J Appl Phycol (2012) 24:965–971 DOI 10.1007/s10811-011-9718-8
Competition between the cyanobacterium Microcystis aeruginosa and the diatom Cyclotella sp. under nitrogen-limited condition caused by dilution in eutrophic lake Yoshimasa Amano & Kosei Takahashi & Motoi Machida Received: 15 January 2011 / Revised and accepted: 8 September 2011 / Published online: 23 September 2011 # Springer Science+Business Media B.V. 2011
Abstract The improvement of water quality in Lake Tega, Japan, has been carried out by dilution, causing the shift of dominant species from Microcystis aeruginosa to Cyclotella sp. in summer. The disappearance of Microcystis blooms would be related to dilution, but the detail effect has not been understood yet. In this study, the effect of nitrate concentration on the competition between M. aeruginosa and Cyclotella sp. was investigated through the singlespecies and the competitive culture experiments. The single-species culture experiment indicated that the half saturation constants for M. aeruginosa and Cyclotella sp. were 0.016 and 0.234 mg N L−1, representing that M. aeruginosa would possess a higher affinity to nitrate. On the other hand, the maximum growth rate for Cyclotella sp. was obtained as 0.418 day−1, which did not represent a significant difference with 0.366 day−1 obtained for M. aeruginosa. The competitive culture experiment revealed that Cyclotella sp. completely dominated over M. aeruginosa at the nitrate concentrations of 0.5 and 2.5 mg N L−1. The dominance of Cyclotella sp. could be attributed to the difference in the abilities of nitrate storage as well as nitrate uptake. One of the possibilities for the disappearance of Microcystis blooms caused by dilution as observed in Lake Tega could be due to the decrease in nitrate concentration, and the lower N:P ratio seemed not to relate to Microcystis blooms. Y. Amano (*) : K. Takahashi : M. Machida Graduate School of Engineering, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba-shi, Chiba 263-8522, Japan e-mail:
[email protected] Y. Amano : M. Machida Safety and Health Organization, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba-shi, Chiba 263-8522, Japan
Keywords Competition . Cyclotella sp. . Dilution . Eutrophic lake . Microcystis aeruginosa . Nitrogen-limited condition
Introduction There have been many articles regarding to the improvement of water quality in eutrophic lakes, e.g., removal of nutrients by chemicals and/or aquatic plants, dredging, nutrient loading control, and so on (Gelin and Ripl 1978; Zhang and Prepas 1996; Lane et al. 2001; Pendleton et al. 2001). Dilution is one of the effective methods for the improvement of water quality as well as suppression of water blooms, and several attempts have been put into practice (Oglesby 1969; Welch et al. 1972; Welch and Patmont 1980; Welch 1981; Hosper and Meyer 1986; Hu et al. 2010). In Tega-numa (Lake Tega, Japan), which had the worst water quality from 1974 to 2000 in Japan, an attempt for the improvement of water quality has been carried out by dilution transferring river water into the lake in 2000. After that, the dominant species of phytoplankton have shifted from cyanobacteria (mainly, Microcystis aeruginosa) to diatoms (mainly, Cyclotella sp.) in summer. According to the data reported by Chiba Prefectural Government (2011), the cell densities for cyanobacteria and diatoms were 1.4×105 cells mL−1 (84% in total cells) and 1.6×104 cells mL−1 (9%) as the mean value from 1998 to 1999, whereas the mean cell density from 2005 to 2009 was 2.8×102 cells mL−1 (1%) for cyanobacteria and 4.1×104 cells mL−1 (74%) for diatoms during summer (July, August, and September). The dilution supplies one order of magnitude greater water with 4.7×105 m3 day−1, 1/10 lower phosphorus concentration of 0.06 mg P L−1 and 1/2 lower nitrogen concentration of 2.4 mg N L−1 than those for the existent
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8.7 mg L−1 (1.6 mg P L−1) and Na2SiO3 9H2O of 111 mg L−1 (11 mg Si L−1) was used to culture M. aeruginosa and Cyclotella sp. Both species were grown in a 200 mL of WC medium in a 500 mL Erlenmeyer flask at a pH of 8.0, which reflects the pH in Lake Tega. The cultivation temperature and light intensity were set as 20°C and 27 μmol photons m−2 s−1 with a light and dark cycle of 14 h:10 h. Each flask was randomly displaced and stirred by hand several times a day.
river inflows (Chiba Prefectural Government 2011; Amano et al. 2010). This implies that the change in dominant species from M. aeruginosa to Cyclotella sp. would be related to the change in dilution rate, phosphorus and/or nitrogen concentration. Tatsumoto et al. (2007) experimentally examined the influence of dilution rate (defined as the inflow volume of medium per unit time divided by the total volume of culture medium) on the change in dominant species from M. aeruginosa to Cyclotella sp., and the results clearly showed that the change in dilution rate did not affect the shift of dominant species. In the previous studies (Amano et al. 2009, 2010), the effect of phosphorus concentration with a high silicon concentration (11 mg Si L−1) on the competition between M. aeruginosa and Cyclotella sp. was investigated, and the results showed that the reduction of phosphorus concentration led to the dominance of M. aeruginosa, and Cyclotella sp. never dominated over M. aeruginosa, even phosphorus concentration was reduced to 0.01 mg P L−1. Consequently, our previous studies concluded that the phosphorus concentration was not the influential factor for the shift of dominant species as observed in Lake Tega. The effects of dilution for the enhancement of lake water quality are generally understood as mainly a decrease and/ or increase in concentrations of various nutrients, which limit and/or prompt the algal growth. The dilution for water quality improvement has been demonstrated in many lakes mentioned above. Many studies showed that the nutrient concentrations, such as nitrogen and phosphorus, were lowered by dilution, but some reports still observed the appearance of water blooms although others had successful results. Thus, the relationship between the appearance of water blooms and the change in water quality caused by dilution still remains unclear. In this study, the effect of nitrogen concentration on the competition between M. aeruginosa and Cyclotella sp. was experimentally examined. The single-species culture experiment was conducted to reveal the growth kinetics for both species, while the competitive culture experiment was done to examine their competitive abilities. The experimental results obtained in this study were related to the conditions in Lake Tega after the dilution, and the mechanisms of transition for algal species were discussed.
