Production of superoxide anion and hydrogen peroxide associated with cell growth of Chattonella antiqua

We determined the effects of various light spectra (white, green, blue, and red) on the growth rate, biochemical composition, and fatty acid content of Tisochrysis lutea (Haptophyta, Isochrysidales) maintained in batch cultures. The growth rate peaked with white and blue light, and the lowest rate was observed with green and red light. The chlorophyll a content differed significantly between light spectra and growth phases—higher values were recorded with blue and red light in both growth phases. The proximal composition varied significantly with growth phases and light spectrum. In the exponential growth phase, protein content was significantly greater with blue light and in the stationary phase with green light. The level of carbohydrates in the exponential growth phase was significantly higher for white light, but unchanged in the stationary growth phase between light spectra. The lipid percentages were similar in the exponential phase but differed significantly in the stationary growth phase. The lipid percentages peaked in the stationary growth phase with red and green light. The highest eicosapentaenoic acid (EPA) levels were seen in white light in the exponential growth phase and under green light in the stationary growth phase. Docosahexaenoic acid (DHA) levels were greatest in the exponential growth phase with red light and in the stationary growth phase with green light. Blue light increased the DHA content in both growth phases. We conclude that T. lutea alters its metabolic pathways and experience shifts in growth rate, proximate composition, and fatty acid content, depending on the type of light used.

The lipid and biochemical composition of the haptophyte Isochrysis galbana TK1 was examined. Cultures were grown at 15 °C and 30 °C, and harvested in the exponential and early stationary growth phases. Carbohydrate and protein content varied at the two culture temperatures and growth phases. The highest protein content was found at the exponential growth phase at 15 °C, and the highest carbohydrate content was found at the stationary phase at the same culture temperature. Lipid accumulated in the stationary growth phase and its content was higher at 30 °C than at 15 °C regardless of the growth phase. The neutral lipids were the major class of lipid found in all the cultures. The stationary phase culture had a higher proportion of neutral lipids than the exponential phase culture and the proportion decreased slightly when culture temperature was increased from 15 °C to 30 °C. Phospholipid levels remained constant at the two temperatures, but slightly decreased in the stationary phase. Glycolipids in the exponentially growing cells were higher than those from stationary growth phase and increased with temperature. Polyunsaturated fatty acids (PUFAs) predominated in glycolipids and phospholipids. Cells grown at 15 °C contained higher proportion of 18:3 (n–3) and 22:6 (n–3) with a corresponding decrease in 18:2 (n–6), monounsaturated and saturated fatty acids.

We studied the effects of expected end-of-the-century p CO2 (1000 ppm) on the photosynthetic performance of a coastal marine cyanobacterium Synechococcus sp. PCC7002 during the lag, exponential, and stationary growth phases. Elevated p CO2 significantly stimulated growth, and enhanced the maximum cell density during the stationary phase. Under ambient p CO2 conditions, the lag phase lasted for 6 days, while elevated p CO2 shortened the lag phase to two days and extended the exponential phase by four days. The elevated p CO2 increased photosynthesis levels during the lag and exponential phases, but reduced them during the stationary phase. Moreover, the elevated p CO2 reduced the saturated growth light (Ik) and increased the light utilization efficiency ( α ) during the exponential and stationary phases, and elevated the phycobilisome:chlorophyll a (Chl a ) ratio. Furthermore, the elevated p CO2 reduced the particulate organic carbon (POC):Chl a and particulate organic nitrogen (PON):Chl a ratios during the lag and stationary phases, but enhanced them during the exponential phase. Overall, Synechococcus showed differential physiological responses to elevated p CO2 during different growth phases, thus providing insight into previous studies that focused on only the exponential phase, which may have biased the results relative to the effects of elevated p CO2 in ecology or aquaculture.

Changes in bacterial cells induced by high pressure at subzero temperature

Raphidophytes (class Raphidophyceae) produce high levels of reactive oxygen species (ROS), yet little is known regarding cellular scavenging mechanisms needed for protection against these radicals. Enzymatic activities of the antioxidants superoxide dismutase (SOD) and catalase (CAT) were measured in conjunction with the production of superoxide (O2•−) and hydrogen peroxide (H2O2) in batch cultures of five different raphidophytes species during early exponential, late‐exponential, and stationary growth phases. The greatest concentrations of O2•− per cell were detected during exponential growth with reduced levels in stationary phases in raphidophytes Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara, Chattonella marina (Subrahman.) Y. Hara et Chihara, and Chattonella antiqua (Hada) Ono (strain 18). Decreasing trends from exponential to stationary phases for SOD activity and H2O2 per cell were observed in all species tested. Significant correlations between O2•− per cell and SOD activity per cell over growth phase were only observed in three raphidophytes (Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua strain 18), likely due to different cellular locations of externally released O2•− radicals and intracellular SOD enzymes measured in this study. CAT activity was greatest at early exponential phase for several raphidophytes, but correlations between H2O2 per cell and CAT activity per cell were only observed for Fibrocapsa japonica Toriumi et Takano, Chattonella antiqua (strain 18), and Chattonella subsalsa Biecheler. Our results suggest that SOD and CAT play important protective roles against ROS during exponential growth of several raphidophytes, while other antioxidant pathways may play a larger role for scavenging ROS during later growth.

