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.

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.

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.

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.

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.

Deuterium and its impact on living organisms.

The rare stable isotope of hydrogen, deuterium, has fascinated researchers since its discovery in the 1930s. Subsequent large-scale production of deuterium oxide, commonly known as heavy water, became a starting point for further research. Deuterium exhibits unique physicochemical properties as well as having the strongest kinetic isotope effects among all other elements. Moreover, a broad variety of morphological and physiological changes have been observed in deuterium-treated cells and organisms, including changes in fundamental processes such as cell division or energy metabolism. Even though our understanding of such alterations is still insufficient, it is evident that some of them make growth in a deuterium-enriched environment a challenging task. There seems to be certain species-specific limits to their tolerance to heavy water, where some organisms are unable to grow in heavy water whilst others have no difficulties. Although the effects of deuterium on living organisms are, in general, negative, some of its applications are of great biotechnological potential, as is the case of stable isotope-labelled compounds or deuterated drugs.