Marine Pollution Bulletin 92 (2015) 52–58
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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Effect of orthophosphate and bioavailability of dissolved organic phosphorous compounds to typically harmful cyanobacterium Microcystis aeruginosa Jihua Li a,1, Zhongwei Wang a,b,1, Xin Cao a, Zhengfang Wang a, Zheng Zheng a,⇑ a b
Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, PR China Dalian Environmental Monitoring Centre, Dalian 116023, PR China
a r t i c l e
i n f o
Article history: Available online 24 January 2015 Keywords: Microcystis aeruginosa Dissolved organic phosphorus Phosphorus depletion Alkaline phosphatase
a b s t r a c t Results show that Microcystis aeruginosa can utilize both dissolved organic phosphorus (DOP) and orthophosphate (DIP) even under low phosphorus (P) conditions to sustain its growth. Total P concentrations decreased markedly in all three P source treatments. Alkaline phosphatase activity (APA) in the different P sources tested changed in response to the DOP and DIP. The APA of DOP groups remained low after decreasing significantly, but the APA in the DIP treatments remained high during the period of culture. Changes in APA at different PO3 4 –P concentrations in a culture medium revealed negative correlations between APA and DIP. However, a positive relationship was observed between APA and DOP under low P concentrations. These findings indicate that M. aeruginosa can regulate its physiological metabolism to acclimate to low ambient DIP environments. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Harmful algal blooms (HABs) are a critical issue for the global freshwater environment (Huang et al., 2014). HABs have occurred in more than half of the freshwater systems in China; the cyanobacterium Microcystis aeruginosa is one of the dominant species causing the bloom, which produces toxins and cyanobacterial metabolites (Pan et al., 2006). These blooms threaten animal life, human health, and social and economic sustainability (Li et al., 2011; Paerl et al., 2011). Anthropogenic nutrient over-enrichment of these aquatic ecosystems has been linked to the proliferation of blue-green algae (Paerl et al., 2001). Nitrogen (N), and phosphorous (P), are the key nutrients of concern in most aquatic ecosystems; N is needed for protein synthesis and P is needed for phospholipids DNA, RNA, and the principal nucleotide cofactors required for energy transfer in cells (Paerl 2008; Conley et al., 2009). Specifically, P is considered the primary limiting nutrient for algae and its deficiency can significantly influence the production of Chl-a and the rate of photosynthesis (Schindler et al., 2008; Zhang et al., 2011). Hence, P loading restrictions have been implemented to reverse its detrimental effects on eutrophic freshwater lakes (Passy et al., 2013). In natural waters, dissolved phosphorus (DP) ⇑ Corresponding author. 1
E-mail address: [email protected] (Z. Zheng). The first two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.marpolbul.2015.01.001 0025-326X/Ó 2015 Elsevier Ltd. All rights reserved.
comprises orthophosphate (DIP) (Diaz et al., 2008) and dissolved organic phosphorus (DOP) derived from the degradation of glycolipids, glycoproteins, and antibiotics (Bai et al., 2014). Although DIP is considered the most important P source for microalgae, DOP is available for the production of both phytoplankton and bacteria in some freshwater systems (Rinker and Powell, 2006). In these systems, DOP concentrations can exceed DIP concentrations based on two possible reasons. On one hand, an abundance of organic P was discharged into the water bodies because of the fast development of agriculture and medicine worldwide (Bai et al., 2014). On the other hand, organic P is abundant in lake sediments and it can be transferred from sediments into the overlying water. Thus, regeneration of P from dissolved organic matter is a potentially important source of bioavailable P. Wang et al. (2010) demonstrated that DOP could be mineralized for supplementing P sources for the growth of bloom algae when the DIP concentration is insufficient, particularly at the beginning of cyanobacterial bloom formation in some lakes. Alkaline phosphatase (AP), which contributes most significantly to phosphate regeneration in aquatic systems, plays an important role in utilizing DOP compounds (Ammerman and Azam, 1991; Huang et al., 2005). In phosphate-depleted waters, phytoplankton can synthesize cell-surface AP, which leads to the utilization of DOP. The use of AP is therefore likely to represent a temporal strategy used by phytoplankton to overcome serious P depletion. To our knowledge, however, the bioavailability of
