Botanical Studies (2012) 53: 125-133.
The effects of temperature on the growth of and ammonia uptake by marine microalgae
Shih-Yuan CHEN, Li-Ying PAN, Min-Jhe HONG, and An-Chin LEE*
Department of Aquatic Biosciences, College of Life Science, National Chiayi University, Chiayi 600, Tai^wan
(Received June 8, 2010; Accepted September 21, 2011)
ABSTRACT. Four species of microalgae were evaluated at different temperatures for their ability to remove ammonia from intensive marine fish/shrimp culture systems. Growth rates were highest on the first of three days of culturing for marine microalgae Nannochloropsis oculata, Isochrysis aff. galbana, Chaetoceros muel-leri, and Tetraselmis chui. This rate was used to compare both the growth and the total ammonium nitrogen (TAN) uptake efficiencies of these organisms at different temperatures. Their dry weights (DWs) per cell were 10.3 pg for Nannochloropsis oculata, 40.4 pg for Isochrysis aff. galbana, 39.7 pg for Chaetoceros muelleri, and 369 pg for Tetraselmis chui at 25°C. Temperatures for their respective optimal growth rates were 26, 28, 33, and 25°C. There were no significant differences in growth rates among these four microalgal species at 25°C. Temperatures for maximal biomass production were 30°C for both Nannochloropsis oculata and Iso-chrysis aff. galbana, 35°C for Chaetoceros muelleri, and 20~30°C for Tetraselmis chui. Temperatures for maximal specific TAN uptake were respectively 25, 25, 20~30, and 25°C. Overall, Tetraselmis chui was the most efficient at TAN uptake among the four species. Our results suggest that T. chui is a good choice for re­moving ammonia from indoor intensive marine culture systems.
Keywords: Algal production; Ammonia; Growth rate; Marine microalgae.
The growth of microalgae can be affected by many factors, such as nutrients, light intensity, salinity, pH, and temperature. Temperature stress, in particular, influences the growth rate and chemical composition of microalgae and may limit nutrient interactions. Temperature has a major effect on the phase transition of membrane lipids, the kinetics of cellular enzymes, and active transport systems across membranes (Quinn and Williams, 1983; Wheeler, 1983). Microalgal growth rates can be stimulated and cells become smaller at higher temperatures (Rijssel and Gieskes, 2002). Although a high growth temperature was related to a significant decrease in protein content and increases in lipids and carbohydrates in Spirulina species (Tomaselli et al., 1988; Oliveira et al., 1999), the response of microalgal chemical compositions to high and low growth temperatures varies from species to species (Thompson et al., 1992a; Renaud et al., 1995).
Ammonia is formed as the principal product of protein metabolism in aquatic organisms. Ammonia exists in two forms that are convertible in water, NH3 and NH4+, the sum of which is called total ammonia nitrogen (TAN). The ratio of NH3/NH4+ varies with water pH, ionic strength,

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Tel: 886-5-2717849; Fax: 886-5-2717847.
pressure, and temperature. The proportion of un-ionized ammonia (NH3), the more-toxic form to aquatic organ­isms, increases with water pH (Randall and Tsui, 2002; Ip and Chew, 2010). Ammonia can be removed by bacte­rial biofilters (Tseng et al., 1996; Grommen et al., 2002), heterotrophic bacterial biofilters (Avnimelech et al., 1992; Avnimelech, 1999), seaweed biofilters (Cohen and Neroi, 1991; Neori et al., 1996 and 2000), and microalgae (Tseng et al., 1991; Voltolina et al., 2005; de Godos et al., 2010). A bacterial biofilter converts ammonia into nitrate in two steps through the action of ammonium- and nitrite-oxidizing bacteria. However, the end product, nitrate, still remains in the water. Although nitrate is relatively harm-less to aquatic organisms (Russo and Thurston, 1991), levels considered toxic to them (1000∼3000 mg-L-1) should be avoided (Lawson, 1995). Heterotrophic bacteria can absorb ammonia as a precursor for cellular protein synthesis, but the C/N ratio imposes certain limitations on their growth. In addition to ammonia removal, microalgae can also produce biomass which can be used in several ways to improve the economic efficiency of aquaculture systems (Tenore, 1976; Arieli et al., 1993; Muller-Feuga et al., 2003; Lubzens and Zmora, 2003; Plaza et al., 2010; Rodriguez-Meizoso et al., 2010).
