Cultivation of the microalgae Chlamydomonas reinhardtii and Desmodesmus quadricauda in highly deuterated media: Balancing the light intensity.

The production of organic deuterated compounds in microalgal systems represents a cheaper and more versatile alternative to more complicated chemical synthesis. In the present study, we investigate the autotrophic growth of two microalgae, Chlamydomonas reinhardtii and Desmodesmus quadricauda, in medium containing high doses of deuterated water, D2O. The growth of such cultures was evaluated in the context of the intensity of incident light, since light is a critical factor in the management of autotrophic algal cultures. Deuteration increases the light sensitivity of both model organisms, resulting in increased levels of singlet oxygen and poorer photosynthetic performance. Our results also show a slowdown in growth and cell division processes with increasing D2O concentrations. At the same time, impaired cell division leads to cell enlargement and accumulation of highly deuterated compounds, especially energy-storing molecules. Thus, considering the specifics of highly deuterated cultures and using the growth conditions proposed in this study, it is possible to obtain highly deuterated algal biomass, which could be a valuable source of deuterated organic compounds.

The Effect of Variable Light Source and Light Intensity on the Growth of Three Algal Species.

Light is the essential energy source for autotrophically growing organisms, including microalgae. Both light intensity and light quality affect cell growth and biomass composition. Here we used three green algae-Chlamydomonas reinhardtii, Desmodesmus quadricauda, and Parachlorella kessleri-to study the effects of different light intensities and light spectra on their growth. Cultures were grown at three different light intensities (100, 250, and 500 µmol m-2 s-1) and three different light sources: fluorescent lamps, RGB LEDs, and white LEDs. Cultures of Desmodesmus quadricauda and Parachlorella kessleri were saturated at 250 µmol m-2 s-1, and further increasing the light intensity did not improve their growth. Chlamydomonas reinhardtii cultures did not reach saturation under the conditions used. All species usually divide into more than two daughter cells by a mechanism called multiple fission. Increasing light intensity resulted in an increase in maximum cell size and division into more daughter cells. In Parachlorella kessleri cells, the concentration of photosynthetic pigments decreased with light intensity. Different light sources had no effect on algal growth or photosynthetic pigments. The results show a species-specific response of algae to light intensity and support the use of any white light source for their cultivation without negative effects on growth.

Analysis of Commitment Point Attainment in Algae Dividing by Multiple Fission.

This work represents a detailed guide for commitment point analysis in microalgae dividing by multiple fission. The method is based on allowing the committed cells to divide in favorable conditions in the dark. This protocol offers a strategy to monitor cell cycle progression, both in control cultures and cultures treated with compounds affecting cell cycle length and/or progression. As the variety of such compounds is wide, our aim was to make the protocol easily modifiable to various research aims. The technique is easy to follow, low-cost, does not require any special equipment and offers reliable results in a reasonable time. The protocol offers step-by-step instructions, explains the theory behind these steps and offers solutions to some of the problems that may arise during the procedure.

Supra-Optimal Temperature: An Efficient Approach for Overaccumulation of Starch in the Green Alga Parachlorella kessleri.

Green algae are fast-growing microorganisms that are considered promising for the production of starch and neutral lipids, and the chlorococcal green alga Parachlorella kessleri is a favorable model, as it can produce both starch and neutral lipids. P. kessleri commonly divides into more than two daughter cells by a specific mechanism-multiple fission. Here, we used synchronized cultures of the alga to study the effects of supra-optimal temperature. Synchronized cultures were grown at optimal (30 °C) and supra-optimal (40 °C) temperatures and incident light intensities of 110 and 500 µmol photons m-2 s-1. The time course of cell reproduction (DNA replication, cellular division), growth (total RNA, protein, cell dry matter, cell size), and synthesis of energy reserves (net starch, neutral lipid) was studied. At 40 °C, cell reproduction was arrested, but growth and accumulation of energy reserves continued; this led to the production of giant cells enriched in protein, starch, and neutral lipids. Furthermore, we examined whether the increased temperature could alleviate the effects of deuterated water on Parachlorella kessleri growth and division; results show that supra-optimal temperature can be used in algal biotechnology for the production of protein, (deuterated) starch, and neutral lipids.