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J. Li et al. / Marine Pollution Bulletin 92 (2015) 52–58
DIP and DOP to M. aeruginosa, as well as their AP regulations, has yet to be elucidated. To fill the research gap, this study investigates the potential ecological significance of DOP and DIP to algal blooms and evaluates the relationship between AP and P utilization. DIP and two different DOP compounds were used to test their bioavailability to M. aeruginosa. A variety of growth parameters of M. aeruginosa were analyzed and the changes in Total P (TP) in culture solutions were monitored under different P sources. Relationships between different DOP compounds and alkaline phosphatase activity (APA) were investigated during M. aeruginosa growth. Changes in APA at different orthophosphate (PO3 4 –P) concentrations in the culture media were also studied. 2. Materials and methods 2.1. Preparation of M. aeruginosa culture M. aeruginosa (FACHB-905) was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. The culture was centrifuged at 4000 rpm for 8 min at the exponential growth stage; the pelleted cells were washed three times with sterile P-free BG11 medium (Rippka et al., 1979). Subsequently, the cells were incubated in P-free medium from 5 d to 7 d to deplete the intracellular P stores. 2.2. Experimental set up After P starvation, the cells were re-inoculated into a 500 mL glass flasks filled with 250 mL BG11 medium, which contained three different phosphorus compounds: b-glycero-phosphate (bGP), adenosine triphosphate (ATP–P), and orthophosphate (Table 1). Among the three P compounds, b-GP and ATP–P are two different DOP sources while orthophosphate represents DIP. Every P compound was added at different five concentrations with a gradient of 0.1, 0.5, 1.0, 2.0, and 4.0 mg L1. Each experiment was conducted in triplicate. Hence, a total of 45 flasks were used in this study. To provide the maximal possible sterility and avoid any contamination of samples, the procedures were conducted under laminar air-flow chambers. All materials were sterilised by autoclaving before assembly and operation. To investigate the potential ecological significance of DIP and DOP for algal blooms, the growth of M. aeruginosa at the same initial algae concentration (3.0 106
cells mL1) was studied. The cultures were incubated in a light–dark cycle of 12 h:12 h with an intensity of 1600 lx provided by cool white fluorescent tubes at 25 ± 1 °C. All compounds were obtained from the Sigma Chemical Company (St. Louis, MO, USA). The culture flasks were shaken twice every day and their position was changed randomly.
2.3. Cell counts, APA, TP, and PO3 4 concentrations analyses All the experiments were conducted in triplicates. During the incubation period, 30 mL samples were collected every 2 d and used for test indicators analyses. Cell counts were performed on a compound microscope equipped with a hemocytometer after the samples were preserved with acid Lugol’s solution (Ma et al., 2009). Each sample was measured at least three times, with a maximum deviation of approximately 20%. TP and PO3 4 concentrations in the culture medium were measured using molybdenum–antimony spectrophotometric method (Li et al., 2015). APA was assayed according to the method described by Kruskopf and Du Plessis (2004). Specifically, a sample was harvested by centrifugation (8000 rpm) and homogenized at 4 °C in sodium phosphate buffer (0.2 mol/L, pH 7.4). The mixture was incubated for 5 min at 4 °C and centrifuged at 10,000 rpm for 5 min at 4 °C. Subsequently, the APA in the supernatant was measured using the colorimetric method with p-nitrophenyl phosphate (pNPP, Sigma) as the substrate.
2.4. Growth rates, P utilization efficiency and data analysis Specific growth rate (l) and mean doubling time (G) were calculated according to the equation, l ¼ ðInN2 InN1 Þðt2 t1 Þ1 G ¼ In2l1 , where N1 and N2 are the cell density at times t2 and t1, respectively. The maximum cell number (Nmax) is the cell density at t1 when the growth rate was lower than 5%. P utilization efficiency (g) was analyzed according to the equation, g = (C0Ct)/C0 100%, where C0 and Ct are the P concentrations in the media at initial time and at the end time, respectively. The differences in the growth responses of M. aeruginosa to different P factors were analyzed by ANOVA. Minitab software was used to conduct statistical analyses. The level of significance used was p < 0.01.