Uptake rates of nitrate and ammonium were studied as a function of nitrate or ammonium concentrations with cultures of Chaetoceros gracilis and Isochrysis galbana (Eppley et al., 1969). Although the half-saturation constant
Botanical Studies, Vol. 53, 2012
(the concentration at one-half the maximum uptake rate) for the uptake of nitrate (0.30.1 fiM) was compared to that of ammonium (0.50.3 fiM) in C. gracilis, ammonium is the preferred nitrogenous nutrient of many marine phytoplankton species (McCarthy, 1980). Since free ammonia is toxic to algae, it does not accumulate in their cells but is stored in the system through an ammonia-binding reaction. Glutamate dehydrogenase and glutamate synthetase activities are higher in green algae, which are peculiar in having a highly effective pathway to detoxify ammonia (Klochenko et al., 2003). Microalgae exhibit an active, and most likely a passive, ammonium uptake. The proportion of ammonia in marine systems can increase to > 10% of TAN. The concentration gradient of ammonia across a cell membrane can be maintained by ammonia protonation within the cell and equilibration between ammonium and ammonia outside the cell (Henderson, 1971). However, nitrate is apparently taken up by an active transport system in algae, and this transport system appears to be ATP-driven rather than directly dependent on an electrochemical gradient (Falkowski, 1975; Stewart, 1980; Wheeler, 1983). Therefore, the uptake rate of ammonium is less temperature-dependent than that of nitrate.
Aquatic organisms in Taiwan widely consume Nan-nochloropsis oculata, Isochrysis aff. galbana, C. muelleri, and Tetraselmis chui as live food (Su et al., 1997). The am­monia produced from intensive marine fish/shrimp farms deteriorates water quality but can be removed by these microalgae, which are then consumed by bivalves as food (Forrest et al., 2009). However, information on the relative efficiencies of ammonia uptake among these four species of microalgae is scant. The aim of this study was to evalu­ate the efficiency of ammonia uptake by these four species of microalgae at various temperatures and finally, to select a candidate for removing ammonia from intensive marine fish/shrimp culture systems.
Artificial seawater (ASW) preparation and microalgae maintenance
ASW was prepared according to APHA et al. (1995) guidelines, and its composition is given in Table 1. ASW (33 g-L-1) was freshly prepared and aerated for 3 days. Tetraselmis chui (Chlorophyta), I. aff. Galbana (Haptophyta), C. muelleri (Bacillariophyta), and N. ocu-lata (Eustigmatophyceae) were a gift from the Tungkang Biotechnology Research Center, Fisheries Research Insti­tute, Pingtung, Taiwan. They were maintained in Walne's Enriched Seawater (WES) medium (Walne, 1974) as modified by Su (1999) at a continuous light intensity of 10 [iEm-2s-1 and 15°C.
Preparation of inoculated microalgae
Microalgae were acclimated to various temperatures (i.e., 15, 20, 25, 30, and 35°C) for 1 week prior to the experiment. Twenty milliliters of microalgae was inocu-
lated in 250-mL flasks with 180 mL of WES medium at a continuous light intensity of 180 fiE m-2 s-1 with shaking (125 rpm, Orbital shaker TS-520) until the microalgae grew to OD750nm values of 0.2~0.3. One hundred milliliters of microalgae was transferred to 1-L flasks with 900 mL of WES medium aerated at a level of 3 L-min-1 and a con­tinuous light intensity of 180 fiE m-2 s-1. Preparation was completed after these microalgae had grown to OD750nm values of 0.5~0.6.
