Physiological, Transcriptome, and Metabolome Analyses Reveal the Tolerance to Cu Toxicity in Red Macroalgae Gracilariopsis lemaneiformis.

Heavy metal copper (Cu) will inevitably impact the marine macroalgae Gracilariopsis lemaneiformis (G. lemaneiformis), which is a culture of economic importance along China's coastline. In this study, the detoxification mechanism of Cu stress on G. lemaneiformis was revealed by assessing physiological indicators in conjunction with transcriptome and metabolome analyses at 1 d after Cu stress. Our findings revealed that 25 µM Cu stimulated ROS synthesis and led to the enzymatic oxidation of arachidonic acid residues. This process subsequently impeded G. lemaneiformis growth by suppressing photosynthesis, nitrogen metabolism, protein synthesis, etc. The entry of Cu ions into the algae was facilitated by ZIPs and IRT transporters, presenting as Cu2+. Furthermore, there was an up-regulation of Cu efflux transporters HMA5 and ABC family transporters to achieve compartmentation to mitigate the toxicity. The results revealed that G. lemaneiformis elevated the antioxidant enzyme superoxide dismutase and ascorbate-glutathione cycle to maintain ROS homeostasis. Additionally, metabolites such as flavonoids, 3-O-methylgallic acid, 3-hydroxy-4-keto-gama-carotene, and eicosapentaenoic acid were up-regulated compared with the control, indicating that they might play roles in response to Cu stress. In summary, this study offers a comprehensive insight into the detoxification mechanisms driving the responses of G. lemaneiformis to Cu exposure.

Impacts of microplastic and seawater acidification on unicellular red algae: Growth response, photosynthesis, antioxidant enzymes, and extracellular polymer substances.

Microplastics (MPs) pollution and seawater acidification have increasingly become huge threats to the ocean ecosystem. Their impacts on microalgae are of great importance, since microalgae are the main primary producers and play a critical role in marine ecosystems. However, the impact of microplastics and acidification on unicellular red algae, which have a unique phycobiliprotein antenna system, remains unclear. Therefore, the impacts of polystyrene-MPs alone and the combined effects of MPs and seawater acidification on the typical unicellular marine red algae Porphyridium purpureum were investigated in the current study. The result showed that, under normal seawater condition, microalgae densities were increased by 17.75-41.67 % compared to the control when microalgae were exposed to small-sized MPs (0.1 µm) at concentrations of 5-100 mg L-1. In addition, the photosystem II and antioxidant enzyme system were not subjected to negative effects. The large-sized MPs (1 µm) boosted microalgae growth at a low concentration of MPs (5 mg L-1). However, it was observed that microalgae growth was significantly inhibited when MPs concentration increased up to 50 and 100 mg L-1, accompanied by the remarkably reduced Fv/Fm value and the elevated levels of SOD, CAT enzymes, phycoerythrin (PE), and extracellular polysaccharide (EPS). Compared to the normal seawater condition, microalgae densities were enhanced by 52.11-332.56 % under seawater acidification, depending on MPs sizes and concentrations, due to the formed CO2-enrichment condition and appropriate pH range. PE content in microalgal cells was significantly enhanced, but SOD and CAT activities as well as EPS content markedly decreased under acidification conditions. Overall, the impacts of seawater acidification were more pronounced than MPs impacts on microalgae growth and physiological responses. These findings will contribute to a substantial understanding of the effects of MPs on marine unicellular red microalgae, especially in future seawater acidification scenarios.

The response of Pyropia haitanensis to inorganic arsenic under laboratory culture.