Single-species culture experiment was used to examine the growth characteristics of both species under nitrogenlimited condition. Both species were individually grown for 7 days in the WC medium at the nitrate concentration of 0.1 mg N L−1 to adapt them to the nitrate-deficient circumstance. Then 200 mL of the WC medium in 500 mL Erlenmeyer flasks, in which nitrate concentrations ranged from 0.1 to 5 mg N L−1, were inoculated into the nitrate-deficient cultures for each species. The initial cell density was 5,000 cells mL−1. The cultivation conditions were the same as above. An aliquot of each sample was taken every 2–3 days during the experiment, and the cell counting for the determination of cell density was performed twice for each sample. The experiment was done in duplicates, and the result is represented as the mean value ± standard deviation. The growth rate, μ (day−1), based on the exponential growth phases of M. aeruginosa and Cyclotella sp., was obtained by following equations:
Materials and methods
m ¼ mmax
Microcystis aeruginosa (strain: UTEX 2061) and Cyclotella sp. (strain: CCAP 1070/4) were obtained from the Culture Collection of Algae, the University of Texas, and from the Culture Collection of Algae and Protozoa, respectively. Wright’s cryptophytes (WC) medium (Guillard and Lorenzen 1972) with NaNO3 of 85 mg L−1 (14 mg N L−1), K2HPO4 of
Growth characteristics of M. aeruginosa and Cyclotella sp.
dC ¼ mC; dt
m¼
lnðC2 C1 Þ ; t2 t1
ð1Þ
ð2Þ
where C1, C2, t1 and t2 were the cell density (cells mL−1) for M. aeruginosa and/or Cyclotella sp. at the elapsed time of t1 (day), and the cell density at t2 (day). The maximum growth rate, μmax (day−1), and saturation constant, KN (mg N L−1), were estimated using each μ value based on the following equations: N ; KN þ N
ð3Þ
N N m þ max ; ¼ KN m mmax
ð4Þ
where N is the initial nitrate concentration (mg N L−1) for the single-species culture experiment.
J Appl Phycol (2012) 24:965–971
967 0.5
Competitive ability of M. aeruginosa and Cyclotella sp. under nitrogen-limited condition Growth rate (day −1)
0.4
The competitive culture experiment was conducted to examine the competitive ability of both M. aeruginosa and Cyclotella sp. under nitrate-limited condition. A set of 200 mL of the WC medium in 500 mL Erlenmeyer flasks with the nitrate concentration of 0.1, 1.0 and 2.5 mg N L−1 was inoculated into the mixed nitrate-deficient medium of M. aeruginosa and Cyclotella sp. The nitrate concentration of 2.5 mg N L−1 reflected the concentration of Lake Tega water after the dilution was undertaken. The initial cell density was 5,000 cells mL−1 for each species. The other experimental conditions were the same as above.
0.3
0.2
0.1
0
0
2
4
6
Nitrate concentration ( mg N L −1)
Fig. 2 Growth rate of Cyclotella sp. in nitrate-limited conditions
Results Growth characteristics under nitrate-limited condition M. aeruginosa and Cyclotella sp. were grown in all media in the single-species culture experiment. The exponential phases for the growth of M. aeruginosa were observed during days 1–7 for N=0.1 and 0.25 mg L−1, days 1–10 for N=0.5 mg L−1, days 1–13 for N=1.0, days 1–15 for 2.5 mg L−1, and days 1–18 for N=5.0 mg L−1. On the other hand, the exponential growth of Cyclotella sp. was achieved during days 2–7 for N=0.1 and 1.0 mg L−1, days 2–9 for N = 0.25, 0.5 and 5.0 mg L−1 , and days 4–14 for N=2.5 mg L−1. The cell densities during the exponential growth phase for each species were substituted to Eq. (2), and the growth rates of M. aeruginosa and Cyclotella sp. as a function of nitrate concentration are illustrated in Figs. 1 and 2. The growth rate for M. aeruginosa was faster for all nitrate concentrations, even when the concentration was as
0.5
Growth rate (day −1)
0.4
0.3
0.2
0.1
0
0
2
4
6
Nitrate concentration ( mg N L −1)
Fig. 1 Growth rate of M. aeruginosa in nitrate-limited conditions
low as 0.10 mg N L−1. In contrast, the growth rate of Cyclotella sp. in the early stage was slower than that of M. aeruginosa. The half saturation constant for M. aeruginosa was estimated as 0.016 mg N L−1, which was much lower (P0.05) between both species. Competitive ability of M. aeruginosa and Cyclotella sp. under nitrate limitation The result of the competitive culture experiment between M. aeruginosa and Cyclotella sp. under nitrate-limited conditions is shown in Fig. 3a–c. M. aeruginosa and Cyclotella sp. could not grow at 0.1 mg N L−1, with no significant difference (P>0.05) between both species at the end of the experiment (day 23; Fig. 3a). At 0.5 mg N L−1, the cell density for M. aeruginosa (4.5×104 cells mL−1) was significantly lower (P
