Skeletonema costatum was submitted to two experiments using UV lights and CO2with the purpose of observing changes in the lipids profile and the synthesis of polyunsaturated aldehydes (PUA) after cell disruption. When cells receive CO2 supply, it was noticed that the production of PUA was significantly lower. The same was observed when the culture was treated with a dose of 45.9 kJ·m-2 of ultra-violet A/B ray. The premise to all experiments was the production of 2,4-heptadienal compared to the supply of EPA as substrate. As a result, the same synthesis rate was observed both when the CO2 treatment was applied and in the experiment control. On the other hand, the culture subjected to ultraviolet radiation showed a 68% greater demand with the utilization of the substrate. These observations suggested that EPA was consumed before cell disruption and was probably exuded to the surrounding environment as a sign of stress. Changes in cell morphology could be observed by the migration of the chloroplast nearby the cell wall, where PUA was produced, indicating a defense strategy.
The ocean covers 70% of the planet’s surface and presents an extraordinary microbial diversity, constituting the largest ecosystem in the world [
The rising of CO2 level in the atmosphere is causing ocean’s acidification [
This report compares how cells respond to the influence of UV light and the increase of CO2, by changing concentrations of polyunsaturated fatty acids (PUFAs) and producing oxylipins, under the influence of the environmental conditions in the intra-specific response.
Phytoplancton samples were collected in a Coquimbo Bay (29˚55'S and 71˚19'W) in a campaign between September to December 2009 using a network mesh of 23 µm. Cells of Skeletonema costatum was isolated and cultivated in a f/2 + Si [
The cultures were performed in triplicate in a controlled environment at 20˚C, light intensity of 50 µmol/m2/s and constant aeration in Erlenmeyer of 2 L. For the treatment carried out with addition of CO2 cultivation was supplied with a flow of 0.5 mm³/s of carbon dioxide with a purity of 99%, for 24 h in parallel aeration. For the test with ultraviolet cultures received photosynthetic active radiation PAR (700 - 400 nm) while were subjected to UV A/B, of wavelength 400 - 280 nm. A filter Schott WG 305 was used to prevent radiation at frequencies lower than 280 nm. Equation (1) calculates the doses of the UV light the irradiated [
J・m−2= W・m−2・s (1)
The final dose was calculates in 45.9 KJ・m−2, concerning the radiation of 15.3 KJ・m−2 found at 1 m depth accumulated over three days, as described [
To descript the change profile of fatty acids during the curve growth, atransesterification methodology was applied [
A standard sample of 50 mL with 7.4 × 104 cells/mL obtained from each culture was concentrated in a glass fiber filter (What man GB) under a reduced pressure of 700 mbar. The concentrated biomass was washed with 1 mL of the derivatized reagent (25 mM PFBHA. HCl in 100 mM Tris/HCl, pH 7.0). The sample was transferred to a 4 mL vial that was exposed to ultrasound for 1 min at 4˚C. As internal standard 5 µL of benzaldehyde solution (1 mM) was added, the sample remained at room temperature for 30 min to complete the enzymatic reaction and after then it was stored at −20˚C. For the extraction of the derivatized compounds 0.5 mL of methanol and 1 mL of hexane were added and homogenate in vortex, 2 drops of sulfuric acid (5 mM) was added and homogenate again. The upper layer of the sample was removed pipetting and sodium sulfate was added to remove water. The sample was filtrated with a Millipore® membrane of 0.2 µm and transferred to a 1 mL conical vial and dried by a nitrogen gas flow. At the end, 50 µL of hexane was added and further analyzed in a GC-MS [
A capillary GC column (50 m × 0.32 mm i.d. HP-1) fitted with a cold on-column injector was directly coupled to a mass spectrometer (Thermo Finnigan). Ionization was by electron impact at 70 V, 250˚C. The GC oven temperature was maintained at 30˚C for 5 min and then increased at 5˚C・min−1 to 250˚C. Tentative identifications were made by comparison with mass spectra databases (NIST 2002).
To compare concentrations of PUA and fatty acids between strains and treatments a one way ANOVA was performed and the a posteriori Tukey test was applied when necessary to indicate the significant differences (p < 0.05).
The three pre-selected strains subjected to a semiquantitative analysis by SPME-PDMS presented a pronounced difference of PUA synthesis
Fatty acids presented a tendency of change the profile during de growing curve as describe in
Abundance of fattyacids | ||||||
---|---|---|---|---|---|---|
Day 3 | Day 5 | Day 7 | ||||
Mean | S.D. | Mean | S.D. | Mean | S.D. | |
C14:0 | 38.36% | 3.44% | 34.31% | 5.92% | 22.98% | 18.99% |
C16:0 | 11.31% | 2.63% | 7.15% | 6.95% | 14.40% | 12.26% |
C18:0 | 5.35% | 7.69% | 0.00% | 0.00% | 0.00% | 0.00% |
C16:1 | 25.39% | 2.51% | 30.07% | 4.85% | 25.97% | 20.72% |
C18:2 | 0.52% | 0.45% | 0.17% | 0.30% | 1.71% | 0.94% |
C18:3 | 7.57% | 2.59% | 11.99% | 1.55% | 15.94% | 3.45% |
C20:5 | 10.11% | 2.44% | 15.43% | 5.16% | 17.01% | 3.08% |
C22:6 | 1.39% | 0.28% | 0.87% | 0.10% | 2.00% | 0.64% |
Through the methodology of derivatization was possible to trap and identify some fatty acids used in the metabolic pathway of oxylipins. In cells culture grown with CO2 and UV light was observed a decrease in the total fatty acid concentration. The abundance of EPA was significantly lower (p < 0.05) in the treatment whit CO2, especially with cells challenged with UV light (p < 0.01) as presented in
The total production of aldehydes was accessed and in both treatments. The amount of PUA produced was significant lower (p < 0.05) in compeer to control (
In the present work, a screening was conducted to select strains that produce the larger amount of aldehydes. From these three microalgae analyzed the strain A did not produce the molecules of interest. However, these algae (Skeletonema costatum) could be a good source of food for plankton consumption, because the low toxicityeven after cell disruption. About the two other strains of algae tested, it was observed a very similar profile of aldehydes which leads us to believe that it was the same species. Moreover the microalgae that not produce aldehydes could be another species of Skeletonema sp. and more accurate analysis should be conducted for the identification that isolated strain.
In cultures that grow with supply of carbon dioxide, the cells density does not surpass the control culture. In a work made by Blanchemain and Grize [
The effect of CO2 on the lipid composition was also studied in the others microalgae. In strains of Chlamidomonasreinhardtii was observed that increasing concentrations of PUFA occurred when CO2 concentrations was reduced from 2% to 0.03% [
With regard to the physiology of the defense and the production of oxylipins after the cell wall damage was possible to observe the same proportions between the aldehydes produced. We can assume that the path of enzymes involved in the synthesis of PUA remained active with no direct effect by UV light in their functions. Kouwenber and Lantoine [
The result of this experiment with indirect evidence suggests that microalgae synthesize and release oxylipins before cell disruption respond to a stress signal, as described by Vidoudez and Pohnert [
Despite cultures were not axenic, the PUA that was produced by and identified in Skeletonema costatum corroborated with the literature [
L. F. M. P. thanks EWOS Innovation for the financial support at this study. Special thanks to E. Uribe, A. Quiroz, and J. Pino whose help was very important to the present work. J. Troncosois acknowledged the improving comments to the manuscript.