Growth characteristics and chemical compositions of Phaeodactylum tricornutum under different nitrog.doc

蔡卓平等：不同氮浓度下三角褐指藻生长特性和化学组成 1639growth characteristics and chemical compositions of phaeodactylum tricornutum under different nitrogen concentrationscai zhuoping, duan shunshan*institute of hydrobiology, jinan university, guangzhou 510632, chinaabstract: the marine diatom phaeodactylum tricornutum is a phytoplankton species that contains a high content of bioactive compounds and thus it can be served as an important food source and feed additive in the successful rearing of aquatic animals like shrimp and fish. while its overproliferation along the coastal areas of china in recent years has brought about many negatively environmental and economic consequences around. in order to investigate the effects of different nitrogen concentrations on the growth characteristics and chemical compositions in phaeodactylum tricornutum, algal cell density, specific growth rate, biomass, and the contents of soluble sugar, protein and chlorophyll a have been examined in laboratory cultures by employing low nitrogen (ln, 44 moll-1 nano3), medium nitrogen (mn, 880 moll-1 nano3) and high nitrogen (hn, 4400 moll-1 nano3) concentrations. our results indicated that the growth curve, specific growth rate and cell biomass of phaeodactylum tricornutum were significantly affected by nitrogen concentrations. they were 149.50104 cellsml-1, 0.37 d-1, and 209.33 gml-1 under ln condition, whereas they were 951.67104 cellsml-1, 0.67 d-1, and 806.00 gml-1 under hn condition at the 10th day during the stationary phase. although hn treatment significantly enhanced the algal cell number and cell yield in the stationary phase, it did not show remarkable differences in the growth curve and specific growth rate in the early stages as compared with mn treatment. besides, the contents of soluble sugar, protein and chlorophyll a were obviously increased with the increasing of nitrogen concentrations, which were approximately 2.5, 1.5 and 15 times higher in hn treatment than those in ln treatment, respectively. it can be concluded from the present investigation that higher nitrogen concentrations can accelerate the growth and division as well as biosynthesis of some chemical compositions of phaeodactylum tricornutum, and therefore this should be taken into account for the prevalence of red tides dominated by this marine microalga in recent years. key words: nitrogen concentrations; growth characteristics; chemical compositions; phaeodactylum tricornutumclc number: x173 document code: a article id: 1672-2175（2007）06-1633-04as long as algal growth is not overprolific in the water, algae are of benefit to marine culture operations because they are primary producers in the oceans, ensuring the aquatic food supply from those producers is sustainable and plentiful1. sometimes, unfortunately, some species of algae proliferate quickly in such large numbers that they can give brownish yellow or reddish color to the entire body of water depending on the algae involved, which is best known as red tides 2-3. it has been well documented that red tides have increased in frequency, intensity and geographic distribution during the last decades4-5 and that they have brought about many negatively environmental and economic consequences6-8. the marine diatom phaeodactylum tricornutum is a phytoplankton species that contains a high content of bioactive compounds and polyunsaturated fatty acids (pufas), such as eicosapentaenoic acid (epa) and docosahexaenoic acid (dha), and thus it can be served as an important food source and feed additive in the successful rearing of aquatic animals like shrimp and fish9-11. however, earlier observations have revealed that phaeodactylum tricornutum was responsible for the red tides occurred in shangdong province in 1995, and that in 2006 a red tide occurred along the coast of hainan province also due to the overproliferation of this marine microalga, which potentially destroyed the natural marine ecosystems around and inflicted significantly negative impacts on the society, with a tremendous economic loss. on the other hand, some studies implicated that nitrogen, an essential element for plant growth, could be involved in phytoplankton proliferation, which led to the occurrence of red tides around the globe12-13. likewise, some studies also implicated that excessive n nutrient enrichment via industrial and agricultural inputs could be a major basis in the increased prevalence of red tides14-15. in the current study, marine diatom phaeodactylum tricornutum was subject to varying n concentrations in laboratory cultures. the objectives