Table 1 Molecular formula, weight and structure of the inorganic and organic P compounds used in the experiment. Compound class
Compound (acronym) (chemical formula)
Molecular weight (g mol1)
Inorganic
Orthophosphate (K2HPO4–P) (K2HPO4)
174
Organic
b-Glycero-phosphate (b-GP) (C3H7Na2O6P)
216
Organic
Adenosine triphos-phate (ATP–P) (C10H14N5Na2O13P3)
551
Structure
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J. Li et al. / Marine Pollution Bulletin 92 (2015) 52–58
3. Results 3.1. Growth of M. aeruginosa The effects of DIP and DOP sources under different P concentrations on the availability to M. aeruginosa after culturing for 14 d are shown in Fig. 1. Algal cells in the medium treated with DIP increased rapidly during the inoculation period (Fig. 1a). However, the cells of P0.1 reached a peak of approximately 8.91 106 cells mL1 on Day 10–Day 12 of the test. The l values and Nmax increased as P concentration increased (Table 2). In the P4.0 treatment, the maximum l value and Nmax were 0.164 d1 and 2.42 107 cells mL1, respectively. M. aeruginosa with different P source treatments of 0.1 mg L1 exhibited similar variations (Table 2). M. aeruginosa cultured in b-GP and ATP–P grew well, which suggests that M. aeruginosa can utilize DOP compounds in different molecular forms (Fig. 1 b and c). The growth changes in cell numbers under b-GP and ATP–P treatments displayed a similar pattern: increased on the Day 7 of incubation, and then remained at a constant level. The maximum l and Nmax with b-GP were 0.068 d1 and 6.93 106 cells mL1 when the concentration of b-GP was 0.5 mg L1. However, the maximum l values and Nmax were 0.078 d1 and 6.32 106 cells mL1 when the concentration of ATP–P was 1.0 mg L1 (Table 2). Table 3 shows that the growth response of P forms was significant (p < 0.01). However, the growth response was insignificant under the interaction between P concentration and P forms.
3.2. Changes in P concentration in culture medium The total P (TP) concentrations in the culture medium were monitored to determine the bioavailability of DIP and DOP to M.
aeruginosa. Fig. 2 shows the variations in TP in the solutions with DIP, ATP–P, and b-GP treatments. Among the DIP groups, the P concentrations of P0.1, P1.0, P2.0, and P4.0 systems decreased sharply during the first 3 d of incubation and remained low; the TP removal efficiencies were 89.58%, 54.35%, and 52.19%, respectively. However, the TP concentration in the P0.5 group decreased significantly until Day 7 of incubation; the TP removal efficiency in the culture medium was 56.54%. Similarly, the P concentrations of all ATP–P groups decreased significantly during the first 3 d of incubation and fluctuated within a narrow range afterwards. The removal efficiencies of P0.1, P0.5, P1.0, P2.0, and P4.0 systems were 89.58%, 54.35%, 52.19%, 59.07%, 56.29%, respectively. In the b-GP systems, the P concentrations of P0.1, P1.0, P2.0, and P4.0 groups decreased during Day 5 except that of the P0.5 group.
3.3. Alkaline phosphatase activity Fig. 4 shows the APA of M. aeruginosa under DOP and DIP of incubation. Overall, the changes in APA treated with DOP and DIP were different, except that all the initial values were low at the beginning of culture because of stock culture conditions. In the DIP systems, the APA of P0.1 system increased faster than the four other systems in 3 d; a decline in APA was observed on Day 7, but the APA values maintained a relatively high level afterwards. Among the five DIP systems, the APA of P0.5 group did not exhibit the tendency to increase until Day 7, which corresponded to the change in P concentrations in the culture medium. However, the APA values in all b-GP treatments demonstrated an increasing trend within 5 d and then displayed a significant decreasing trend before fluctuating within a narrow range. In the ATP–P systems, the APA values increased steadily in 5 d, followed by a gradual decline.
(a) K2HPO4-P
6
-1
Cell numbers (10 cells ml )
24 P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
20 16 12 8 4 0
2
4
6
8
10
12
14
16
Days (d)
(b) 7.5
(c) -GP
6.5 6.0
6.0
ATP-P
5.5
6
5.5 5.0 4.5 P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
4.0 3.5 3.0 2.5
6.5
-1
Cell numbers (10 cells ml )
Cell numbers (106 cells ml-1)
7.0
0
2
4
6
8
Days (d)
10
12
5.0 4.5 P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
4.0 3.5 3.0
14
16
0
2
4
6
8
10
12
14
16
Days (d)
Fig. 1. Growth curve of M. aeruginosa under different phosphorus sources: (a) K2HPO4, (b) b-GP and (c) ATP–P with the phosphorus concentrations (0.1, 0.5, 1.0, 2.0 and 4.0 mg/L).