To Divide or Not to Divide? How Deuterium Affects Growth and Division of Chlamydomonas reinhardtii.

Extensive in vivo replacement of hydrogen by deuterium, a stable isotope of hydrogen, induces a distinct stress response, reduces cell growth and impairs cell division in various organisms. Microalgae, including Chlamydomonas reinhardtii, a well-established model organism in cell cycle studies, are no exception. Chlamydomonas reinhardtii, a green unicellular alga of the Chlorophyceae class, divides by multiple fission, grows autotrophically and can be synchronized by alternating light/dark regimes; this makes it a model of first choice to discriminate the effect of deuterium on growth and/or division. Here, we investigate the effects of high doses of deuterium on cell cycle progression in C. reinhardtii. Synchronous cultures of C. reinhardtii were cultivated in growth medium containing 70 or 90% D2O. We characterize specific deuterium-induced shifts in attainment of commitment points during growth and/or division of C. reinhardtii, contradicting the role of the "sizer" in regulating the cell cycle. Consequently, impaired cell cycle progression in deuterated cultures causes (over)accumulation of starch and lipids, suggesting a promising potential for microalgae to produce deuterated organic compounds.

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.

Characterization of Growth and Cell Cycle Events Affected by Light Intensity in the Green Alga Parachlorella kessleri: A New Model for Cell Cycle Research.

Multiple fission is a cell cycle variation leading to the production of more than two daughter cells. Here, we used synchronized cultures of the chlorococcal green alga Parachlorella kessleri to study its growth and pattern of cell division under varying light intensities. The time courses of DNA replication, nuclear and cellular division, cell size, total RNA, protein content, dry matter and accumulation of starch were observed at incident light intensities of 110, 250 and 500 µmol photons m-2s-1. Furthermore, we studied the effect of deuterated water on Parachlorella kessleri growth and division, to mimic the effect of stress. We describe a novel multiple fission cell cycle pattern characterized by multiple rounds of DNA replication leading to cell polyploidization. Once completed, multiple nuclear divisions were performed with each of them, immediately followed by protoplast fission, terminated by the formation of daughter cells. The multiple fission cell cycle was represented by several consecutive doublings of growth parameters, each leading to the start of a reproductive sequence. The number of growth doublings increased with increasing light intensity and led to division into more daughter cells. This study establishes the baseline for cell cycle research at the molecular level as well as for potential biotechnological applications, particularly directed synthesis of (deuterated) starch and/or neutral lipids as carbon and energy reserves.

Starch Production in Chlamydomonas reinhardtii through Supraoptimal Temperature in a Pilot-Scale Photobioreactor.

An increase in temperature can have a profound effect on the cell cycle and cell division in green algae, whereas growth and the synthesis of energy storage compounds are less influenced. In Chlamydomonas reinhardtii, laboratory experiments have shown that exposure to a supraoptimal temperature (39 °C) causes a complete block of nuclear and cellular division accompanied by an increased accumulation of starch. In this work we explore the potential of supraoptimal temperature as a method to promote starch production in C. reinhardtii in a pilot-scale photobioreactor. The method was successfully applied and resulted in an almost 3-fold increase in the starch content of C. reinhardtii dry matter. Moreover, a maximum starch content at the supraoptimal temperature was reached within 1-2 days, compared with 5 days for the control culture at the optimal temperature (30 °C). Therefore, supraoptimal temperature treatment promotes rapid starch accumulation and suggests a viable alternative to other starch-inducing methods, such as nutrient depletion. Nevertheless, technical challenges, such as bioreactor design and light availability within the culture, still need to be dealt with.