Up to now, complicated organoarsenicals were mainly identified in marine organisms, suggesting that these organisms play a critical role in arsenic biogeochemical cycling because of low phosphate and relatively high arsenic concentration in the marine environment. However, the response of marine macroalgae to inorganic arsenic remains unknown. In this study, Pyropia haitanensis were exposed to arsenate [As(V)] (0.1, 1, 10, 100 µM) or arsenite [As(III)] (0.1, 1, 10 µM) under laboratory conditions for 3 d. The species of water-soluble arsenic, the total concentration of lipid-soluble and cell residue arsenic of the algae cells was analyzed. As(V) was mainly transformed into oxo-arsenosugar-phosphate, with other arsenic compounds such as monomethylated, As(III), demethylated arsenic and oxo-arsenosugar-glycerol being likely the intermediates of arsenosugar synthesis. When high concentration of As(III) was toxic to P. haitanensis, As(III) entered into the cells and was transformed into less toxic organoarsenicals and As(V). Transcriptome results showed genes involved in DNA replication, mismatch repair, base excision repair, and nucleotide excision repair were up-regulated in the algae cells exposed to 10 µM As(V), and multiple genes involved in glutathione metabolism and photosynthetic were up-regulated by 1 µM As(III). A large number of ABC transporters were down-regulated by As(V) while ten genes related to ABC transporters were up-regulated by As(III), indicating that ABC transporters were involved in transporting As(III) to vacuoles in algae cells. These results indicated that P. haitanensis detoxifies inorganic arsenic via transforming them into organoarsenicals and enhancing the isolation of highly toxic As(III) in vacuoles.

Efficient open cultivation of cyanidialean red algae in acidified seawater.

Microalgae possess high potential for producing pigments, antioxidants, and lipophilic compounds for industrial applications. However, their open pond cultures are often contaminated by other undesirable organisms, including their predators. In addition, the cost of using freshwater is relatively high, which limits the location and scale of cultivation compared with using seawater. It was previously shown that Cyanidium caldarium and Galdieria sulphuraria, but not Cyanidioschyzon merolae grew in media containing NaCl at a concentration equivalent to seawater. We found that the preculture of C. merolae in the presence of a moderate NaCl concentration enabled the cells to grow in the seawater-based medium. The cultivation of cyanidialean red algae in the seawater-based medium did not require additional pH buffering chemicals. In addition, the combination of seawater and acidic conditions reduced the risk of contamination by other organisms in the nonsterile open culture of C. merolae more efficiently than the acidic condition alone.

Pyropia yezoensis genome reveals diverse mechanisms of carbon acquisition in the intertidal environment.

Changes in atmospheric CO2 concentration have played a central role in algal and plant adaptation and evolution. The commercially important red algal genus, Pyropia (Bangiales) appears to have responded to inorganic carbon (Ci) availability by evolving alternating heteromorphic generations that occupy distinct habitats. The leafy gametophyte inhabits the intertidal zone that undergoes frequent emersion, whereas the sporophyte conchocelis bores into mollusk shells. Here, we analyze a high-quality genome assembly of Pyropia yezoensis to elucidate the interplay between Ci availability and life cycle evolution. We find horizontal gene transfers from bacteria and expansion of gene families (e.g. carbonic anhydrase, anti-oxidative related genes), many of which show gametophyte-specific expression or significant up-regulation in gametophyte in response to dehydration. In conchocelis, the release of HCO3- from shell promoted by carbonic anhydrase provides a source of Ci. This hypothesis is supported by the incorporation of 13C isotope by conchocelis when co-cultured with 13C-labeled CaCO3.

Effects of Cadmium on Bioaccumulation, Bioabsorption, and Photosynthesis in Sarcodia suiae.

This study investigated the changes in bioaccumulation, bioabsorption, photosynthesis rate, respiration rate, and photosynthetic pigments (phycoerythrin, phycocyanin, and allophycocyanin) of Sarcodia suiae following cadmium exposure within 24 h. The bioabsorption was significantly higher than the bioaccumulation at all cadmium levels (p < 0.05). The ratios of bioabsorption/bioaccumulation in light and dark bottles were 2.17 and 1.74, respectively, when S. suiae was exposed to 5 Cd2+ mg/L. The chlorophyll a (Chl-a) concentration, oxygen evolution rate (photosynthetic efficiency), and oxygen consumption rate (respiratory efficiency) decreased with increasing bioaccumulation and ambient cadmium levels. The levels of bioaccumulation and bioabsorption in light environments were significantly higher than those in dark environments (p < 0.05). In addition, the ratios of phycoerythrin (PE)/Chl-a, phycocyanin (PC)/Chl-a, and allophycocyanin (APC)/Chl-a were also higher in light bottles compared to dark bottles at all ambient cadmium levels. These results indicated that the photosynthesis of seaweed will increase bioaccumulation and bioabsorption in a cadmium environment.

Physiological damages of Sargassum cymosum and Hypnea pseudomusciformis exposed to trace metals from mining tailing.