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J. Li et al. / Marine Pollution Bulletin 92 (2015) 52–58 Table 2 Mean values of specific growth rate (l), mean doubling time (G) in days and maximum cell numbers (Nmax) of M. aeruginosa under different phosphorus sources. Parameters
l G Nmax
DIP
ATP–P
b-GP
P0.1
P0.5
P1.0
P2.0
P4.0
P0.1
P0.5
P1.0
P2.0
P4.0
P0.1
P0.5
P1.0
P2.0
P4.0
0.083 3.2 8.91
0.135 2.7 18.53
0.146 2.6 20.56
0.154 2.6 22.41
0.164 2.5 24.17
0.047 3.8 5.60
0.068 3.4 6.93
0.043 3.8 4.97
0.051 3.7 6.03
0.052 3.7 5.85
0.076 3.3 6.19
0.045 3.8 5.35
0.078 3.2 6.32
0.075 3.3 5.94
0.069 3.4 5.87
Table 3 Two-way ANOVA: cell number versus P form and P concentration. Source
df
SS
MS
F
P
P form P concentration Interaction Error Total
2 4 8 75 89
523.76 25.09 77.44 1059.56 1685.85
261.882 6.272 9.680 14.127 –
18.54 0.44 0.69 – –
0.000 0.776 0.703 – –
4. Discussion The roles of the bioavailability of various P compounds in phytoplankton have been studied in recent years (Herbes et al., 1974; Ammerman and Azam, 1991; Scanlan et al., 1997). Although DIP can be directly utilized by algal cells and is considered the most important P source for microalgae (Herbes et al., 1974), many authors have demonstrated that phytoplankton is also capable of utilizing DOP via enzymatic hydrolysis (Cembella et al., 1984; Huang and Hong, 1999). The present study has shown that the cyanobacteria, M. aeruginosa, can utilize DOP apart from DIP even
under low P conditions (Fig. 1). The capability of M. aeruginosa to use two different DOP compounds by inducing AP might be an important characteristic that allows the algae to sustain growth in DIP-deficient environments (Huang et al., 2005). The abilities that M. aeruginosa uses to utilize DOP under DIP-deficient conditions may be the principal features that allow the algae to form blooms in aquatic systems. In the present study, P forms may be the key factor in the growth of M. aeruginosa (Table 3). For example, M. aeruginosa in the b-GP and ATP–P treatments exhibited similar growth curves compared with that treated. However, the l and Nmax were higher in media treated with ATP–P than those treated with b-GP, suggesting that M. aeruginosa can easily utilize DOP with low molecular weight (Table 2). It has been reported that algae could accumulate and store large quantities of P in phosphate-rich environments (Watanabe et al., 1987). Huang et al. (2005) have shown that algae can accumulate excess phosphorus in the first 2 d to 3 d of incubation (Huang et al., 2005). Our results revealed that M. aeruginosa exhibited a rapid uptake when cultured in orthophosphate media, consistent with the report by Wu et al. (2009). Changes in TP concentration in the culture medium indicate the bioavailability of DIP and
(a) 4.5 -1
P concentration (mg L )
4.0
K2HPO4-P
P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0
2
4
6
8
10
Days (d)
(b) 4.5
(c) 4.5 -GP
4.0
P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
3.0 2.5
-1
3.5
P concentration (mg L )
-1
P concentration (mg L )
4.0
2.0 1.5 1.0 0.5
ATP-P
P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
0.0 0
2
4
6
Days (d)
8
10
0
2
4
6
8
10
Days (d)
Fig. 2. Variation of TP in culture media under different phosphorus sources: (a) K2HPO4, (b) b-GP and (c) ATP–P with the phosphorus concentrations (0.1, 0.5, 1.0, 2.0 and 4.0 mg/L).
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J. Li et al. / Marine Pollution Bulletin 92 (2015) 52–58
-1
-1
4-Nitrophenol (ug mg Pro h )
(a) 0.35 P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
0.30 0.25
K2HPO4-P
0.20 0.15 0.10 0.05 0.00
0
1
2
3
4
5
6
7
8
9
10 11
Days (d)
(c) 0.40 -1
4-Nitrophenol (ug mg Pro h )
-1
P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
-GP
0.24 0.20 0.16 0.12 0.08 0.04 0.00
0.30 0.25 0.20 0.15 0.10 0.05 0.00
0
1
2
3
4
5
6
7
8
9
ATP-P
P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
0.35
-1
-1
4-Nitrophenol (ug mg Pro h )
(b) 0.28
10 11
0
1
2
3
4
Days (d)
5
6
7
8
9
10 11
Days (d)
Fig. 3. Alkaline phosphatase activity of M. aeruginosa under different phosphorus sources.