of the experiments presented here were to examine the effects of n concentrations on the growth characteristic of phaeodactylum tricornutum, and to characterize the changes of some chemical compositions, including soluble sugar, protein and chlorophyll a under various n concentrations, thus trying to gather some basic physiological and biochemical information of this microalga.1 materials and methods1.1 algal culture conditions the marine diatom phaeodactylum tricornutum, obtained from the institute of hydrobiology, jinan university, guangzhou, china, was cultured in glass flasks with artificial seawater added with sterile f/2 enrichment solution for microalgal cultures16. the cells were grown in a plant growth chamber at constant irradiance (5000 lx) and temperature (20 ) in a 12 h/12 h (light/dark) photoperiod cycle. the initial algal density for inoculation was approximately 14104 cellml-1. there were three nitrogen concentrations formed by adding different amount of nano3 into the medium as listed in table 1 (ln, with a lower nitrogen concentration; mn, complete nutrients; hn, with the higher nitrogen concentration), and each treatment consisted of three replications. during the maintenance and experimental stages, the glass flasks containing algal cells were shaken by hand three times every day.table 1 different n concentrations used in the experimentnutrient elementlow concentration(ln)medium concentration(mn)high concentration(hn)nitrogen (moll-1)44 moll-1880 moll-14400 moll-11.2 determination of cell growthcell density was monitored and recorded daily with the help of a microscope using a hemocytometer. the specific growth rate was calculated using the following equation: = (ln x2lnx1)/(t2t1), where x2 and x1 are the numbers of algal cells at t2 and t1 days, respectively17. certain algal samples were taken and filtered through the membrane filters; then cell biomass in the steady-stage was determined after drying the filters and the collected algal pellet for 72 h at 80 18.1.3 measurement of chemical compositionsall the procedures for analyzing the chemical compositions in this study were modified from the method as described by li19. a 10 ml algal sample was collected in centrifuge tubes and centrifuged at 5000 g for 15 min at 4 to obtain the cell pellet. soluble sugar was measured using the phenol method and performed with a spectrophotometer at 485 nm wavelength, with sugar as the standard calibration. after algal cells were disrupted by ultrasonication in an ice bath, protein was assayed by reading the optical densities of the extracts on a spectrophotometer at 260 nm and 280 nm. similarly, cell suspension was concentrated, followed by extraction with 95% ethanol. then the extract was kept in darkness at 4 oc till the algal cells became colorless, and the chlorophyll a was analyzed spectrophotometrically.2 results and discussion 2.1 effects of n concentrations on the growth of phaeodactylum tricornutumfig. 1 growth of phaeodactylum tricornutum in different n concentrations over time.ln: low n concentration; mn: medium n concentration; hn: high n concentration. vertical bars indicate standard errors of the means of three repeatsgrowth curves of the marine mircoalga phaeodactylum tricornutum in various n levels during the whole experimental period are represented in fig. 1. in the first two days, cells of phaeodactylum tricornutum grew slowly, and there were not apparent differences in cell density among three n concentrations. however, the algal cells grew very fast thereafter, especially for the cells in mn and hn treatments. it was demonstrated that there were extremely significant effects of n concentration on the cell density after day 2. the highest cell density under ln condition appeared after algal growth for 4 days (about 170104 cellsml-1), and then it began to remain steady. whereas the cell density under mn and hn conditions was peaked at day 8 (reaching to 900104 and 950104 cellsml-1, respectively), after when it also began to keep stationary. it indicated that high nitrogen concentration could accelerate the proliferation of algal cells. interestingly, the tendency of the growth of phaeodactylum tricornutum was similar under mn and hn conditions, exhibiting a shape similar to “s”. but slightly higher cell density in hn condition was observed in the stationary phase, which suggested that although 880 moll-1 nitrogen was sufficient for the early growth of phaeodactylum tricornutum in this laboratory culture, higher n concentration could still possess a pronounced effect on the algal proliferation in the steady-stage. table 2 cell density, specific growth rate and biomass of phaeodactylum