Positive effects of continuous low nitrate levels on growth and photosynthesis of Alexandrium tamarense (Gonyaulacales, Dinophyceae)

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1 lackwell Science, LtdOxford, UKPREPhycological Research Japanese Society of Phycology Original rticlegrowth and photosynthesis of lexandrium tamarensey. Shi et al. Phycological Research 25; 53: 4348 Positive effects of continuous low nitrate levels on growth and photosynthesis of lexandrium tamarense (Gonyaulacales, Dinophyceae) Yanjun Shi, 1 Hanhua Hu 2 * and Wei Cong 1 1 State Key Laboratory of iochemical Engineering, Institute of Process Engineering, Chinese cademy of Sciences, eijing 18, and 2 Institute of Hydrobiology, Chinese cademy of Sciences, Wuhan 4372, China SUMMRY The growth and photosynthesis of lexandrium tamarense (Lebour) alech in different nutrient conditions were investigated. Low nitrate level (.882 mmol/l) resulted in the highest average growth rate from day to day 1 (8 1 2 s ml -1 d -1 ), but the lowest yield (542 s ml -1 ) in three nitrate level cultures. High nitrate-grown s showed lower levels of orophyll a-specific and a -specific light-saturated photosynthetic rate (P m a and P m ), dark respiration rate (R d and R d ) and orophyll a-specific apparent photosynthetic efficiency (a a ) than was seen for low nitrate-grown s; whereas the s became light saturated at higher irradiance at low nitrate condition. When cultures at low nitrate were supplemented with nitrate at.7938 mmol/l in late exponential growth phase, or with nitrate at.7938 mmol/l and phosphate at.72 mmol/l in stationary growth phase, the yield was drastically enhanced, a 79 times increase compared with non-supplemented control culture, achieving s ml -1 and 52 3 s ml -1, respectively; however, supplementation with nitrate in the stationary growth phase or with nitrate and phosphate in the late exponential growth phase increased the yield by no more than 2 times. The results suggested that continuous low level of nitrate with sufficient supply of phosphate may facilitate the growth of. tamarense. Key words: lexandrium tamarense, growth; nitrate, phosphate, photosynthesis, red tide bloom. INTRODUCTION mong the factors controlling the development of red tide blooms, the nutrient loading has been generally considered to be the most important (Hallegraeff 1993). In field situations, nitrogen and phosphorus are usually subject to temporal and spatial variation in ambient concentrations and rate of supply. Populations in coastal areas may encounter changes in the availability of nutrients during river inputs, rainfall and coastal upwelling. Dinoflagellates are able to migrate vertically to and from the nutricline that also brings transients in nutrient availability (Flynn 22a). lexandrium tamarense (Lebour) alech is known as the main causative organism of paralytic shellfish poisoning in mammals, including humans. Paralytic shellfish poisoning causes economic and public health problems. looms of. tamarense have been recorded in many oceanic regions and have attracted concern from around the world (Glibert et al. 1988; Ichimi et al. 21; Weise et al. 22). ecause strong relationships appear to exist between algal blooms and nutrient enrichment, numerous studies have been carried out on the nutrients that affect growth and toxin production of. tamarense and other dinoflagellate species, and have especially focused on nitrogen and phosphorus supply (oyer et al. 1987; Flynn et al. 1994; Yamamoto & Tarutani 1999; John & Flynn 2). Most studies agreed that nitrogen or phosphorus restriction reduced population growth of dinoflagellates, although its effects on toxin productivity are more complicated (Siu et al. 1997; Flynn 22b; Wang & Hsieh 22). Glibert et al. (1988) investigated photosynthetic responses of. tamarense during growth in a natural bloom and in batch culture; then concluded that the decline of the bloom was a result of the decrease of nitrogen in the water column. However, in field studies, Ichimi et al. (21) observed that phosphorus could have been a limiting factor in the growth of. tamarense and that the cyst formation was induced by depletion of phosphorus source. There is much uncertainty regarding the relationships between nutrient supply and the development of specific species or species groups. Some success has been achieved in linking changes in the N : P ratios with increase in *To whom correspondence should be addressed. hanhuahu@ihb.ac.cn Communicating editor:. Flores-Moya. Received 8 September 23; accepted 8 July 24.