(a) 100 P Utilization Efficiency (%)
P4.0 P2.0
90
P1.0 P0.5
80
R2=0.9884
70
60
P0.1
K2HPO4-P
50 8
10
12
14
16
18
20
22 6
24
26
-1
Maximum cell numbers X m (10 cells ml )
(c) 100
90 P4.0
80
70
P0.1
R2=0.8273
P Utilization Efficiency (%)
P Utilization Efficiency (%)
(b) 100
P0.5
P2.0
P1.0 P0.1
60
50
-GP
5.0
5.5
6.0
6.5
P4.0
-1
Maximum cell numbers X m (10 cells ml )
P2.0
P1.0
80 70 60 R2=0.7750 50 P0.5 40 30 5.2
7.0 6
90
ATP-P 5.4
5.6
5.8
6.0
6.2 6
6.4 -1
Maximum cell numbers X m (10 cells ml )
Fig. 4. Relationship between the P utilization efficiency and the maximum cell numbers of M. aeruginosa.
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J. Li et al. / Marine Pollution Bulletin 92 (2015) 52–58
(b)
-1
0.25 0.20 0.15 0.10 0.05 0.00 0-0.05(4) 0.05-0.1(4) 0.10-0.5(9) 0.5-1.0(4)
0.30
-GP
-1
0.30
4-Nitrophenol (ug mg Pro h )
K2HPO4-P
-1
-1
4-Nitrophenol (ug mg Pro h )
(a) 0.35
0.25 0.20 0.15 0.10 0.05 0.00 0-0.01(5)
> 1.0(4)
0.01-0.05(6)
4-Nitrophenol (ug mg Pro h )
0.40
-1
-1
0.45
> 0.1(8)
-1
PO4 -P(mg L )
PO4 -P(mg L )
(c)
0.05-0.1(6)
3-
-1
3-
ATP-P
0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00
0-0.005(4) 0.005-0.01(4) 0.01-0.05(8) 0.05-0.1(5) 3-
> 0.1(4)
-1
PO4 -P(mg L ) Fig. 5. Changes of alkaline phosphatase activity at different PO3 4 –P concentrations.
DOP compounds to M. aeruginosa (Fig. 2). In the current study, the TP concentrations in DOP source treatments decreased significantly in the initial three days, but TP concentrations in the media treated with DIP decreased faster, which indicates a higher uptake and storage capacity compared with those in the DOP treatments. When P was exhausted in the culture media, M. aeruginosa utilized the internal P storage to maintain its growth. The constantly low TP concentrations in the solutions indicate that the cell numbers will reach the highest level in a few days. Hence, the changes in TP in the culture media showed the existence of a relationship with the Nmax of M. aeruginosa during incubation (Zhao et al., 2012). We further tested the relationship between the g and the Nmax of M. aeruginosa through nonlinear fit (Fig. 4). Nmax of M. aeruginosa exhibited a good linear response to DIP (p < 0.01, R2 = 0.9884), indicating that P dominated the primary productivity in the system (Liang et al., 2014). Moreover, Nmax and b-GP as well as ATP–P, obtained a significantly positive fit (p < 0.01, R2 = 0.8273; and p < 0.01, R2 = 0.7750, respectively). These results are supported by those of previous studies, particularly those conducted on P-limited rivers (Heiskary and Markus, 2001; Rier and Stevenson, 2006; Royer et al., 2008). The production of AP has been proposed as a mechanism to allow cyanobacteria to overcome serious phosphorus depletion (Scanlan and Wilson, 1999; Hoppe, 2003). When P content in cells decreases, phytoplankton can produce AP in the substrate quickly to hydrolyze DOP. The APA is generally recommended as an indicator of P status to compensate for the ambient P deficiency (Jamet et al., 2001). In the present study, intracellular APA was low under all of the three P source treatments during the adaptation period. Subsequently, the activities of AP increased significantly in 3 d,
which promoted DOP decomposition by M. aeruginosa during the incubation period. These data indicated that M. aeruginosa could utilize DOP because DOP was hydrolyzed to DIP gradually via AP. The APA also remained low before P was depleted in the DOP treatments, a result that is consistent with that of Huang and Hong (1999). The values of APA in the DIP treatment remained relatively high when the P concentration was very low (Fig. 3). Some authors have proven that DIP negatively correlates with algal APA (Huang et al., 2005). However, studies on the relationship between DOP and APA during the