same division number, or prolonged with increasing division number. It appeared that different regulatory mechanisms operated through these two phases, both of which were inconsistent with gating of cell division at any constant time interval. No evidence was found to support any hypothetical timer, suggested to be triggered at the time of daughter cell release.

Bioaccumulation and toxicity of selenium compounds in the green alga Scenedesmus quadricauda.

BACKGROUND: Selenium is a trace element performing important biological functions in many organisms including humans. It usually affects organisms in a strictly dosage-dependent manner being essential at low and toxic at higher concentrations. The impact of selenium on mammalian and land plant cells has been quite extensively studied. Information about algal cells is rare despite of the fact that they could produce selenium enriched biomass for biotechnology purposes. RESULTS: We studied the impact of selenium compounds on the green chlorococcal alga Scenedesmus quadricauda. Both the dose and chemical forms of Se were critical factors in the cellular response. Se toxicity increased in cultures grown under sulfur deficient conditions. We selected three strains of Scenedesmus quadricauda specifically resistant to high concentrations of inorganic selenium added as selenite (Na2SeO3) - strain SeIV, selenate (Na2SeO4) - strain SeVI or both - strain SeIV+VI. The total amount of Se and selenomethionine in biomass increased with increasing concentration of Se in the culturing media. The selenomethionine made up 30-40% of the total Se in biomass. In both the wild type and Se-resistant strains, the activity of thioredoxin reductase, increased rapidly in the presence of the form of selenium for which the given algal strain was not resistant. CONCLUSION: The selenium effect on the green alga Scenedesmus quadricauda was not only dose dependent, but the chemical form of the element was also crucial. With sulfur deficiency, the selenium toxicity increases, indicating interference of Se with sulfur metabolism. The amount of selenium and SeMet in algal biomass was dependent on both the type of compound and its dose. The activity of thioredoxin reductase was affected by selenium treatment in dose-dependent and toxic-dependent manner. The findings implied that the increase in TR activity in algal cells was a stress response to selenium cytotoxicity. Our study provides a new insight into the impact of selenium on green algae, especially with regard to its toxicity and bioaccumulation.

Accumulation, activity and localization of cell cycle regulatory proteins and the chloroplast division protein FtsZ in the alga Scenedesmus quadricauda under inhibition of nuclear DNA replication.

Synchronized cultures of the green alga Scenedesmus quadricauda were grown in the absence (untreated cultures) or in the presence (FdUrd-treated cultures) of 5-fluorodeoxyuridine, the specific inhibitor of nuclear DNA replication. The attainment of commitment points, at which the cells become committed to nuclear DNA replication, mitosis and cellular division, and the course of committed processes themselves were determined for cell cycle characterization. FdUrd-treated cultures showed nearly unaffected growth and attainment of the commitment points, while DNA replication(s), nuclear division(s) and protoplast fission(s) were blocked. Interestingly, the FdUrd-treated cells possessed a very high mitotic histone H1 kinase activity in the absence of any nuclear division(s). Compared with the untreated cultures, the kinase activity as well as mitotic cyclin B accumulation increased continuously to high values without any oscillation. Division of chloroplasts was not blocked but occurred delayed and over a longer time span than in the untreated culture. The FtsZ protein level in the FdUrd-treated culture did not exceed the level in the untreated culture, but rather, in contrast to the untreated culture, remained elevated. FtsZ structures were both localized around pyrenoids and spread inside of the chloroplast in the form of spots and mini-rings. The abundance and localization of the FtsZ protein were comparable in untreated and FdUrd-treated cells until the end of the untreated cell cycle. However, in the inhibitor-treated culture, the signal did not decrease and was localized in intense spots surrounding the chloroplast/cell perimeter; this was in agreement with both the elevated protein level and persisting chloroplast division.