ROS and calcium signaling mediated pathways involved in stress responses of the marine microalgae Dunaliella salina to enhanced UV-B radiation.

UV-B ray has been addressed to trigger common metabolic responses on marine microalgae, however, the upstream events responsible for these changes in marine microalgae are poorly understood. In the present study, a species of marine green microalgae Dunaliella salina was exposed to a series of enhanced UV-B radiation ranging from 0.25 to 1.00 KJ·m-2 per day. The role of ROS and calcium signaling in the D. salina responses to UV-B was discussed. Results showed that enhanced UV-B radiation markedly decreased the cell density in a dose-dependent manner, but the contents of protein and glycerol that were essential for cell growth increased. It suggested that it was cell division instead of cell growth that UV-B exerted negative effects on. The subcellular damages on nuclei and plasmalemma further evidenced the hypothesis. The nutrient absorption was affected with UV-B exposure, and the inhibition on PO43- uptake was more serious compared to NO3- uptake. UV-B radiation promoted reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) contents, decreased the redox status and altered the antioxidant enzyme activities. The addition of the ROS scavenger and the glutathione biosynthesis precursor N-acetyl-l-cysteine (NAC) alleviated the stress degree, implying ROS-mediated pathway was involved in the stress response to UV-B radiation. Transient increase in Ca2+-ATPase was triggered simultaneously with UV-B exposure. Meanwhile, the addition of an intracellular free calcium chelator aggravated the damage of cell division, but exogenous calcium and ion channel blocker applications did not, inferring that endogenously initiated calcium signaling played roles in response to UV-B. Cross-talk analysis showed a relatively clear relationship between ROS inhibition and Ca2+-ATPase suppression, and a relation between Ca2+ inhibition and GPx activity change was also observed. It was thus presumed that ROS-coupled calcium signaling via the glutathione cycle was involved in the response of marine microalgae to UV-B stimuli.

UV-C mediated rapidcarotenoid induction and settling performance of Dunaliellasalina and Haematococcus pluvialis.

Microalgae are primary producers of organic pigments carotenoids in aquatic environments. However, commercial-scale microalgae application for high value carotenoids production is rarely economical due to the cost-effectiveness of carotenoid induction and microalgal harvesting process. Here, we present a novel approach, using a small dose of externally applied UV-C radiation, to rapidly induce unsaturated fatty acids and carotenoid biosynthesis in Dunaliella salina and Haematococcus pluvialis, and also to significantly promote their swimming cells settling for primary dewatering. The amount of total carotenoids and ß-carotenoid were doubled in 24 h on D. salina upon 50 mJ/cm(2) of UV-C radiation, whereas the astaxanthin yield of H. pluvialis was increased five times in 48 h at 30 mJ/cm(2) . Meanwhile, 95% of algal cells of D. salina and H. pluvialis settled in 15 h and 2 h, respectively. This novel technique represents a convenient, time-saving and cost-effective method for commercial microalgal carotenoids production.

Long-term adaptive response to high-frequency light signals in the unicellular photosynthetic eukaryote Dunaliella salina.

Productivity of microalgal cultivation processes is tightly related to photosynthetic efficiency, and therefore to light availability at the cell scale. In an agitated, highly turbid suspension,the light signal received by a single phytoplankton cell moving in a dense culture is a succession of flashes. The growth characteristics of microalgae under such dynamic light conditions are thus fundamental information to understand nonlinear properties of the photosynthetic process and to improve cultivation process design and operation. Studies of the long term consequences of dynamic illumination regime on photosynthesis require a very specific experimental set-up where fast varying signals are applied on the long term. In order to investigate the growth response of the unicellular photosynthetic eukaryote Dunaliella salina (Chlorophyceae) to intermittent light exposure, different light regimes using LEDs with the same average total light dose were applied in continuous cultures. Flashing light with different durations of light flashes (△t of 30 s, 15 s, 2 s and 0.1 s) followed by dark periods of variable length (0.67 ≤ L:D ≤ 2) yielding flash frequencies in the range 0.017-5 Hz, were compared to continuous illumination. Specific growth rate, photosynthetic pigments, lipid productivity and elemental composition were measured on two duplicates for each irradiance condition. The different treatments of intermittent light led to specific growth rates ranging from 0.25 to 0.93 day(-1) . While photosynthetic efficiency was enhanced with increased flash frequency, no significant differences were observed in the particular carbon and chlorophyll content. Pigment analysis showed that within this range of flash frequency, cells progressively photoacclimated to the average light intensity.