The damages of Mariana's mining mud in the physiology of the brown algae Sargassum cymosum and its main epiphytic, the red algae Hypnea pseudomusciformis, were evaluated by controlled essays. Seaweeds were exposed to presence or absence of mud, isolated or in biological association, for 5 and 15 days. Measured parameters were growth rates, biochemical descriptors, and the chemical investigation of concentration and metal profile of the mud dissolved in seawater. Results showed that the highest values for metals were Al > Fe > Mn > Zn in both exposure periods. The mud also affected the growth rate with lethality in both isolated and associative treatments with H. pseudomusciformis after 15 days. According to our redundancy analysis (RDA), the profile and concentration of all metallic elements can induce different physiological responses of the organisms. We were able to observe a higher physiological adaptive ability of S. cymosum against the long-term presence of metals by the synthesis of phenolic compounds, while the deviation of metabolic routes in H. pseudomusciformis can be addressed as the main responsible for its lethality. Moreover, the presence of Hypnea in associative treatments reduces Sargassum's detoxification ability. The present results reinforce the importance of biological interaction studies in a context of physiological resilience against mining mud pollution and mutual influences of species over the individual ability to avoid oxidative stress.

Lability in sulfur acidic cultivation medium explains unstable effects of CDK inhibitors on Cyanidioschyzon merolae cell proliferation.

We previously showed that nuclear DNA replication (NDR) is regulated by a checkpoint monitoring the occurrence of organelle DNA replication (ODR) in a unicellular red alga Cyanidioschyzon merolae. These analyses depended on the use of chemical CDK inhibitors such as CDK2 inhibitor II and roscovitine, but subsequent analyses yielded conflicting results depending on the experimental conditions. In the present study, we identified significantly short half-lives of the used chemicals in the sulfur acidic cultivation medium, which reconciles the discrepancy among these results.

Participation of actin filaments, myosin and phosphatidylinositol 3-kinase in the formation and polarisation of tetraspore germ tube of Gelidium floridanum (Rhodophyta, Florideophyceae).

This study aimed to examine the evidence of direct interaction among actin, myosin and phosphatidylinositol 3-kinase (PI3K) in the polarisation and formation of the tetraspore germ tube of Gelidium floridanum. After release, tetraspores were exposed to cytochalasin B, latrunculin B, LY294002 and BDM for a period of 6 h. In control samples, formation of the germ tube occurred after the experimental period, with cellulose formation and elongated chloroplasts moving through the tube region in the presence of F-actin. In the presence of cytochalasin B, an inhibitor of F-actin, latrunculin B, an inhibitor of G-actin, and BDM, a myosin inhibitor, tetraspores showed no formation of the germ tube or cellulose. Spherical-shaped chloroplasts were observed in the central region with a few F-actin filaments in the periphery of the cytoplasm. Tetraspores treated with LY294002, a PI3K inhibitor, showed no formation of the tube at the highest concentrations. Polarisation of cytoplasmic contents did not occur, only cellulose formation. It was concluded that F-actin directs the cell wall components and contributes to the maintenance of chloroplast shape and elongation during germ tube formation. PI3K plays a fundamental role in signalling for the asymmetric polarisation of F-actin. Thus, F-actin regulates the polarisation and germination processes of tetraspores of G. floridanum.

The impact of elevated atmospheric CO2 on cadmium toxicity in Pyropia haitanensis (Rhodophyta).

Cadmium is one of the major heavy metal pollutions in coastal waters, and it is well known that cadmium at trace concentration is toxic to macroalgae. Change in marine carbonate system and ocean acidification caused by elevated atmospheric CO2 also alter physiological characteristics of macroalgae. However, less research is focused on the combined impacts of elevated CO2 and cadmium pollution on the growth and physiology in macroalgae. In this study, the maricultivated macroalga Pyropia haitanensis (Rhodophyta) was cultured at three levels of Cd2+ (control, 4 and 12 mg L-1) and two concentrations of CO2, the ambient CO2 (AC, 410 ppm) and elevated CO2 (HC, 1100 ppm). The results showed that 12 mg L-1 Cd2+ significantly suppressed the relative growth rate and superoxide dismutase activity in AC-grown P. haitanensis, while such inhibition extents by Cd2+ were alleviated in HC-grown algae. Cd2+ had no effects on efficiency of electron transport (α) and maximum electron transport rate (ETRmax), but α was increased by elevated CO2. Cd2+ dramatically suppressed the maximum net photosynthesis oxygen evolution rate (NPRm) and the minimum saturation irradiance (Ik) when the algal thalli were grown at AC, while such suppression of NPRm by Cd2+ was much decreased when the thalli were grown at HC. Collectively, our results suggested that elevated CO2 would alleviate Cd2+ toxicity on P. haitanensis.