Comparing Biochemical and Raman Microscopy Analyses of Starch, Lipids, Polyphosphate, and Guanine Pools during the Cell Cycle of Desmodesmus quadricauda.

Photosynthetic energy conversion and the resulting photoautotrophic growth of green algae can only occur in daylight, but DNA replication, nuclear and cellular divisions occur often during the night. With such a light/dark regime, an algal culture becomes synchronized. In this study, using synchronized cultures of the green alga Desmodesmus quadricauda, the dynamics of starch, lipid, polyphosphate, and guanine pools were investigated during the cell cycle by two independent methodologies; conventional biochemical analyzes of cell suspensions and confocal Raman microscopy of single algal cells. Raman microscopy reports not only on mean concentrations, but also on the distribution of pools within cells. This is more sensitive in detecting lipids than biochemical analysis, but both methods-as well as conventional fluorescence microscopy-were comparable in detecting polyphosphates. Discrepancies in the detection of starch by Raman microscopy are discussed. The power of Raman microscopy was proven to be particularly valuable in the detection of guanine, which was traceable by its unique vibrational signature. Guanine microcrystals occurred specifically at around the time of DNA replication and prior to nuclear division. Interestingly, guanine crystals co-localized with polyphosphates in the vicinity of nuclei around the time of nuclear division.

Cell Cycle Arrest by Supraoptimal Temperature in the Alga Chlamydomonas reinhardtii.

Temperature is one of the key factors affecting growth and division of algal cells. High temperature inhibits the cell cycle in Chlamydomonas reinhardtii. At 39 °C, nuclear and cellular divisions in synchronized cultures were blocked completely, while DNA replication was partly affected. In contrast, growth (cell volume, dry matter, total protein, and RNA) remained unaffected, and starch accumulated at very high levels. The cell cycle arrest could be removed by transfer to 30 °C, but a full recovery occurred only in cultures cultivated up to 14 h at 39 °C. Thereafter, individual cell cycle processes began to be affected in sequence; daughter cell release, cell division, and DNA replication. Cell cycle arrest was accompanied by high mitotic cyclindependent kinase activity that decreased after completion of nuclear and cellular division following transfer to 30 °C. Cell cycle arrest was, therefore, not caused by a lack of cyclin-dependent kinase activity but rather a blockage in downstream processes.

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.

Effects of cyclin-dependent kinase activity on the coordination of growth and the cell cycle in green algae at different temperatures.

The progression of the cell cycle in green algae dividing by multiple fission is, under otherwise unlimited conditions, affected by the growth rate, set by a combination of light intensity and temperature. In this study, we compared the cell cycle characteristics of Desmodesmus quadricauda at 20 °C or 30 °C and upon shifts between these two temperatures. The duration of the cell cycle in cells grown under continuous illumination at 20 °C was more than double that at 30 °C, suggesting that it was set directly by the growth rate. Similarly, the amounts of DNA, RNA, and bulk protein content per cell at 20 °C were approximately double those of cells grown at the higher temperature. For the shift experiments, cells grown at either 20 °C or 30 °C were transferred to darkness to prevent further growth, and then cultivated at the same or the other temperature. Upon transfer to the lower temperature, fewer nuclei and daughter cells were produced, and not all cells were able to finish the cell cycle by division, remaining multinuclear. Correspondingly, cells placed in the dark at the higher temperature divided faster into more daughter cells than the control cells. These differences correlated with shifts in the preceding cyclin-dependent kinase activity, suggesting that cell cycle progression was not related to growth rate or cell biomass but correlated with cyclin-dependent kinase activity.

Stable isotope compounds - production, detection, and application.

Stable isotopes are used in wide fields of application from natural tracers in biology, geology and archeology through studies of metabolic fluxes to their application as tracers in quantitative proteomics and structural biology. We review the use of stable isotopes of biogenic elements (H, C, N, O, S, Mg, Se) with the emphasis on hydrogen and its heavy isotope deuterium. We will discuss the limitations of enriching various compounds in stable isotopes when produced in living organisms. Finally, we overview methods for measuring stable isotopes, focusing on methods for detection in single cells in situ and their exploitation in modern biotechnologies.