2 44 Y. Shi et al. blooms and in linking the Si : N and Si : P ratios with species changes (Hodgkiss & Ho 1997). In the present study, we report the growth and photosynthetic characteristics of. tamarense grown under nitrate and phosphate sufficient and deficient conditions in batch cultures, and especially focus on the effect of supplementation with nitrate or/and phosphate on the growth of. tamarense initially grown in low nitrate medium. ln (s ml -1 ) MTERILS ND METHODS ln (s ml -1 ) Time (d) Fig. 1. Growth curves of lexandrium tamarense cultured under different NaNO 3 () with middle P and different NaH 2 PO 4 () with middle N: ( ) control (low N) with.882 mmol/l NaNO 3 ; ( and ) with.882 (middle N), and mmol/l NaNO 3 (high N), respectively; (,, and ) with.36 (low P),.36 (middle P), and.18 mmol/l NaH 2 PO 4 (high P), respectively. lexandrium tamarense was obtained from the Institute of Oceanology, Chinese cademy of Science (Qingdao, China). It was cultured at 22 C and 6 mmol photon m -2 s -1 in a 14:1 h LD photoperiod plant growth chamber. The stock culture was grown to its late exponential phase at a concentration of about 1 s ml -1 for use as inoculum. Experiments were conducted in 25 ml Erlenmeyer flasks with 1 ml artificial seawater enriched with f/2 solution (Harrison et al. 198) except nitrate or phosphate was added as designated. NaNO 3 was used to prepare the media that contained.882 (low N),.882 (middle N) and mmol/l NO 3 3 (high N) with.36 mmol/l PO 4 in each culture in triplicate; NaH 2 PO 4 was used to prepare the media that contained.36 (low P),.36 (middle P) and.18 mmol/l PO 3 4 (high P) with.882 mmol/l NO 3 in each culture in triplicate. t low N culture, NO3 or NO3 and PO 3 4 were supplemented to the culture media to gain middle N with middle P or middle N with high P cultures in late exponential growth phase or in stationary growth phase. Cell density was monitored on alternate days by enumeration with a phytoplankton-counting chamber (.1 ml). Specific growth rates (m) were calculated using the equation m= (lnx t - lnx )/t, where X is the initial density, X t is the density after t days. verage growth rate from day to day 1 was calculated using the equation r = (X 1 - X )/1, where X 1 and X represent the density at day 1 and, respectively. Chlorophyll a contents were determined according to Jeffrey and Humphrey (1975), with 9% acetone extracts. Exponentially growing s were harvested and resuspended in artificial seawater enriched with f/2. Their photosynthetic activity was assayed by measuring the rate of O 2 evolution under different irradiances using a Clark-type O 2 electrode (Hansatech Instruments Ltd, UK). The temperature was kept at 22 C by a circulating water bath. Data were treated by non-linear fitting technique using model P = P m tanh (a I/P m ) + R d (Henley 1993), where P is the photosynthesis rate, and I is the light level. P m, light-saturated photosynthesis; a, the initial slope at limiting irradiances, was calculated to assess the photosynthetic efficiency; R d, dark respiration rate. I c, the light intensity at which net photosynthesis rate is zero, was calculated as R d /a. I k, the light intensity at which photosynthesis is initially saturated, was calculated as P m /a. Data are represented as mean and standard deviations obtained from the duplicates (n = 3). Statistical significance of the data was analyzed using a t-test. RESULTS Effect of NO 3 and PO 4 3 levels on growth Growth curves at different nitrate and phosphate levels are shown in Figure 1. Cell density increased at a logarithmic rate from day to day 8. Cell growth exhibited similar patterns at the three nitrate levels, the specific growth rate is not significantly different (P >.5), but yield is markedly different in the three nitrate concentrations (P <.5). t low N level, the average growth rate from day to day 1 is the highest, whereas the maximum density was obtained at middle N

3 Growth and photosynthesis of lexandrium tamarense 45 Table 1. Maximum density, specific growth rate, average growth rate and orophyll a content of lexandrium tamarense (Lebour) alech cultured under different NaNO 3 with middle P and different NaH 2 PO 4 with middle N levels Culture conditions Growth rate Maximum density NaNO 3 NaH 2 PO 4 m day 1 (1 3 s ml -1 ) (mmol/l) (mmol/l) (d -1 ) (1 2 s ml -1 d -1 ) Chlorophyll a content (1-5 mg -1 ) Low N ±.2 8 ± ± ±.37 Middle N ± ± ± ±.51 High N ± ± ± ±.78 Low P ± ± ± ±.29 Middle P ± ± ± ±.51 High P ± ± ± ±.66 Table 2. Maximum density of lexandrium tamarense (Lebour) alech cultured at low nitrate level (.882 mmol/l NaNO 3 ) supplemented with nitrate or phosphate at different times Time Supplementation Maximum density (1 3 s ml -1 ) NaNO 3 (mmol/l) NaH 2 PO 4 (mmol/l) t day ± ±.33 t day ± ±.94 The start concentration of phosphate was.36 mmol/l (middle P). The total nitrate concentration added to the medium