Transcriptional response of the extremophile red alga Cyanidioschyzon merolae to changes in CO2 concentrations.

Cyanidioschyzon merolae (C. merolae) is an acidophilic red alga growing in a naturally low carbon dioxide (CO2) environment. Although it uses a ribulose 1,5-bisphosphate carboxylase/oxygenase with high affinity for CO2, the survival of C. merolae relies on functional photorespiratory metabolism. In this study, we quantified the transcriptomic response of C. merolae to changes in CO2 conditions. We found distinct changes upon shifts between CO2 conditions, such as a concerted up-regulation of photorespiratory genes and responses to carbon starvation. We used the transcriptome data set to explore a hypothetical CO2 concentrating mechanism in C. merolae, based on the assumption that photorespiratory genes and possible candidate genes involved in a CO2 concentrating mechanism are co-expressed. A putative bicarbonate transport protein and two α-carbonic anhydrases were identified, which showed enhanced transcript levels under reduced CO2 conditions. Genes encoding enzymes of a PEPCK-type C4 pathway were co-regulated with the photorespiratory gene cluster. We propose a model of a hypothetical low CO2 compensation mechanism in C. merolae integrating these low CO2-inducible components.

Comparative transcriptional profiling of Gracilariopsis lemaneiformis in response to salicylic acid- and methyl jasmonate-mediated heat resistance.

Culturing the economically important macroalga Gracilariopsis lemaneiformis (Rhodophyta) is limited due to the high temperatures in the summertime on the southern Chinese coast. Previous studies have demonstrated that two phytohormones, salicylic acid (SA) and methyl jasmonate (MJ), can alleviate the adverse effects of high-temperature stress on Gp. lemaneiformis. To elucidate the molecular mechanisms underlying SA- and MJ-mediated heat tolerance, we performed comprehensive analyses of transcriptome-wide gene expression profiles using RNA sequencing (RNA-seq) technology. A total of 14,644 unigenes were assembled, and 10,501 unigenes (71.71%) were annotated to the reference databases. In the SA, MJ and SA/MJ treatment groups, 519, 830, and 974 differentially expressed unigenes were detected, respectively. Unigenes related to photosynthesis and glycometabolism were enriched by SA, while unigenes associated with glycometabolism, protein synthesis, heat shock and signal transduction were increased by MJ. A crosstalk analysis revealed that 216 genes were synergistically regulated, while 18 genes were antagonistically regulated by SA and MJ. The results indicated that the two phytohormones could mitigate the adverse effects of heat on multiple pathways, and they predominantly acted synergistically to resist heat stress. These results will provide new insights into how SA and MJ modulate the molecular mechanisms that counteract heat stress in algae.

Increased iron availability resulting from increased CO2 enhances carbon and nitrogen metabolism in the economical marine red macroalga Pyropia haitanensis (Rhodophyta).

Ocean acidification caused by rising CO2 is predicted to increase the concentrations of dissolved species of Fe(II) and Fe(III), leading to the enhanced photosynthetic carbon sequestration in some algal species. In this study, the carbon and nitrogen metabolism in responses to increased iron availability under two CO2 levels (390 µL L-1 and 1000 µL L-1), were investigated in the maricultivated macroalga Pyropia haitanensis (Rhodophyta). The results showed that, elevated CO2 increased soluble carbonhydrate (SC) contents, resulting from enhanced photosynthesis and photosynthetic pigment synthesis in this algae, but declined its soluble protein (SP) contents, resulting in increased ratio of SC/SP. This enhanced photosynthesis performance and carbon accumulation was more significant under iron enrichment condition in seawater, with higher iron uptake rate at high CO2 level. As a key essential biogenic element for algae, Fe-replete functionally contributed to P. haitanensis photosynthesis. Increased SC fundamentally provided carbon skeletons for nitrogen assimilation. The significant increase of carbon and nitrogen assimilation finally contributed to enhanced growth in this alga. This was also intuitively reflected by respiration that provided energy for cellular metabolism and algal growth. We propose that, in the predicted scenario of rising atmospheric CO2, P. haitanensis is capable to adjust its physiology by increasing its carbon and nitrogen metabolism to acclimate the acidified seawater, at the background of global climate change and simultaneously increased iron concentration due to decreased pH levels.