Growth rate of microalgae and their TAN uptake at different temperatures
In order to apply this approach in the field, only am­monia and phosphate were added in the experiment on microalgal growth and TAN uptake. Fifty milliliters of mi-croalgae was inoculated in 1-L flasks with 950 mL of ASW containing 13 mg-L-1 TAN-N and 4 mg-L-1 PO43--P aer­ated at 5 L-min-1 under a continuous light intensity of 180 [iE-m-2-s-1. The cell densities of T. chui, I. aff. galbana, C. muelleri, and N. oculata at OD750nm values of 0.5~0.6 were approximately 1.2 x 106, 6 x 106, 7 x 106, and 1.9 x 107 cells/mL, respectively. Therefore, their initial respective concentrations were 6 x 104, 2.9 x 105, 3.4 x 105, and 9.3 x 105 cells-mL-1. After 1 day of culture, the microalgae were sampled to determine their cell densities and dry weights, as well as the concentration of TAN remaining in the cul-ture. The experiments were performed in duplicate at 15, 20, 25, 30, and 35°C
Analytical methods
Growth rate and cell density. After the microalgae were sampled, 0.5% povidone iodine was added to immo­bilize them. The cell density of the microalgae was quanti­fied with a 0.1-mm-deep Neubauer chamber in duplicate. The growth rate was calculated as:
μ = {log2(N2) - log2(N1)}/(t – t0);
where μ is the growth rate (divisions day-1), and N1 and N2 represent initial (t0) and at t time cell densities (cells-mL-1) in the medium, respectively.
Table 1. Composition of artificial seawater (36 g L-1).


Concentration (g-L-1)


CHEN et al. ― Growth and ammonia uptake by microalgae
Table 2. Dry weight (pg) per cell ot marine microalgae at various temperatures. Data represent the mean the standard deviation. Different letters indicate significant differences in a specific species of microalgae among different temperatures (p<0.05).
Temperature (°C)



(Dry weight (pg) cell-1)



Nannochloropsis oculata





Isochrysis aff. galbana
Chaetoceros muelleri
Tetraselmis chui
Dry weight. This assay was based on a method de­scribed by D'Souza and Kelly (2000). The dry weight of the microalgae was determined by filtering 100 mL of microalgae onto precombusted glass-fiber filters (What­man GF/C for T. chui, I. aff. galbana, and C. muelleri; GF/ F for N. oculata, 47 mm in diameter) and washing with 0.5 M ammonia formate to remove residual salts from the culture. The filters were then dried at 60°C until a constant weight was reached.
TAN remaining and specific uptake. Twenty mil-liliters of microalgal medium was sampled and centrifuged at 14,000 xg for 15 min at 4°C. After centrifugation, the supernatant was removed and used to determine the TAN concentration. TAN determination was according to the phenol hypochlorite method in APHA et al. (1995). Spe­cific TAN uptake (qt) was calculated by using the equation below:
Figure 1. Growth rates of four microalgae species on the first, second, and third days of culture at 25°C. Each bar represents the mean value with the standard deviation. Different letters in­dicate a significant difference in a specific species of microalgae among different days culture (p < 0.05).
where Co and Ct (mg-L-1) represent the TAN concentration initial and at t time in the medium. V(L) is the volume of the medium, M(g) is the biomass (g).
Data analysis
The mean values of growth rates, and of TAN removal at studied conditions were compared by variance analysis (ANOVA) using SigmaStat statistical software from SPSS (SPSS, Chicago, IL, USA), ver. 10. Duncan's test for pair-wise comparisons was used at the 5% significance level.
Dry weights per cell of marine microalgae
Dry weights per cell of I. aff. galbana and C. muelleri were temperature-independent at temperatures of < 30°C (Table 2). Among these four microalgal species, only C. muelleri could grow at 35°C. The dry weight of C. muel-leri at 35°C was much higher than those at < 30°C. The dry weight of N. oculata at 20, and 25°C was significantly lower than those at 15, and 30°C. However, the dry weight of T. chui at 30°C was significantly higher than those at < 25°C.
Temperature effect on the growth rates (p) and biomass production
Microalgae in log-phase were inoculated into ASW only with
13 mg-L-1 TAN-N and 4 mg-L-1 PO43-- P at 25°C. The 3-day microalgal culture growth rates are given in Figure 1. First-day growth rates for N. oculata and C. mu-elleri were significantly higher than those for the second and third days. First-day growth rates of I. aff. galbana and T. chui were comparable to those for the second day but significantly higher than those for the third day. There-fore, first-day growth rates for the four microalgal species could be used to evaluate their growth rates and their TAN uptake rates at different temperatures.