Arsenic (As) metabolism in freshwater algae at different growth phases has rarely been documented. To address this gap, this study was conducted to assess the intra- and extracellular As metabolism, along with speciation changes, in Microcystis aeruginosa across three growth phases. The treatment involved varying concentrations of As (0, 0.4, 0.6, 0.8 and 1 mg/L, in the form of arsenate, iAsV) under three phosphorus levels (0.02 mg/L as low, 0.1 mg/L as medium, and 0.5 mg/L as high P in the form of phosphate). The findings revealed that extracellular iAsV remained the dominant As species during the lag and exponential growth phases of M. aeruginosa in the growth media, while intracellular trivalent As (iAsIII) emerged as the pronounced species during the exponential growth phase, but also exhibited a significant negative correlation with the P levels. Moreover, elevated P levels had promoted the formation of intra- and extracellular dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) in the exponential growth phase. During the stationary growth phase, intracellular iAsV was found to decrease with the increasing P levels. During the whole growth phases, P had consistently reduced algal As absorption levels. The significant promotion of algal As absorption in response to iAsV was observed only during the lag growth phase. The As bioaccumulation exhibited a correlational relationship with the algal reproduction. Both low and high P levels (0.02 and 0.5 mg/L) decreased the accumulation of As in algae cells during the exponential and stationary growth phases. The transformation and release rate of As were concomitantly influenced by P, and exhibited the same trends within the growth phase. These trends differed between the exponential and stationary growth phases, with an inhibitory effect being present during the former, while a promotional effect was observed during the latter. This study provides insight into potential As hazards in freshwater lakes with algae bloom.

Photosynthesis and respiration in marine phytoplankton: Relationship with cell size, taxonomic affiliation, and growth phase

Diatoms are capable of accumulating substantial amounts of triacylglycerides in their cells, which differ in the composition of fatty acids depending on the conditions of cultivation, making them attractive subjects in biotechnology. In the present study, we characterized the structural features of lipid bodies in the diatom Entomoneis cf. paludosa (W. Smith) Reimer strain 8.0727-B and revealed the peculiarities of fatty acid composition in cultures during the stationary and exponential growth phases. Laser scanning confocal microscopy revealed an increased number of lipid bodies in the cytoplasm during the stationary phase of culture growth. Electron microscopy of ultrathin sections showed that an extreme increase in the number and size of plastoglobules in the cells occurs in the stationary phase of culture growth. The gas chromatography with mass spectrometric detection method revealed differences in the fatty acid composition depending on the growth phase. The studied strain can be recommended as a source of hexadecanoic and octadecanoic fatty acids from the culture during the stationary growth phase, as well as eicosapentaenoic fatty acid from the culture during the exponential growth phase.

Changes in cell wall polysaccharides of Silene vulgaris callus during culture

Anabaena circinalis (Kutz.) Rabenhorst was isolated during a taste and odor episode characterized by high concentrations of geosmin, with raw water containing up to 45 ng geosmin/L. This species has been successfully cultured with sustained geosmin synthesis on both sterile river water and defined medium. Gas chromatography of cellular extracts and closed-loop stripping of growth media indicated that this organism produces geosmin and not 2-methylisoborneol (MIB). With cultures we examined the changes in cell-associated geosmin, chlorophyll and released geosmin during exponential and stationary growth phases of Anabaena. Cell-associated geosmin samples were collected daily from cultures and filtered onto a polycarbonate filter, extracted in acetone, and quantified by capillary gas Chromatograph using flame ionization detection. Media-associated geosmin was quantified from algae-free filtrates from each culture concentrated by closed-loop stripping and analyzed as above. Cell-associated geosmin averaged at 8 × 10−6 ng geosmin/cell in the exponential phase and dropped to 2.5 × 10−6 ng geosmin/cell in the stationary phase of growth. The loss in cell-associated geosmin was accompanied by an increase in geosmin released into the media. Media-associated geosmin reached 12 ng/mL in the stationary phase. Apparently cell lysis in the stationary phase caused the release of cell-associated geosmin into the media. Cell-associated geosmin was closely correlated with filament number (average r2 from 4 experiments =0.96) during the exponential growth phase and was correlated with chlorophyll a (average r2 from 3 experiments =0.95).

