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.
- MeSH
- buněčné dělení * MeSH
- buněčný cyklus * MeSH
- Viridiplantae MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
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.
- MeSH
- biomasa * MeSH
- bioreaktory MeSH
- buněčný cyklus MeSH
- Chlamydomonas reinhardtii metabolismus MeSH
- fotobioreaktory * MeSH
- kultivační média MeSH
- mikrořasy MeSH
- průmyslová mikrobiologie metody MeSH
- škrob metabolismus MeSH
- světlo MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- buněčná stěna chemie MeSH
- buněčný cyklus * MeSH
- časové faktory MeSH
- Chlorophyta cytologie růst a vývoj MeSH
- guanin analýza MeSH
- lipidová tělíska metabolismus MeSH
- lipidy analýza MeSH
- mikroskopie * MeSH
- polyfosfáty analýza MeSH
- Ramanova spektroskopie * MeSH
- škrob analýza MeSH
- velikost buňky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
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.
- MeSH
- bílkoviny řas metabolismus MeSH
- buněčné kultury metody MeSH
- Chlamydomonas reinhardtii cytologie fyziologie MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- down regulace MeSH
- fyziologický stres MeSH
- kontrolní body buněčného cyklu * MeSH
- regulace genové exprese u rostlin MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- bleomycin toxicita MeSH
- Chlamydomonas reinhardtii účinky léků genetika MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- cytostatické látky toxicita MeSH
- DNA rostlinná účinky léků MeSH
- dvouřetězcové zlomy DNA MeSH
- kofein farmakologie MeSH
- kontrolní body buněčného cyklu MeSH
- mutageny toxicita MeSH
- replikace DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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 Parachlorellakessleri (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 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 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.
- MeSH
- biotechnologie metody MeSH
- Chlorophyta růst a vývoj metabolismus MeSH
- deuterium aplikace a dávkování škodlivé účinky analýza MeSH
- hmotnostní spektrometrie metody MeSH
- izotopové značení metody MeSH
- izotopy analýza chemie MeSH
- magnetická rezonanční spektroskopie MeSH
- racionální návrh léčiv * MeSH
- Ramanova spektroskopie metody MeSH
- rostliny účinky léků MeSH
- savci MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
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.
- MeSH
- biomasa * MeSH
- Chlorella vulgaris MeSH
- fotobioreaktory * MeSH
- mikrořasy MeSH
- podnebí MeSH
- Publikační typ
- časopisecké články MeSH