was.882 mmol/l, which is equal to the level of middle N. The total phosphate concentration added to the medium was.18 mmol/l, which is equal to the level of high P. level. Cell density at low P level decreased significantly at later growth phase. Moreover, the average growth rate from day to day 1 and yield had the lowest value when s were grown at low P condition. Cells grown at high P concentration showed the highest yield, which increased by 3 times compared with middle P. s shown in Table 1, growth of. tamarense was considerably influenced by nitrate and phosphate levels. Middle N (.882 mmol/l) with high P (.18 mmol/l) concentration culture resulted in the maximum density and the highest average growth rate from day to day 1 at the three phosphate levels. Obviously, to maintain a high growth rate and yield of. tamarense a desired nitrate concentration with a high phosphate level should be considered. Effect of NO 3 and PO 4 3 supplementation on growth s shown in Table 1, the average growth rate from day to day 1 in low N level is the highest among all conditions. We concluded that a lower nitrate level might induce faster propagation of s; therefore, we designated a supplementation experiment to investigate its effect on growth characteristics of. tamarense formerly cultured in low N levels (summarized in Table 2). Maximum density was increased markedly when the culture was supplemented with a suitable amount of NO 3 or NO3 and PO 4 3 at selected times. When NO 3 was added at day 1 and day 14, the density of the culture supplemented at day 1 reached a maximum level of s ml -1, representing a 7 times increase, as compared with the control culture with no supplementation. However, the density reached a maximum level of 52 3 s ml -1, when the culture was supplemented with NO3 and PO 4 3 at day 14, representing a 9 times increase, as compared with the control culture with no supplementation. The difference in enhancement among the supplementation at different times was significant (P <.5). s NO 3 was added at day 1 when the population was still increasing, life was prolonged to grow into stationary phase; meanwhile, division was promoted and density was enhanced drastically. However, as NO3 was added at day 14 when the population growth slowed down and the stationary phase was marked, a short lag phase was observed, followed by an increase in division and density (Fig. 2). Effect of NO 3 and PO 4 3 levels on photosynthesis Representative -specific and orophyll a-specific photosynthesis/irradiance curves for different NO3 and PO 3 4 level cultures are shown in Figures 3 and 4. Photosynthesis becomes light saturated in low nutrient grown s (low N or low P) at higher irradiance than in high nutrient grown s, and I c was higher in low nutrient grown s than in higher nutrient grown s. The photosynthetic parameters are summarized in Tables 3 and 4. High N grown s showed the lowest levels of orophyll a-specific and -specific lightsaturated photosynthetic rate (P a m and P m ), dark

4 46 Y. Shi et al. Table 3. Parameters for photosynthetic-light responses (P-I) curves of lexandrium tamarense (Lebour) alech cultured under different NaNO 3 with middle P levels Photosynthetic parameters Low N Middle N High N P m mmol O 2 (1 7 s h) -1 1 ± ± ± 6.6 P a m mmol O 2 (mg a h) ± ± ± 7.4 a mmol O 2 (1 7 s h) -1 (mmol photons m -2 s -1 ) -1.8 ±.1 1. ±.1.8 ±.1 a a mmol O 2 (mg a h) -1 (mmol photons m -2 s -1 ) ± ±.1.7 ±.1 R d mmol O 2 (1 7 s h) ± ± ± 6. R a d mmol O 2 (mg a h) ± ± ± 6.7 I k mmol photons m -2 s I c mmol photons m -2 s ln (s ml -1 ) day 1 mmol O 2 (1 7 s h) ln (s ml -1 ) day 14 mmol O 2 (mg Chla h) Time (d) Fig. 2. The effect of NaNO 3 or/and NaH 2 PO 4 supplementation at day 1 () or day 14 () on the growth of lexandrium tamarense cultured at low nitrate (.882 mmol/l NaNO 3 ) with middle P level: ( ) control (low N) with.882 mmol/l NaNO 3 ; ( and ) with.7938 mmol/l NaNO 3 supplements at day 1, and day 14, respectively; ( and ) with.7938 mmol/l NaNO 3 and.72 mmol/l NaH 2 PO 4 supplements at day 1, and day 14, respectively. respiration rate (R d a and R d ) and orophyll a- specific apparent photosynthetic efficiency (a a ) in three level nitrate-grown s. Obviously, the orophyll a-specific parameters declined significantly in PFDs (mmol photon m 2 s 1 ) Fig. 3. Representative curves of -specific () and orophyll a-specific () photosynthesis versus irradiance for low N ( ), middle N ( ), and high N ( ) with middle P cultures. contrast to -specific ones in high N culture s. This difference might largely result from the increase in orophyll a content per for high N grown s (Table 1). High P grown s showed the highest values of P m a, a a and R d a, and the lowest values of a and R d. It was related to the lowest orophyll a content in high P grown s (Table 1). Cells exhibited higher physiological activity and rapid metabolic rate in high P culture.