Simple processes for optimized growth and harvest of Ettlia sp. by pH control using CO2 and light irradiation.

Microalgae cultures show wide range of pH depending on the availability of light and CO2 for their strain specific photosynthesis. Thus, the modulation of light irradiation and CO2 supply can be applied for the pH control of microalgae cultures. The optimal pH of Ettlia sp. YC001, for phototrophic growth and auto-flocculation was investigated by controlling light irradiation and 10% CO2 supply. Ettlia sp. YC001 showed the highest biomass productivity, 96.7 mg L(-1) d(-1) , at pH 8.5. The flocculating activity of Ettlia sp. YC001 showed a sigmoid pattern with pH increase and was above 70% at pH 10.5. Based on these differentiated optimal pH regimes for the growth and flocculation, an integrated process consisting of cultivation and settling vessels was proposed. The integrated process demonstrated that high flocculation activity of Ettlia sp. YC001 could be achieved in the settling vessel with various hydraulic retention times by only irradiation of light to maintain high pH while maintaining the optimal growth in cultivation vessel with the light irradiation and CO2 supply at pH 8.5. Thus, the proposed strategy for pH control would provide a simple, cost-effective, and flexible design and operation for microalgae cultivation-harvest systems.

Red light and carbon dioxide differentially affect growth, lipid production, and quality in the microalga, Ettlia oleoabundans.

Ettlia oleoabundans, a freshwater unicellular green microalga, was grown under different light qualities ± carbon dioxide-enriched air to determine the combined effects on growth and lipid production of this oleaginous species. Keeping total light intensity constant, when a portion of the cool white was replaced by red, volumetric lipid yield increased 2.8-fold mainly due to the greater yield of oleic acid, a desirable biodiesel precursor. Only 30 min of red light treatment was sufficient to increase lipid yield and quality to the same level as cultures provided red light for >14 days, indicating the potential role of red light in stimulating lipid production of this species. Carbon dioxide enrichment via air sparging enhanced exponential growth, carbon conversion efficiency, and nutrient consumption. Together, these results showed that light quality plays an important role in microalgal lipid production. Adjustment in light quality and gas delivery efficiency with carbon dioxide enrichment improved lipid yield and quality in this and possibly other oleaginous algal species.

Intracellular glycerol accumulation in light-limited Dunaliella tertiolecta culture is determined by partitioning of glycerol across the cell membrane.

Dunaliella accumulates intracellular glycerol to counterbalance the extracellular salinity. In N-limited chemostat cultures of D. tertiolecta, total glycerol production (sum of intracellular and extracellular) and intracellular glycerol content were proportional to the salinity of the culture medium. In the light-limited D. tertiolecta culture, total glycerol output (sum of intracellular and extracellular) was relatively constant at different salinities (0.5 and 2.0 M), while the intracellular glycerol content was proportional to the culture medium salinity, that is, the cells released less glycerol into the culture medium, rather than de novo synthesis of glycerol at high culture medium salinity. The study implies different regulatory mechanisms in the accumulation of intracellular glycerol in N-limited and light-limited D. tertiolecta in response to salinity.

Expression of the high light-inducible Dunaliella LIP promoter in Chlamydomonas reinhardtii.

The development of highly inducible promoters is critical for designing effective transformation systems for transgenic analyses. In this study, we investigated the promoter of the light-inducible protein gene (LIP) of the marine alga Dunaliella sp. LIPs are homologs of the early light-induced proteins (ELIPs) of Arabidopsis thaliana. DNA sequence analysis revealed that the LIP promoter contains several light-responsive motifs. Constructs containing progressive truncations of the LIP promoter fused with a Renilla luciferase gene were introduced into Chlamydomonas reinhardtii to identify the light-responsive region in the promoter. Transcription from the LIP promoter was stimulated by high light (HL) in a light intensity-dependent manner. In contrast, oxidative stress induced by chemicals had little effect on the LIP promoter, which implies that the LIP promoter is exclusively induced by high light. Truncation of the promoter to a -100 base pair (bp) region abrogated light inducibility, which suggests the presence of a negative cis-regulatory element upstream of the -100 bp fragment. The LIP promoter can be utilized in transgenic research to specifically select and propagate transgenic microalgae under high-light conditions.

Enhanced growth and lipid accumulation by a new Ettlia texensis isolate under optimized photoheterotrophic condition.