growth of M. aeruginosa are few, particularly those on the relationship between extracellular DOP and intracellular APA. Fig. 5 shows the changes in APA at different PO3 4 –P concentrations in the culture media. Our results revealed negative correlations between the APA of M. aeruginosa and DIP. Conversely, the values of APA decreased as the DIP concentrations increased in the media. Nausch (1998) pointed out that P in cells might inhibit the induction of APA when its concentration reaches a certain level, but P-deficiency may cause M. aeruginosa to induce APA. The chemical composition of P is also crucial for phosphatase generation (Hoppe, 2003). In the current study, the APA in the b-GP treatment increased within the PO3 4 –P concentration range of 0 mg L1–0.1 mg L1 and decreased afterwards. However, other variation patterns of APA were observed in the ATP–P system. The APA increased when the PO3 4 –P concentration was in the range of 0 mg L1–0.05 mg L1 and > 0.1 mg L1, but decreased in 0.05 mg L1–0.1 mg L1. In general, a negative correlation between APA and DOP under low P concentrations was observed. Although there is no conclusive evidence that smaller molecular DOP is transported directly into algal cells, a report has demonstrated that the transportation and metabolism of DOP by phytoplankton was
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J. Li et al. / Marine Pollution Bulletin 92 (2015) 52–58
apparently not mediated by AP; other results also have revealed the complex correlations between APA and DOP (Hernandez et al., 2002; Huang et al., 2005). In summary, the present study has suggested the following: (1) M. aeruginosa is capable of utilizing both DIP and DOP even under low P conditions to sustain its growth; (2) M. aeruginosa can accumulate and store cellular P to ensure that its growth rates do not immediately decline; (3) The l values and Nmax in the ATP–P treatments can be higher than those in b-GP systems possibly because of the low molecular weight of ATP–P; (4) The changes in APA at different PO3 4 –P concentrations in the culture medium can reveal negative correlations between APA and DIP, although a positive relationship can be observed between APA and DOP under low P concentrations. However, the relationship between APA and different DOP compounds needs to be further studied. The findings from future studies, together with those from the present study, may be important to the full understanding of the HABs mechanism, particularly in DIP-deficient lakes.
Acknowledgements The authors thank the students from Fudan University for helping with lab experiments. This study is financially supported by the Major Science and Technology Program for Water Pollution Control and Treatment (No. 2012ZX07102-004).
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Kruskopf, M.M., Du Plessis, S., 2004. Induction of both acid and alkaline phosphatase activity in two green-algae (chlorophyceae) in low N and P concentrations. Hydrobiologia 513, 59–70. Li, Y., Cao, W.Z., Su, C.X., Hong, H.S., 2011. Nutrient sources and composition of recent algal blooms and eutrophication in the northern Jiulong River, Southeast China. Mar. Pollut. Bull. 63, 249–254. Li, J., Yang, X., Wang, Z., Shan, Y., Zheng, Z., 2015. Comparison of four aquatic plant treatment systems for nutrient removal from eutrophied water. Bioresource Technol. 179, 1–7. Liang, X.Q., Zhu, S.R., Ye, R.Z., Guo, R., Zhu, C.Y., Fu, C.D., Tian, G.M., Chen, Y.X., 2014. Biological thresholds of nitrogen and phosphorus in a typical urban river system of the Yangtz delta. China. Environ. Pollut. 192, 251–258. Ma, H., Cui, F., Liu, Z., Fan, Z., 2009. Pilot study on control of phytoplankton by zooplankton coupling with filter-feeding fish in surface water. Water Sci. Technol. 