Figure 2 shows the microalgal growth rates at different temperature levels. The f values for N. oculata and T. chui were significantly greater than those for I. aff. galbana and C. muelleri at 20°C, while values of the latter were signifi­cantly greater than the former at 30°C. Based on polyno­mial regression data, respective optimal temperatures for N. oculata, I. aff. galbana, C. muelleri, and T. chui growth were 26, 28, 33, and 25°C.
Botanical Studies, Vol. 53, 2012
Figure 3 shows the biomass production in dry weight (DW, mg-L-1) per liter of microalgae at various tem­peratures. Biomass values of T. chui, I. aff. galbana, C. muelleri, and N. oculata were 85, 43, 39, and 33 DW mg-L-1-day-1 at 25°C, respectively. The N. oculata and I. aff. galbana biomass production levels at 30°C were sig­nificantly higher than those at 1525°C. T. chui biomass production at 15°C was significantly lower than it was be­tween 20 and 30°C, where there was no significant differ­ence. The biomass production of C. muelleri at 35°C was much higher than levels at 15~30°C.
Effect of temperature on microalgal TAN uptake
The specific TAN uptake rates of microalgae at different temperatures are given in Figure 4. The optimal temperature for specific TAN uptake by N. oculata, I. aff. galbana, and T. chui was 25°C. However, there was no significant change in the specific TAN uptake rates by C. muelleri between 20 and 30°C. The specific TAN uptake rates by N. oculata and T. chui were higher than those for I. aff. galbana and C. muelleri at 25°C. Figure 5 shows the efficiency of the daily TAN uptake per liter for the four microalgal species at various temperatures. The daily TAN uptake rate for T. chui was much greater than for the other three microalgal species at all temperatures tested. The temperatures for optimal TAN uptake by T. chui, I. aff. galbana, and C. muelleri were 25, 25, and 35°C. Howev­er, TAN uptake by N. oculata at 25°C was comparable to that at 30°C. TAN uptake by T. chui at 25°C was roughly 2-fold that at other temperatures and 3-fold that of other microalgal species. Although the specific TAN uptake rate (111 mg TAN-N-g-1 dry weight-day-1) of T. chui was smaller than that (130 mg TAN-N-g-1 dry weight-day-1) for N. oculata at 25°C, the biomass produced by the former (85 mg dry weight-L-1-day-1) was much greater than for the latter (33 mg dry weight-L-1-day-1). T. chui was thus more efficient at daily TAN uptake per liter due to this higher biomass production.
Effects of temperature on the dry weight per cell of microalgae
It is generally recognized that microalgal cell sizes are inversely proportional to their growth rate, which increases with temperature in a certain range (Atkinson et al., 2003; Sayegh and Montagnes, 2011). Therefore, cell
Figure 2. Effects of temperature on the growth rate of microal-gae. The polynomial regressions of the growth rate (f) against temperature (T) between 15 and 35°C were: Nannochloropsis oculata: i = -1.02 + 0.066T + 0.0077T2 - 0.0002T3, R2 = 0.96; Isochrysis aff. galbana: fi = 10.03 - 1.683T + 0.091T2 - 0.00153, R2 = 0.99; Chaetoceros muelleri: fi = 3.364 - 0.44T + 0.023T2 -0.0003T3, R2 = 0.98; and Tetraselmis chui: fi = 3.78 - 0.74T + 0.05T2 - 0.001T3, R2 = 0.99.
Figure 3. Effects of temperature on the daily algal biomass production per liter of microalgae. Each bar represents the mean value with the standard deviation. Different letters indicate sig­nificant differences in a specific species of microalgae among different temperatures (p < 0.05).
Figure 4. Effects of temperature on specific total ammonia nitrogen (TAN) uptake of microalgae. Each bar represents the mean value with the standard deviation. Different letters indicate significant differences in a specific species of microalgae among different temperatures (p < 0.05).
CHEN et al. ― Growth and ammonia uptake by microalgae
cultures. These smaller sizes may be due to nitrogen nutri­ent limitation. Lourenco and Barbarino (1998) reported that the amount of total inorganic nitrogen T. gracilis ac­cumulated at the mid-log phase was 2.5-fold that of its sta­tionary phase. The discrepancy in T. chui dry weight may have been due to the microalgae being in different phases.