A green microalgae, named as Ettlia texensis was obtained from local freshwater in Turkey. The effects of autotrophic, photoheterotrophic and heterotrophic cultivations on biomass and lipid production were studied. Searching the preferences of the carbon and nitrogen source revealed that this strain could grow photoheterotrophically well with glucose and yeast extract. In the optimized medium, the highest biomass productivity and total lipid content achieved were 0.97 g/L d and 26% of dry weight basis, respectively. Moreover, the major fatty acid methyl esters were C16:0; C18:1; C18:2 and C18:3. In a scale-up attempt, productions were accomplished in a 3 L stirred tank bioreactor. The final biomass and lipid productivities obtained in bioreactor with 250 rpm agitation rate were 0.92 g/L d and 322 mg/L d, respectively. The biochemical compositions were monitored simultaneously by the FTIR spectroscopy during the production in bioreactor. E. texensis could be potent candidate for commercial production in the bioreactor photoheterotrophically.

At high temperature lipid production in Ettlia oleoabundans occurs before nitrate depletion.

Temperature and light intensity effects on biomass and lipid production were investigated in Ettlia oleoabundans to better understand some fundamental properties of this potentially useful but poorly studied microalgal species. E. oleoabundans entered dormant state at 5 °C, showed growth at 10 °C, and when exposed to light at 70 µmol photons per square meter per second at 10 °C, cells reached a biomass concentration of >2.0 g L(-1) with fatty acid methyl esters of 11.5 mg L(-1). Highest biomass productivity was at 15 °C and 25 °C regardless of light intensity, and accumulation of intracellular lipids was stimulated by nitrate depletion under these conditions. Although growth was inhibited at 35 °C, at 130 µmol photons per square meter per second lipid content reached 10.37 mg L(-1) with fatty acid content more favorable to biodiesel dominating; this occurred without nitrate depletion. In a two-phase temperature shift experiment at two nitrate levels, cells were shifted after 21 days at 15 °C to 35 °C for 8 days. Although after the shift growth continued, lipid productivity per cell was less than that in the 35 °C cultures, again without nitrate depletion. This study showed that E. oleoabundans grows well at low temperature and light intensity, and high temperature can be a useful trigger for lipid accumulation independent of nitrate depletion. This will prove useful for improving our knowledge about lipid production in this and other oleaginous algae for modifying yield and quality of algal lipids being considered for biodiesel production.

Enhancement of carotenoid biosynthesis in the green microalga Dunaliella salina with light-emitting diodes and adaptive laboratory evolution.

There is a particularly high interest to derive carotenoids such as ß-carotene and lutein from higher plants and algae for the global market. It is well known that ß-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased ß-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.

Cell survival after UV radiation stress in the unicellular chlorophyte Dunaliella tertiolecta is mediated by DNA repair and MAPK phosphorylation.

Ultraviolet radiation (UVR) induces damage in a variety of organisms, and cells may adapt by developing repair or tolerance mechanisms to counteract such damage; otherwise, the cellular fate is cell death. Here, the effect of UVR-induced cell damage and the associated signalling and repair mechanisms by which cells are able to survive was studied in Dunaliella tertiolecta. UVR did not cause cell death, as shown by the absence of SYTOX Green-positive labelling cells. Ultrastructure analysis by transmission electron microscopy demonstrated that the cells were alive but were subjected to morphological changes such as starch accumulation, chromatin disaggregation, and chloroplast degradation. This behaviour paralleled a decrease in F(v)/F(m) and the formation of cyclobutane-pyrimidine dimers, showing a 10-fold increase at the end of the time course. There was a high accumulation of the repressor of transcriptional gene silencing (ROS1), as well as the cell proliferation nuclear antigen (PCNA) in UVR-treated cells, revealing activation of DNA repair mechanisms. The degree of phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinases was higher in UVR-exposed cells; however, the opposite occurred with the phosphorylated extracellular signal-regulated kinase (ERK). This confirmed that both JNK and p38 need to be phosphorylated to trigger the stress response, as well as the fact that cell division is arrested when an ERK is dephosphorylated. In parallel, both DEVDase and WEHDase caspase-like enzymatic activities were active even though the cells were not dead, suggesting that these proteases must be considered within a wider frame of stress proteins, rather than specifically being involved in cell death in these organisms.

Growth of the microalgae Neochloris oleoabundans at high partial oxygen pressures and sub-saturating light intensity.