60, 737–743. Nausch, M., 1998. Alkaline phosphatase activities and the relationship to inorganic phosphate in the Pomeranian Bight (southern Baltic Sea). Aquat. Microb. Ecol. 16, 87–94. Paerl, H., 2008. Chapter 10: Nutrient and other environmental controls of harmful cyanobacterial blooms along the freshwater–marine continuum. Cyanobacterial Harmful Algal Blooms: State Sci. Res. needs 619, 217–237. Paerl, H.W., Fulton, R.S., Moisander, P.H., Dyble, J., 2001. Harmful freshwater algal blooms, with an emphasis on cyanobacteria. The Scientific World J. 1, 76–113. Paerl, H.W., Xu, H., McCarthy, M.J., Zhu, G., Qin, B., Li, Y., Gardner, W.S., 2011. Controlling harmful cyanobacterial blooms in a hyper-eutrophic lake (Lake Taihu, China): the need for a dual nutrient (N & P) management strategy. Water Res. 45, 1973–1983. Pan, G., Zhang, M.M., Chen, H., Zou, H., Yan, H., 2006. Removal of cyanobacterial blooms in Taihu Lake using local soils. I. Equilibrium and kinetic screening on the flocculation of microcystis aeruginosa using commercially available clays and minerals. Environ. Pollut. 141, 195–200. Passy, P., Gypens, N., Billen, G., Garnier, J., Thieu, V., Rousseau, V., Callens, J., Parent, J.Y., Lancelot, C., 2013. A model reconstruction of riverine nutrient fluxes and eutrophication in the Belgian coastal zone since 1984. J. Marine Syst. 128, 106– 122. Rier, S.T., Stevenson, R.J., 2006. Response of periphytic algae to gradients in nitrogen and phosphorus in streamside mesocosms. Hydrobiologia 561, 131–147. Rinker, K.R., Powell, R.T., 2006. Dissolved organic phosphorus in the Mississippi River plume during spring and fall 2002. Mar. Chem. 102, 170–179. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stanier, R.Y., 1979. Generic assignments, strain histories and properties of pure culture of cyanobacteria. J. Microbiol. 111, 11–61. Royer, T.V., David, M.B., Gentry, L.E., Mitchell, C.A., Starks, K.M., Heatherly, T., Whiles, M.R., 2008. Assessment of chlorophyll-a as a criterion for establishing nutrient standards in the streams and rivers of illinois. J. Environ. Qual. 37, 437– 447. Scanlan, D.J., Wilson, W.H., 1999. Application of molecular techniques to addressing the role of P as a key effecter in marine ecosystems. Hydrobiologya. 401, 149– 175. Scanlan, D.J., Silman, N.J., Donald, K.M., Wilson, W.H., Carr, N.G., Joint, I., Mann, N.H., 1997. An immunological approach to detect phosphate stress in population and single cells of photosynthetic picoplankton. Appl. Environ. Microb. 63, 2411– 2420. Schindler, D.W., Hecky, R.E., Findlay, D.L., Stainton, M.P., Parker, B.R., Paterson, M.J., Beaty, K.G., Lyng, M., Kasian, S., 2008. Eutrophication of lakes cannot be controlled by reducing nitrogen input: results of a 37-year whole-ecosystem experiment. P. Natl. Acad. Sci. USA 105, 11254–11258. Wang, Z.C., Li, D.H., Li, G.W., Liu, Y.D., 2010. Mechanism of photosynthetic response in Microcystis aeruginosa PCC7806 to low inorganic phosphorus. Harmful Algae. 9, 613–619. Watanabe, M., Kohata, K., Kunugi, M., 1987. 31P nuclear magnetic resonances study of intracellular phosphate pools and polyphosphate metabolism in Heterosigam akashiwo (hada) HADA (Raphidophyceae). J. Phycol. 23, 54–62. Wu, Z.X., Shi, J.Q., Li, R.H., 2009. Comparative studies on photosynthesis and phosphate metabolism of Cylindrospermopsis raciborskii with Microcystis aeruginosa and Aphanizomenon flos-aquae. Harmful Algae. 8, 910–915. Zhang, J., Geng, J.J., Ren, H.Q., Luo, J., Zhang, A.Q., Wang, X.R., 2011. Physiological and biochemical responses of Microcystis aeruginosa to phosphite. Chemosphere 85, 1325–1330. Zhao, G.Y., Du, J.J., Jia, Y., Lv, Y.N., Han, G.M., Tian, X.J., 2012. The importance of bacteria in promoting algal growth in eutrophic lakes with limited available phosphorus. Ecol. Eng. 42, 107–111.