Temperature effects on the growth rate (μ) and biomass production
As with many rate processes, the microalgal growth rate is expected to increase with temperature, and rapidly decline above the optimal temperature. Low temperatures usually reduce enzymatic activity, which causes a decrease in the growth rate (Davison, 1991). Minimal growth rates were found at 15°C for all four microalgal species. The temperature tolerance of C. muelleri was the high­est among these microalgae. Renaud et al. (2002) found a similar pattern in Chaetoceros sp., which grew well at 33 and 35°C. However, Isochrysis sp. and a commercial Iso-chrysis sp. (clone T.ISO) grew very slowly at 35°C (Re-naud et al., 1995). Thompson et al. (1992b) also claimed that the microalgal growth response to temperature is species-specific.
The growth rate (0.99 day-1) of I. aff. galbana grown at 20°C in this study was higher than the average (0.65 day-1) of that during 7 days of culture (Valenzuela-Espinoza et al., 1999) and was comparable to that (0.84 day-1) in the comparative study on growth performance and biochemi­cal composition of mixed culture of Isochrysis galbana and Chaetoceros calcitrans with monocultures (Phatar-pekar et al., 2000). The T. chui growth rate (1.63 day-1) at 180 μE-m-2-s-1 light intensity at 20°C in this study was much higher (0.64 day-1) than for T. chui grown at 110 μE-m-2-s-1 light intensity at 18°C (Meseck et al., 2005). Meseck et al. (2005) cultured T. chui with Guillard's f/2-enriched medium, which contains 13 fM ethylenedi-aminetetraacetic acid (EDTA) (Guillard, 1975). Although EDTA can alleviate the toxic effects of some metals (e.g., Cd, Cu, and Mn) and increase the bioavailability of oth­ers (e.g., Fe) by preventing precipitation, it suppressed the growth of oceanic phytoplankton (Muggli and Harrison, 1996; Okauchi and Kawamura, 1997). In fact, we found that the growth rate for T. chui after 1 day of culture in medium containing only ammonium and phosphate was higher than for WES medium (data not shown).
TAN-N uptake by microalgae
Although nitrogen is available to microalgae in various forms, nitrate, ammonium ions, and urea are the dominant forms (Syrett, 1981). Among these, ammonium ions are preferentially taken up, followed by nitrate and urea (Le-vasseur et al., 1990; Levasseur et al., 1993). Less energy is required to take up ammonium, and its more-reduced state may be the reason it is preferred over nitrate (Dortch, 1990). Ammonia is generally thought to directly assimilate into the amino acid, glutamine (Flynn, 1991). However, some species, e.g., Hillea sp. and Prorocentrum minimum,
Figure 5. Effects of temperature on daily total ammonia nitro­gen (TAN) uptake per liter by microalgae. Each bar represents the mean value with the standard deviation. Different letters in­dicate significant differences in a specific species of microalgae among different temperatures (p<0.05).
sizes are larger at low temperatures or above the optimal temperature. N. oculata showed a higher dry weight per cell at 15°C, however, the effect of temperature on dry weights per cell of T. chui, I. aff. galbana, and C. muelleri ,was not significant. Nitrogen sources from ammonium can probably explain this unexpected phenomenon, since the ammonium uptake rate is less temperature-dependent than that of nitrate, which is the sole nitrogen nutrient in many medium formulas.