Growth of algal biomass in laboratory and in large-scale algal photobioreactors in the temperate climate of western Germany.

Growth of Chlorella vulgaris was characterized as a function of irradiance in a laboratory turbidostat (1L) and compared to batch growth in sunlit modules (5-25L) of the commercial NOVAgreen photobioreactor. The effects of variable sunlight and culture density were deconvoluted by a mathematical model. The analysis showed that algal growth was light-limited due to shading by external construction elements and due to light attenuation within the algal bags. The model was also used to predict maximum biomass productivity. The manipulative experiments and the model predictions were confronted with data from a production season of three large-scale photobioreactors: NOVAgreen (<36,000L), IGV (2,500-3,500L), and Phytolutions (28,000L). The analysis confirmed light-limitation in all three photobioreactors. An additional limitation of the biomass productivity was caused by the nitrogen starvation that was used to induce lipid accumulation. Reduction of shading and separation of biomass and lipid production are proposed for future optimization.

Deciphering the relationship among phosphate dynamics, electron-dense body and lipid accumulation in the green alga Parachlorella kessleri.

Phosphorus is an essential element for life on earth and is also important for modern agriculture, which is dependent on inorganic fertilizers from phosphate rock. Polyphosphate is a biological polymer of phosphate residues, which is accumulated in organisms during the biological wastewater treatment process to enhance biological phosphorus removal. Here, we investigated the relationship between polyphosphate accumulation and electron-dense bodies in the green alga Parachlorella kessleri. Under sulfur-depleted conditions, in which some symporter genes were upregulated, while others were downregulated, total phosphate accumulation increased in the early stage of culture compared to that under sulfur-replete conditions. The P signal was detected only in dense bodies by energy dispersive X-ray analysis. Transmission electron microscopy revealed marked ultrastructural variations in dense bodies with and without polyphosphate. Our findings suggest that the dense body is a site of polyphosphate accumulation, and P. kessleri has potential as a phosphate-accumulating organism.

The effect of lanthanides on photosynthesis, growth, and chlorophyll profile of the green alga Desmodesmus quadricauda.

Lanthanides (La, Gd, Nd, Ce) accumulated in the green alga Desmodesmus quadricauda but their intracellular localizations were distinctly different: lanthanum and gadolinium were localized in cytoplasm, while neodymium and cerium were in the chloroplast. The effect of lanthanum and neodymium, as representatives of these two groups, on growth, chlorophyll content and photosynthetic rate at different light intensities was studied. At the lowest light intensity used (50 µmol photons m-2 s-1), in the presence of lanthanides (Nd), growth was enhanced by as much as 36 % over lanthanide free control, and the photosynthetic rate increased by up to 300 %. At high light intensities (238, 460, and 750 µmol photons m-2 s-1), photosynthetic rate increased markedly, but there was no significant difference between rates in the presence or absence of lanthanides. However, growth, measured as a percentage of dry weight, if compared with lanthanide free control, increased at all light intensities (31, 39, and 20 %, respectively). The total amount of chlorophyll after lanthanide treatment increased by up to 21 % relative to the control culture, mainly due to an increase in the level of chlorophyll b. Addition of lanthanides caused a change in the chlorophyll a/b ratio from 4.583 in control cultivation, to 1.05. Possible mechanisms of lanthanide-induced photosynthetic change, alterations in photosynthetic structures, and increases in growth are discussed and compared with findings in higher plants. The hypothesis that the lanthanide effect could be due to formation of lanthanide-pheophytins was not confirmed as lanthanide pheophytins were not found in D. quadricauda. Furthermore, we have shown that the preferential incorporation of heavy isotopes of magnesium, namely 25Mg and 26Mg, into chlorophyll during photosynthesis that occurred in controls was diminished in the presence of lanthanides.

