Shuhei Ota

Diatoms function as major primary producers, accumulating large amounts of biomass in most aquatic environments. Given their rapid responses to changes in environmental conditions, diatoms are used for the biological monitoring of water quality and for performing ecotoxicological tests in aquatic ecosystems. However, the molecular basis for their toxicity to chemical compounds remains largely unknown. Here, we sequenced the genome of a freshwater diatom, Mayamaea pseudoterrestris NIES-4280, which has been proposed as an alternative strain of Navicula pelliculosa UTEX 664 for performing the Organisation for Economic Co-operation and Development ecotoxicological test. This study shows that M. pseudoterrestris has a small genome and carries the lowest number of genes among freshwater diatoms. The gene content of M. pseudoterrestris is similar to that of the model marine diatom, Phaeodactylum tricornutum. Genes related to cell motility, polysaccharide metabolism, oxidative stress alleviation, intracellular calcium signalling, and reactive compound detoxification showed rapid changes in their expression patterns in response to copper exposure. Active gliding motility was observed in response to copper addition, and copper exposure decreased intracellular calcium concentration. These findings enhance our understanding of the environmental adaptation of diatoms, and elucidate the molecular basis of toxicity of chemical compounds in algae.

Seafloor resource development in the future is expected to be accompanied by the mining of metal ores, as these sulfide ores contain valuable metals. However, the mining process is detrimental to marine oceanic environments. As a precautionary approach, innovations in the collection of environmental baseline data and new onboard assessment methods for marine environmental impacts are required. Due to the low cell density in open ocean water, techniques for rapid impact assessment of seawater without sample concentration are required. The purpose of this study was to establish a heavy metal impact assessment system for seawater samples of marine microbes using a portable flow cytometer, On-chip Sort. We established a protocol for detecting heavy metal-induced damage to cells via propidium iodide (PI) staining using algal culture strains (Bathycoccus prasinos NIES-2670, Synechococcus sp. NIES-969, Prochlorococcus sp. NIES-2885, and Cyanobium sp. NIES-981) obtained from the marine environment maintained at the Microbial Culture Collection of the National Institute for Environmental Studies, Tsukuba, Japan. Results showed that PI staining could detect the effects of heavy metals on cells. The proportion of PI-positive cells increased with an increase in the concentration of heavy metal mixture or copper exposure. Compared to cyanobacteria, damaged cells of eukaryotic algae were detected. Therefore, the effects of heavy metals on both eukaryotic and prokaryotic algae can be rapidly assessed via PI-based flow cytometry using samples containing low cell densities.

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.

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.

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SUMMARY: Seafloor massive sulfide (SMS) deposits are the target of available metallic resources. The toxic impacts of leachable metals from hydrothermal ore by mining operations in marine environments are a concern. However, ecotoxicological knowledge about marine algae, and particularly open ocean species, is still limited. Here, we evaluated the toxic effects of three leachable metals (i.e. Zn, Cu, and Pb) on seven marine algae, including cyanobacteria and eukaryotes, by a delayed fluorescence method. Cyanobacterial Synechococcus and Cyanobium species were sensitive to Zn and Cu, while eukaryotic algae showed various responses to heavy metal species. The prasinophycean Bathycoccus prasinos NIES‐2670 was sensitive to all metal species; this strain is a potential test strain to detect the leachable metals. A co‐culture experiment showed that the impact on community structure varies depending on leachable metal species. This study demonstrates that surveys across multiple taxonomic groups are necessary to assess the impact of SMS‐mining operations on marine ecosystems as a whole.

Phycology has developed alongside light and electron microscopy techniques. Since the 1950s, progress in the field has accelerated dramatically with the advent of electron microscopy. Transmission electron microscopes can only acquire imaging data on a 2D plane. Currently, many of the life sciences are seeking to obtain 3D images with electron microscopy for the accurate interpretation of subcellular dynamics. Three-dimensional reconstruction using serial sections is a method that can cover relatively large cells or tissues without requiring special equipment. Another challenge is monitoring secondary metabolites (such as lipids or carotenoids) in intact cells. This became feasible with hyperspectral cameras, which enable the acquisition of wide-range spectral information in living cells. Here, we review bioimaging studies on the intracellular dynamics of substances such as lipids, carotenoids and phosphorus using conventional to state-of-the-art microscopy techniques in the field of algal biorefining.