5 Growth and photosynthesis of lexandrium tamarense 47 Table 4. Parameters for photosynthetic-light responses (P-I) curves of lexandrium tamarense (Lebour) alech cultured under different NaH 2 PO 4 with middle N levels Photosynthetic parameters Low P Middle P High P P m mmol O 2 (1 7 s h) ± ± ± 12.4 P a m mmol O 2 (mg a h) ± ± ± 15.7 a mmol O 2 (1 7 s h) -1 (mmol photons m -2 s -1 ) ±.1 1. ±.1.8 ±.1 a a mmol O 2 (mg a h) -1 (mmol photons m -2 s -1 ) ± ± ±.1 R d mmol O 2 (1 7 s h) ± ± ± 1. R a d mmol O 2 (mg a h) ± ± ± 12.7 I k mmol photons m -2 s I c mmol photons m -2 s mmol O 2 (1 7 s h) 1 mmol O 2 (mg Chla h) Fig. 4. Representative curves of -specific () and orophyll a-specific () photosynthesis versus irradiance for low P ( ), middle P ( ), and high P ( ) with middle N cultures. DISCUSSION PFDs (mmol photon m 2 s 1 ) Nitrate and phosphate was the key limiting nutrient for algal growth. In the present study, yield of. tamarense was more significantly decreased in phosphate limitation culture than in nitrate limitation culture. It was probably because the phosphate limitation restricted division, but mild nitrate limitation promoted it to some extent. In addition, high P grown s showed the highest average growth rate at the three phosphate levels. This indicated that sufficient supply of phosphate played an important role in ular metabolism. The patterns of the growth responding to nitrate and phosphate changes were similar to those in other studies, although the highest concentrations of nitrate and phosphate in growth enhancing range were different (Wang & Hsieh 22). This might be because of the artificial seawater enriched with f/2 used in our experiment, while Wang and Hsieh (22) used natural seawater supplemented with K-nutrients. In the present study, more than 2 times increase in yield was observed when phosphate concentration increased from 36 mmol/l to 18 mmol/l, which was much higher than Wang and Hseih reported. Studies have suggested that growth characteristics of algae depend not only on the nutrient concentration, but also on N : P ratios (John & Flynn 2). Our results also confirmed that lower N : P ratio promoted growth and division. t middle N with middle P (N : P = 2) or with high P (N : P = 8.2) conditions both higher average growth rate and higher yield were obtained, while at high N with middle P (N : P = 73.5) or middle N with low P (N : P = 245) condition lower average growth rate and yield were recorded. Photosynthesis was obviously related to the nitrate and phosphate concentrations. Less efficient s need more light to work because of nutrient limitation. Therefore, s under deficient nutrient condition showed higher values of I c and I k, and higher light intensity tends to relieve nutrient limitation to some extent (Geider et al. 1993). The activity of ular metabolism was inhibited by adenosine triphosphate (TP) limitation, H + -TPase limitation and carbon metabolism changes imposed by phosphate limitation. Therefore, the effect of phosphate limitation on photosynthesis is more complicated. The supplementation experiment showed that, in the early stages, the effect of nitrate limitation on growth was not observed as a result of sufficient nitrate for growth on lower density; however, the limitation effect appeared with increasing density and finally resulted in its decrease. When NO 3 was added at day