tricornutum exposed to varying n concentrations on the 10th day treatmentcell density/(104 cellml-1)specific growth rate/d-1cell biomass/(gml-1)ln149.5016.26 c0.370.02b209.3311.37 cmn900.8310.41 b0.650.04a657.3340.51 bhn951.6716.65 a0.670.03a806.0023.52 athe values in the table are the means with standard errors of three replications. values with different letters in the same column are significantly different (p0.05)as it was shown in table 2, cell number, cell biomass and specific growth rate of phaeodactylum tricornutum in the stationary stage were manifestly affected by n concentrations. generally, they were increased with the increasing of n concentrations in the culture. the cell density under hn and mn conditions was about six times higher than that under ln condition (149.5104 cellsml-1). moreover, specific growth rate was doubled in mn and hn conditions, as compared with ln condition. in terms of cell biomass, there was approximately 209 gml-1 for ln, but 657 gml-1 and 806 gml-1 for mn and hm respectively, which confirmed that higher n concentrations could promote the growth and final yield of phaeodactylum tricornutum.2.2 soluble sugar, protein and chlorophyll a in phaeodactylum tricornutumchemical compositions of phaeodactylum tricornutum, including soluble sugar, protein and chlorophyll a in varying n concentrations ware also determined in our experiments. qualitatively, soluble sugar can be used to indicate the status of synthesis, transformation and metabolism of carbohydrate in algae20. protein is thought to be of importance for algal cell division, and chlorophyll a is the main pigment transforming the light energy into chemical energy, which directly represents the photosynthetic ability of algae21. the results obtained from our experiments showed that there were significant effects of n concentrations on the chemical compositions mentioned above. it was evident that the remarkably lowest contents of soluble sugar (45 gmg-1), protein (233 gmg-1) and chlorophyll a (0.25 gmg-1) existed in the ln treatment, in contrasted to mn and hn treatments (fig. 2). it suggested that deficient n concentrations could restrain the biosynthesis and metabolism of these chemical compositions measured. it was worthy to note that no obvious differences in the contents of soluble sugar and chlorophyll a were found between mn and hn treatments. with respect to the content of protein, there were striking differences between mn and hn treatments (274 gmg-1 and 354 gmg-1, respectively), which implied that higher n concentration might have a more considerable effect on the biosynthesis and metabolism of protein.3 conclusionsabcbaaaabfig. 2 contents of soluble sugar, protein and chlorophyll a of phaeodactylum tricornutum grown in the presence of different n concentrations on the 10th day. ln: low n concentration; mn: medium n concentration; hn: high n concentration. each bar denotes the mean of three replicates with standard error. different symbols indicate significant level at p0.05 by duncans new multiple range test.our experimental results have provided the evidence that nitrogen concentration is one of the critical factors influencing the growth and division of phaeodactylum tricornutum. in the present study, ln treatment imposed a remarkable restriction on the cell growth and division of phaeodactylum tricornutum, resulting in decreased cell density, final cell yield as well as specific growth rate, in comparison with mn and hn treatments. on the other hand, nitrogen concentration also could induce distinct changes in the biosynthesis of chemical compositions, such as soluble sugar, protein and chlorophyll a, which were significantly suppressed by low nitrogen concentration. accordingly, nitrogen concentrations should be taken into account for the prevention and treatment of red tides which are dominated by the marine diatom phaeodactylum tricornutum in recent years.acknowledgement: this study was supported by a grant from the national natural science foundation of china (30370231). we thank mr. zhang xiaobing and hu zhangxi in our lab for their assistance in this study and dr. feng yuanjiao for her critical reading of the manuscript.references:1 杜青平, 黄彩娜, 贾晓珊, 等. 1,2,4-三氯苯对3种海洋微藻的毒性效应j.生态环境, 2007, 16(2): 352-357.du qingping, huang caina, jia xiaoshan, et al. the toxic effects of 1, 2, 4-trichlorobenzene on three kinds of ocean tiny algaej. ecology and environment, 2007, 16(2): 352-357.2 hodgkiss i j, ho k c. are changes in n:p ratios in coastal waters the key to increased red tide bloomsj. hydrobiologia, 1997, 352: 141-147. 3 shamsudin l. the blue green algal 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