Highly efficient lipid production in the green alga Parachlorella kessleri: draft genome and transcriptome endorsed by whole-cell 3D ultrastructure.

BACKGROUND: Algae have attracted attention as sustainable producers of lipid-containing biomass for food, animal feed, and for biofuels. Parachlorella kessleri, a unicellular green alga belonging to the class Trebouxiophyceae, achieves very high biomass, lipid, and starch productivity levels. However, further biotechnological exploitation has been hampered by a lack of genomic information. RESULTS: Here, we sequenced the whole genome and transcriptome, and analyzed the behavior of P. kessleri NIES-2152 under lipid production-inducing conditions. The assembly includes 13,057 protein-coding genes in a 62.5-Mbp nuclear genome. Under conditions of sulfur deprivation, lipid accumulation was correlated with the transcriptomic induction of enzymes involved in sulfur metabolism, triacylglycerol (TAG) synthesis, autophagy, and remodeling of light-harvesting complexes. CONCLUSIONS: Three-dimensional transmission electron microscopy (3D-TEM) revealed extensive alterations in cellular anatomy accompanying lipid hyperaccumulation. The present 3D-TEM results, together with transcriptomic data support the finding that upregulation of TAG synthesis and autophagy are potential key mediators of the hyperaccumulation of lipids under conditions of nutrient stress.

Accumulation of energy reserves in algae: From cell cycles to biotechnological applications.

Starch and lipids are key components of algal cells and responsible for buffering variable supplies of energy and carbon that are vital for cell growth and reproduction, particularly DNA replication, nuclear and cellular division. The basic characteristics of energy reserves, their ultrastructure and localization inside the cell, regulation of their synthesis in relation to cell cycle phases, and their control by external factors, including light intensity, temperature, and carbon dioxide are described. Over the last two decades, research in this field has been boosted by possible biotechnological applications of algae for the production of biofuels from energy conserving compounds (bioethanol from starch and biodiesel from lipids). Recent findings on mechanisms that lead to an accumulation of exceptionally high levels of starch and lipids in algae will be summarized in this review. Macroelement (N, S, P) limitation, or depletion in mineral medium, as the most widely used approaches for enhancing both starch and lipid accumulation, are reviewed in detail. Potential biotechnological strategies for the economically viable overproduction of lipid and starch, such as a two-step procedure exploiting the effects of nutrient limitation and depletion, as well as the means and rationale for selecting appropriate strains, are discussed.

Use of lanthanides to alleviate the effects of metal ion-deficiency in Desmodesmus quadricauda (Sphaeropleales, Chlorophyta).

Lanthanides are biologically non-essential elements with wide applications in technology and industry. Their concentration as environmental contaminants is, therefore, increasing. Although non-essential, lanthanides have been proposed (and even used) to produce beneficial effects in plants, even though their mechanisms of action are unclear. Recently, it was suggested that they may replace essential elements. We tested the effect of low concentrations of lanthanides on the common freshwater microalga Desmodesmus quadricauda, grown under conditions of metal ion-deficiency (lower calcium or manganese concentrations). Our goal was to test if lanthanides can replace essential metals in their functions. Physiological stress was recorded by studying growth and photosynthetic activity using a pulse amplitude modulation (PAM) fluorimeter. We found that nutrient stress reduced parameters of growth and photosynthesis, such as maximal quantum yield, relative electron transport rate, photon capturing efficiency and light saturation irradiance. After adding low concentrations of five lanthanides, we confirmed that they can produce a stimulatory effect on microalgae, depending on the nutrient (metal) deprivation. In the case of a calcium deficit, the addition of lanthanides partly alleviated the adverse effects, probably by a partial substitution of the element. In contrast, with manganese deprivation (and at even lower concentrations), lanthanides enhanced the deleterious effect on cellular growth and photosynthetic competence. These results show that lanthanides can replace essential elements, but their effects on microalgae depend on stress and the nutritional state of the microalgae, raising the possibility of environmental impacts at even low concentrations.