Impact of Submarine Groundwater Discharge on Marine Water Quality and Reef Biota of Maui.

Generally unseen and infrequently measured, submarine groundwater discharge (SGD) can transport potentially large loads of nutrients and other land-based contaminants to coastal ecosystems. To examine this linkage we employed algal bioassays, benthic community analysis, and geochemical methods to examine water quality and community parameters of nearshore reefs adjacent to a variety of potential, land-based nutrient sources on Maui. Three common reef algae, Acanthophora spicifera, Hypnea musciformis, and Ulva spp. were collected and/or deployed at six locations with SGD. Algal tissue nitrogen (N) parameters (δ15N, N %, and C:N) were compared with nutrient and δ15N-nitrate values of coastal groundwater and nearshore surface water at all locations. Benthic community composition was estimated for ten 10-m transects per location. Reefs adjacent to sugarcane farms had the greatest abundance of macroalgae, low species diversity, and the highest concentrations of N in algal tissues, coastal groundwater, and marine surface waters compared to locations with low anthropogenic impact. Based on δ15N values of algal tissues, we estimate ca. 0.31 km2 of Kahului Bay is impacted by effluent injected underground at the Kahului Wastewater Reclamation Facility (WRF); this region is barren of corals and almost entirely dominated by colonial zoanthids. Significant correlations among parameters of algal tissue N with adjacent surface and coastal groundwater N indicate that these bioassays provided a useful measure of nutrient source and loading. A conceptual model that uses Ulva spp. tissue δ15N and N % to identify potential N source(s) and relative N loading is proposed for Hawai'i. These results indicate that SGD can be a significant transport pathway for land-based nutrients with important biogeochemical and ecological implications in tropical, oceanic islands.

Mechanisms of metal tolerance in marine macroalgae, with emphasis on copper tolerance in Chlorophyta and Rhodophyta.

Green and red macroalgae are closely related organisms, and with terrestrial plants, and constitute the base of marine food webs in coastal ecosystems. Green and red seaweeds, as all living organisms, require essential metals, such as copper, iron, zinc, which can act as co-factors for several proteins and enzymes; however, these metals in excess can induce stress and impair cell viability. Most important negative effects of metal excess are related to the induction of an oxidative stress condition, characterized by the over-accumulation of Reactive Oxygen Species (ROS). In this respect, copper, abundant in wastewaters disposed to coastal environments from domestic and industrial activities, has been one of the most studied metals. Different investigations have provided evidence that green and red macroalgae display several defenses against copper excess to prevent, or at least reduce, stress and damage, among which are cellular exclusion mechanisms, synthesis of metal-chelating compounds, and the activation of the antioxidant system. Most important defense mechanisms identified in green and red seaweed involve: metal-binding to cell wall and epibionts; syntheses of metallothioneins and phytochelatins that accumulate in the cytoplasm; and the increase in the activity of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione peroxidase and catalase, and greater production of antioxidant metabolites as glutathione and ascorbate in organelles and the cytoplasm. In this review, we go through historical records, latest advances, and pending tasks aiming to expand our current knowledge on defense mechanisms to copper excess in green and red macroalgae, with emphasis on biochemical and molecular aspects.

Effects of sodium bicarbonate concentration on growth, photosynthesis, and carbonic anhydrase activity of macroalgae Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae (Gracilariales, Rhodophyta).