Based on dry weight per cell, the order of the four microalgal species was T. chui > I. aff. galbana > C. mu-elleri > N. oculata at 25°C. The value of 40~51 pg dry weight-cell-1 for I. aff. galbana obtained in this study was comparable to data (36~46 pg dry weight-cell-1) reported by Brown (1991) and by Brown et al. (1998). The cell dry weight of Isochrysis sp. (clone T-ISO) was affected by the irradiance, with 30 and 62 pg dry weight-cell-1 at 50 and 500 fE-m-2-s-1, respectively (Brown et al., 1993a). Brown et al. (1993b) reported a 10 pg-cell-1 dry weight for N. oculata, which is in the 9.8~13 pg-cell-1 range in this study. No significant difference in the dry weights (35.4~41.7 pg-cell-1) for C. muelleri cultured at 15~30°C was observed in this study. The dry weight of C. muel-leri cultured at 35°C, however, sharply increased to 81.4 pg-cell-1. Among these four microalgal species, only C. muelleri could grow at 35°C. High-temperature toler-ance by Chaetoceros species was also reported by Renaud et al. (2002), who observed that its dry weight increased from 25 pg-cell-1 at 25°C to 81.7 pg-cell-1 at 35°C. A sig-nificantly lower dry weight (369 pg-cell-1) for T. chui at 25°C than (539 pg-cell-1) at 30 °C may have been due to a much-higher growth rate (2.0) at 25°C than (1.27) at 30°C. However, Wikfors et al. (1996) determined a 104~135 pg-cell-1dry weight for T. chui harvested in a nitrogen-deficient stationary phase by semi-continuous, E-medium
Botanical Studies, Vol. 53, 2012
failed to grow with ammonium-N because of ammonia's toxic effects in high concentrations (Lourenco et al., 2002). Similarly, I. galbana grown with ammonium-N had a lower growth rate (Lourenco et al., 2002). Valenzuela-Espinoza et al. (1999) reported that 2.16 mg ammonium-N-L-1 was taken up by I. galbana after the first day of incubation. This value is very close to the 2.06 mg TAN-N-L-1 in our data. A similar phenomenon was observed with N. oculata. The amount of ammonium-N (3.73 mg ammonium-N-L-1) taken up by microalgae reported by Su et al. (1997) was comparable to the 4.27 mg TAN-N-L-1 in our study. A rate of 9.4 mg TAN-N-L-1-day-1 taken up by T. chui at 25°C can be approximately expressed as 40 fg am-monium-N-10-6 cells-day-1 which is 2-fold that taken up by Chlorella vulgaris grown in 20 mg ammonium-N-L-1 (Tam and Wong, 1996). The ammonium uptake efficiency of T. chui was comparable to that of Scenedesmus obliquus, which took up 9.27 mg TAN-N-L-1-day-1 in a semi-contin-uous culture with a 30% water exchange rate (Voltolina et al., 2005).
Less than 30% and 21% of the supplied nitrogen is re­spectively recovered as harvested fish and shrimp by aqua-culture (Porter et al., 1987; Hall et al., 1992; Briggs and Funge-Smith, 1994). Most nutrients are released into the environment and contribute to water pollution. In addition to environmental impacts, discharging nutrients causes economic losses and reduces farm profitability. The dis-charged nutrients could be taken up by microalgae which could then be used as aquaculture feed. Although T. chui is more efficient at TAN removal, its growth is greatly limited above 30°C (data not published). Therefore, T. chui is not a practical solution to the problem of TAN removal from outdoor ponds in the summer in Taiwan. Tseng et al. (2006) reported that the water quality of the shrimp ponds was not well controlled by the addition of T. chui. It is very important to maintain the dominant microalgal species when removing TAN from aquacultural systems. Those microalgal species dominant in batch reactors are more stable than those found in continuous-flow reactors (Tseng et al., 1991). In our previous work, we used batch reactors in a recirculating aquaculture system for indoor tilapia culture (Chen et al., 2010). In that study, T. chui took ammonia up so that TAN levels in the tilapia tank water remained at < 3 mg-L-1. Therefore, T. chui is a good candidate for removing ammonia from intensive cul-ture systems.
Acknowledgements. The authors are grateful for the financial support from the National Science Council (NSC97-2313-B-415-003-MY3), Taipei and National Chia-Yi University (NCYU 97T001-05-06-001), Chiayi, Taiwan.
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CHEN et al. ― Growth and ammonia uptake by microalgae
陳士元 潘麗英 洪敏哲 李安進
擬球藻 (Nannochloropsis oculata)、等鞭金藻 (Isochrysis aff. galbana)、牟氏角毛藻 (Chaetoceros
) 和周氏扁藻 (Tetraselmis chui) 在三天的培養中以第一天的生長速率最快。將四種微藻培養
時,每個細胞的乾重分別為10.3、 40.4、39.7 和369 pg。它們的最適成長溫度分別為26、 28、33 和
25°C。在25°C 時,四種微藻的生長速率沒有顯著的差異。四種微藻最大生產量的溫度分別為30、30、
35、和20~30°C,四種微藻最大比總氨氮吸收效率的溫度分別為25、25、20~30 和25°C。在所有測試
的溫度中,周氏扁藻總氨氮吸收的效率在四種微藻中是最高的。而且周氏扁藻在25°C 的總氨氮吸收效