6 48 Y. Shi et al. 1 in the late exponential phase nitrate limitation was relieved, s continued to grow exponentially and density increased significantly. This suggested nitrate concentration at day 1 after supplementation with NO 3 was optimum for growth at that phase; furthermore, a lower N : P ratio was maintained because of a more rapid uptake rate of nitrate than phosphate by. tamarense, which was also favorable for its growth. The increase in density was small when both NO 3 and PO 3 4 were added at day 1. It might be expected that phosphate was not deficient at that time; furthermore, the nitrate availability was adversely 3 affected because of the N : P ratio change by PO 4 addition. However, at day 14 in the stationary phase when physiological activity decreased, density could not be enhanced by NO 3 addition alone, but significantly increased by both NO 3 and PO 3 4 addition. This increase in density suggested that PO 3 4 addition could affect energy metabolism, alter physiological activity and promote photosynthesis. Previous studies on. tamarense reported density of 2 s ml -1 after 2 days in K-nutrients medium (Wang & Hsieh 22). In the present study, higher density (over 4 s ml -1 ) has been achieved during an equivalent nutrient concentration culture by supplementation in initial low N culture treatment. Our results suggest that a continuous low level of nitrate with sufficient supply of phosphate; namely low N : P ratio, might facilitate the growth of. tamarense. n accordingly low N : P ratio was more likely to stimulate the bloom of. tamarense. In eutrophic coastal waters with high nitrate concentration, some dominant species luxury uptake of nitrate resulted in a decrease in N : P ratio, which might promote the propagation of. tamarense (Hodgkiss & Ho 1997). The phosphate nutrient being used up is an important factor linked with the end of the bloom. In addition, our supplementation method supplied a way to markedly enhance the density of. tamarense. CKNOWLEDGMENTS This present study was funded by the National Natural Science Foundation of China (No ) and by the National Key asic Research Special Foundation of China (Project 21C4976). REFERENCES oyer, G. L., Sullivan, J. J., ndersen, R. J., Harrison, P. J. and Taylor, F. J. R Effect of nutrient limitation on toxin production and composition in the marine dinoflagellate Protogonyaulax tamarense. Mar. iol. 96: Flynn, K. J. 22a. How critical is the critical N : P ratio? J. Phycol. 38: Flynn, K. J. 22b. Toxin production in migrating dinoflagellates: a modeling study of PSP producing lexandrium. Harmful lgae 1: Flynn, K., Franco, J. M., Fernandez, P. et al Changes in toxin content, biomass and pigments of the dinoflagellate lexandrium minutum during nitrogen refeeding and growth into nitrogen or phosphorus stress. Mar. Ecol. Prog. Ser. 111: Geider, R. J., La Roche, J., Greene, R. 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The growth and cyst formation of a toxic dinoflagellate, lexandrium tamarense, at low water temperature in northeastern Japan. J. Exp. Mar. iol. Ecol. 261: Jeffrey, S. W. and Humphrey, G. F New spectrophotometric equations for determining orophylls a, b, c 1 and c 2 in higher plants, algae and natural phytoplankton. iochem. Physiol. Pflanz. 167: John, E. H. and Flynn, K. J. 2. Growth dynamics and toxicity of lexandrium fundyense (dinophycease): the effect of changing N/P supply ratios on internal toxin and nutrient levels. Eur. J. Phycol. 35: Siu, G. K. Y., Young, M. L. C. and Chan, D. K. O Environmental and nutritional factors which regulate population dynamics and toxin production in the dinoflagellate lexandrium catenella. Hydrobiologia 352: Wang, D. Z. and Hsieh, D. D. P. 22. Effects of nitrate and phosphate on growth and C2 toxin productivity of lexandrium tamarense CI1 in culture. Marine Poll. ull. 45: Weise,. M., Levasseur, M., Saucier, F. J. et al. 22. The link between precipitation, river runoff, and blooms of the toxic dinoflagellate lexandrium tamarense in the St. Lawrence. Can. J. Fish quat. Sci. 59: Yamamoto, T. and Tarutani, K Growth and phosphate uptake kinetics of the toxic dinoflagellate lexandrium tamarense from Hiroshima ay in the Seto Inland Sea, Japan. Phycol. Res. 47: 2732.

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