Trypanosoma cruzi, the etiologic agent of Chagas disease, cycles through different life stages characterized by defined molecular traits associated with the proliferative or differentiation state. In particular, T. cruzi epimastigotes are the replicative forms that colonize the intestine of the Triatomine insect vector before entering the stationary phase that is crucial for differentiation into metacyclic trypomastigotes, which are the infective forms of mammalian hosts. The transition from proliferative exponential phase to quiescent stationary phase represents an important step that recapitulates the early molecular events of metacyclogenesis, opening new possibilities for understanding this process. In this study, we report a quantitative shotgun proteomic analysis of the T. cruzi epimastigote in the exponential and stationary growth phases. More than 3000 proteins were detected and quantified, highlighting the regulation of proteins involved in different subcellular compartments. Ribosomal proteins were upregulated in the exponential phase, supporting the higher replication rate of this growth phase. Autophagy-related proteins were upregulated in the stationary growth phase, indicating the onset of the metacyclogenesis process. Moreover, this study reports the regulation of N-terminally acetylated proteins during growth phase transitioning, adding a new layer of regulation to this process. Taken together, this study reports a proteome-wide rewiring during T. cruzi transit from the replicative exponential phase to the stationary growth phase, which is the preparatory phase for differentiation.

Antioxidant defence system during exponential and stationary growth phases of Phycomyces blakesleeanus: Response to oxidative stress by hydrogen peroxide

The present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata, in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmol L−1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The short-term assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L−1, respectively. P. subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC50 values for Cu, for cells in the stationary phase, were 443 and 268 μg L−1, respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P. subcapitata. The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples.

Simple SummaryMicroalgae are a huge renewable and eco-friendly source of natural compounds, including vitamins, carbohydrates, pigments, sterols and lipids. They have found applications in different industrial sectors, including the pharmaceutical, nutraceutical and cosmeceutical fields. Isochrysis galbana, a marine flagellate belonging to the Haptophyta phylum, thanks to its nutraceutical composition has been proposed as a shellfish diet (Shellfish Diet 1800®) as well as in the composition of cookies, fresh pasta and yogurt. I. galbana powder, extracts or pure molecules have shown interesting bioactivities, such as antioxidant, antidiabetes and antituberculosis, while the antiproliferative activity is mainly related to lung lymphoblasts. In the current study, we aimed to identify metabolic pathways activated in stressful conditions (stationary growth phase) by transcriptomic and bioactivity-guided fractionation. Overall, the results showed antiproliferative activities against melanoma cells, suggesting new possible applications in cancer prevention and treatment.Haptophytes are important primary producers in the oceans, and among the phylum Haptophyta, the flagellate Isochrysis galbana has been found to be rich in high-value compounds, such as lipids, carotenoids and highly branched polysaccharides. In the present work, I. galbana was cultured and collected at both stationary and exponential growth phases. A transcriptomic approach was used to analyze the possible activation of metabolic pathways responsible for bioactive compound synthesis at the gene level. Differential expression analysis of samples collected at the exponential versus stationary growth phase allowed the identification of genes involved in the glycerophospholipid metabolic process, the sterol biosynthetic process, ADP-ribose diphosphatase activity and others. I. galbana raw extracts and fractions were tested on specific human cancer cells for possible antiproliferative activity. The most active fractions, without affecting normal cells, were fractions enriched in nucleosides (fraction B) and triglycerides (fraction E) for algae collected in the exponential growth phase and fraction E for stationary phase samples. Overall, transcriptomic and bioactivity data confirmed the activation of metabolic pathways involved in the synthesis of bioactive compounds giving new insights on possible Isochrysis applications in the anticancer sector.

Human pancreatic cells of the Capan-1 cell line differentiate in culture. During the exponential growth phase, the cells are undifferentiated, only becoming differentiated during the stationary phase. The formation of domes in this phase is related to the exchange of water and electrolytes. The present study was designed to characterize the localization and expression of alkaline phosphatases (AP) in Capan-1 cells during growth in culture. Biochemical, cytoenzymatic and immunocytochemical methods were employed combined with light and electron microscopic examination. AP essentially of the placental type were expressed progressively during the exponential growth phase, and were seen to be distributed over the surface of the Capan-1 cells. In the stationary phase, the AP became localized on the surface of microvilli. The precipitates of the enzyme reaction highlighted regular four-bodied structures. Biochemical assays showed a progressive increase in activity of this enzyme in cells during both the exponential and stationary growth phases. However, in the stationary phase between days 7 and 8, there was a fall in enzyme activity, with a corresponding increase in this activity in the culture medium. Cytological examination indicated that this fall could be accounted for by loss of AP-positive membranes by vesiculization of apical microvilli and release of microvesicles into the culture medium. Immunoblots showed that Capan-1 cells expressed two types of AP, a placental type (70 kDa) and to a lesser extent a liver type (80 kDa). Expression of the placental type was attributed to a neoplastic derepression of the coding gene, while the liver type was assumed to be a normal gene expression of human duct cells. The placental type AP might thus serve as a marker of transformation, and the liver type as a marker of differentiation.