There is potential for bicarbonate to improve crop yields and economic efficiency of marine algae. However, few studies have focused on the effect of bicarbonate on the growth, photosynthesis, and enzyme activity associated with carbon utilization, especially in commercial macroalgae. Here, the addition of bicarbonate (up to 420 mg L(-1)) to macroalgal cultures has been evaluated for Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae with respect to growth rate, photosynthetic activity, carbonic anhydrase activity, and biochemical composition. The results showed that the effects of NaHCO3 on growth, chlorophyll a, phycoerythrin, photosynthetic oxygen evolution, photochemical parameters of PSI and PSII, carbonic anhydrase activity, and nitrogen content were significant (P < 0.05) and followed the same pattern in the three species. The parameter values were promoted in lower NaHCO3 concentrations (up to 252 or 336 mg L(-1)) and inhibited in higher NaHCO3 concentrations (>336 mg L(-1) for Gp. lemaneiformis and >420 mg L(-1) for the other two species). Moreover, species-specific differences induced by supplementation with bicarbonate were discovered during culture. Optimal concentrations of NaHCO3 used in this study were 252 mg L(-1) for Gp. lemaneiformis and 336 mg L(-1) for G. vermiculophylla and G. chouae. These results suggest that an adequate supplementation of sodium bicarbonate is a viable strategy for promoting growth and photosynthetic activity in some macroalgae as well as for improving biochemical composition. The study will help to accelerate the growth rate of algae and improve the quality of thalli, and will also be useful for enhancing the understanding of carbon utilization in macroalgae.

Pollutant Concentrations and Toxic Effects on the Red Alga Ceramium tenuicorne of Sediments from Natural Harbors and Small Boat Harbors on the West Coast of Sweden.

This investigation set out to analyze the toxicity of surface sediments in a number of natural harbors and small boat harbors on the west coast of Sweden. This was done with the growth inhibition method with Ceramium tenuicorne. Also, concentrations of copper (Cu), lead (Pb), zinc (Zn), irgarol, organotin compounds, and polycyclic aromatic hydrocarbons (PAHs) in the sediments were analyzed. The small boat harbors were heavily polluted by Cu, Zn, butyltins, and PAHs, and to a lesser extent by Pb. The Cu, Pb, Zn, and butyltins probably originated from their past and/or present use in antifouling paints, whereas the PAHs probably had multiple sources, including boat motor exhausts. The measured toxicity of the sediment was generally related to their Cu, Zn, and butyltin content, although other toxic substances than those analyzed here probably contributed to the toxicity in some of the harbors. The natural harbor sediments contained less pollutants and were less toxic than the small boat harbor sediments. Nevertheless, our data indicate that the boating pressure today may be high enough to produce toxic effects even in natural harbors in pristine areas. The strongest relationship between toxicity and the major pollutants was obtained when the sediment toxicity was expressed as gram wet weight per liter compared with gram dry weight per liter and gram total organic carbon per liter. Hence, for pollutants that can be elutriated with natural sea water, sediment toxicity expressed as gram wet weight per liter appears preferable.

Effects of Oxygen Limitation on the Biosynthesis of Photo Pigments in the Red Microalgae Galdieria sulphuraria Strain 074G.

As a consequence of the inhibition of one of the steps in the biosynthesis of the photopigments chlorophyll and phycobilin, the red microalga Galdieria partita excretes coproporphyrinogen III in the medium when growing on glucose. No coproporphyrinogen III was found when the closely related red microalgae G. sulphuraria strain 074G was grown on glucose and excessive amounts of oxygen. When under the same conditions oxygen was limiting, coproporphyrinogen III was present in the medium. We conclude that not glucose but the amount of oxygen in the medium results in the accumulation of coproporphyrinogen III. This is explained by the inactivition of the oxygen-dependent coproporphyrinogen III oxidase that converts coproporhyrinogen III to protoporphyrinogen IX, one of the intermediate steps in the biosynthesis of chlorophyl and phycobilin.

The effect of rapamycin on biodiesel-producing protist Euglena gracilis.

Rapamycin induces autophagy with lipid remodeling in yeast and mammalian cells. To investigate the lipid biosynthesis of Euglena gracilis, rapamycin was supplemented in comparison with two model algae, Chlamydomonas reinhardtii and Cyanidioschyzon merolae. In Euglena, rapamycin induced the reduction of chlorophylls and the accumulation of neutral lipids without deterring its cell proliferation. Its lipidomic profile revealed that the fatty acid composition did not alter by supplementing rapamycin. In Chlamydomonas, however, rapamycin induced serious growth inhibition as reported elsewhere. With a lower concentration of rapamycin, the alga accumulated neutral lipids without reducing chlorophylls. In Cyanidioschyzon, rapamycin did not increase neutral lipids but reduced its chlorophyll content. We also tested fatty acid elongase inhibitors such as pyroxasulfone or flufenacet in Euglena with no significant change in its neutral lipid contents. In summary, controlled supplementation of rapamycin can increase the yield of neutral lipids while the scheme is not always applicable for other algal species.