The effect of partial oxygen pressure on growth of Neochloris oleoabundans was studied at sub-saturating light intensity in a fully-controlled stirred tank photobioreactor. At the three partial oxygen pressures tested (P(O)2= 0.24; 0.63; 0.84 bar), the specific growth rate was 1.38; 1.36 and 1.06 day(-1), respectively. An increase of the P(CO)2from 0.007 to 0.02 bar at P(O2) of 0.84 bar resulted in an increase in the growth rate from 1.06 to 1.36 day(-1). These results confirm that the reduction of algal growth at high oxygen concentrations at sub-saturating light conditions is mainly caused by competitive inhibition of Rubisco. This negative effect on growth can be overcome by restoring the O(2)/CO(2) ratio by an increase in the partial carbon dioxide pressure. In comparison to general practice (P(O(2)) = 0.42 bar), working at partial O(2) pressure of 0.84 bar could reduce the energy requirement for degassing by a factor of 3-4.

Ultraviolet-B radiation improves astaxanthin accumulation in green microalga Haematococcus pluvialis.

Ultraviolet-B (UV-B) radiation has significantly negative effect on cell survival rate (P < 0.01) and positive effect on astaxanthin accumulation (P < 0.01) of Haematococcus pluvialis. H. pluvialis accumulated 3.2 mg/g of astaxanthin when being exposed to 5 W/m(2) of UV-B for 60 min prior to 72 h of high light treatment, which was 122% higher than that of the control. This UV-B treatment also significantly stimulated lipid peroxidation and the value of malondialdehyde and glutathione peroxidase activities were 156 and 166% higher than those of control, respectively (P < 0.01).

Application of terahertz time-domain spectroscopy in intracellular metabolite detection.

Using terahertz time-domain spectroscopy (THz-TDS), we have investigated the THz spectra of astaxanthin and riboflavin and the spectra of two kinds of cell, haenatcoccus plusivalis and bacillus subtilis, which could produce astaxanthin and riboflavin, respectively, during their metabolite process. Riboflavin was found to be much more absorptive to THz radiation and have richer spectral characteristics than astaxanthin. As an intracellular metabolite, riboflavin could be distinguished from the cells by using THz-TDS. The technique has potential applications in high-throughput screening of industrial strains.

Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina.

beta-Carotene is overproduced in the alga Dunaliella salina in response to high light intensities. We have studied the effects of a sudden light increase on carotenoid and fatty acid metabolism using a flat panel photobioreactor that was run in turbidostat mode to ensure a constant light regime throughout the experiments. Upon the shift to an increased light intensity, beta-carotene production commenced immediately. The first 4 h after induction were marked by constant intracellular levels of beta-carotene (2.2 g LCV(-1)), which resulted from identical increases in the production rates of cell volume and beta-carotene. Following this initial phase, beta-carotene productivity continued to increase while the cell volume productivity dropped. As a result, the intracellular beta-carotene concentration increased reaching a maximum of 17 g LCV(-1) after 2 days of light stress. Approximately 1 day before that, the maximum beta-carotene productivity of 30 pg cell(-1) day(-1) (equivalent to 37 mg LRV(-1) day(-1)) was obtained, which was about one order of magnitude larger than the average productivity reported for a commercial beta-carotene production facility, indicating a vast potential for improvement. Furthermore, by studying the light-induced changes in both beta-carotene and fatty acid metabolism, it appeared that carotenoid overproduction was associated with oil globule formation and a decrease in the degree of fatty acid unsaturation. Our results indicate that cellular beta-carotene accumulation in D. salina correlates with accumulation of specific fatty acid species (C16:0 and C18:1) rather than with total fatty acid content.

Semi-continuous cultivation of Haematococcus pluvialis for commercial production.

The objectives of the present study on the growth of Haematococcus pluvialis were to indicate the effects of a long-term semi-continuous cultivation, sterilization, carbon dioxide, and different culture media by using artesian well water. This investigation was an enterprise in order to commercialize the production economically. When the effect of CO(2) was investigated in basal culture medium, the influence of sterilization was also researched in Rudic's culture medium in vertical panel-type photobioreactors for 31 days of semi-continuous cultivation. The maximum cell concentration of 10.55 x 10(5) cells ml(-1), which corresponds to the growth rate of 0.271 day(-1) with the areal productivity of 3.531 g m(-2) day(-1), was found in non-sterilized RM medium on the 24th day of the third run of semi-continuous cultivation at a renewal rate of 50% in a vertical panel-type photobioreactor.