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Effect of orthophosphate and bioavailability of dissolved organic phosphorous compounds to typically harmful cyanobacterium Microcystis aeruginosa Jihua Li a,1, Zhongwei Wang a,b,1, Xin Cao a, Zhengfang Wang a, Zheng Zheng a,⇑ a b
Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, PR China Dalian Environmental Monitoring Centre, Dalian 116023, PR China
a r t i c l e
i n f o
Article history: Available online 24 January 2015 Keywords: Microcystis aeruginosa Dissolved organic phosphorus Phosphorus depletion Alkaline phosphatase
a b s t r a c t Results show that Microcystis aeruginosa can utilize both dissolved organic phosphorus (DOP) and orthophosphate (DIP) even under low phosphorus (P) conditions to sustain its growth. Total P concentrations decreased markedly in all three P source treatments. Alkaline phosphatase activity (APA) in the different P sources tested changed in response to the DOP and DIP. The APA of DOP groups remained low after decreasing significantly, but the APA in the DIP treatments remained high during the period of culture. Changes in APA at different PO3 4 –P concentrations in a culture medium revealed negative correlations between APA and DIP. However, a positive relationship was observed between APA and DOP under low P concentrations. These findings indicate that M. aeruginosa can regulate its physiological metabolism to acclimate to low ambient DIP environments. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Harmful algal blooms (HABs) are a critical issue for the global freshwater environment (Huang et al., 2014). HABs have occurred in more than half of the freshwater systems in China; the cyanobacterium Microcystis aeruginosa is one of the dominant species causing the bloom, which produces toxins and cyanobacterial metabolites (Pan et al., 2006). These blooms threaten animal life, human health, and social and economic sustainability (Li et al., 2011; Paerl et al., 2011). Anthropogenic nutrient over-enrichment of these aquatic ecosystems has been linked to the proliferation of blue-green algae (Paerl et al., 2001). Nitrogen (N), and phosphorous (P), are the key nutrients of concern in most aquatic ecosystems; N is needed for protein synthesis and P is needed for phospholipids DNA, RNA, and the principal nucleotide cofactors required for energy transfer in cells (Paerl 2008; Conley et al., 2009). Specifically, P is considered the primary limiting nutrient for algae and its deficiency can significantly influence the production of Chl-a and the rate of photosynthesis (Schindler et al., 2008; Zhang et al., 2011). Hence, P loading restrictions have been implemented to reverse its detrimental effects on eutrophic freshwater lakes (Passy et al., 2013). In natural waters, dissolved phosphorus (DP) ⇑ Corresponding author. 1
E-mail address: [email protected] (Z. Zheng). The first two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.marpolbul.2015.01.001 0025-326X/Ó 2015 Elsevier Ltd. All rights reserved.
comprises orthophosphate (DIP) (Diaz et al., 2008) and dissolved organic phosphorus (DOP) derived from the degradation of glycolipids, glycoproteins, and antibiotics (Bai et al., 2014). Although DIP is considered the most important P source for microalgae, DOP is available for the production of both phytoplankton and bacteria in some freshwater systems (Rinker and Powell, 2006). In these systems, DOP concentrations can exceed DIP concentrations based on two possible reasons. On one hand, an abundance of organic P was discharged into the water bodies because of the fast development of agriculture and medicine worldwide (Bai et al., 2014). On the other hand, organic P is abundant in lake sediments and it can be transferred from sediments into the overlying water. Thus, regeneration of P from dissolved organic matter is a potentially important source of bioavailable P. Wang et al. (2010) demonstrated that DOP could be mineralized for supplementing P sources for the growth of bloom algae when the DIP concentration is insufficient, particularly at the beginning of cyanobacterial bloom formation in some lakes. Alkaline phosphatase (AP), which contributes most significantly to phosphate regeneration in aquatic systems, plays an important role in utilizing DOP compounds (Ammerman and Azam, 1991; Huang et al., 2005). In phosphate-depleted waters, phytoplankton can synthesize cell-surface AP, which leads to the utilization of DOP. The use of AP is therefore likely to represent a temporal strategy used by phytoplankton to overcome serious P depletion. To our knowledge, however, the bioavailability of
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J. Li et al. / Marine Pollution Bulletin 92 (2015) 52–58
DIP and DOP to M. aeruginosa, as well as their AP regulations, has yet to be elucidated. To fill the research gap, this study investigates the potential ecological significance of DOP and DIP to algal blooms and evaluates the relationship between AP and P utilization. DIP and two different DOP compounds were used to test their bioavailability to M. aeruginosa. A variety of growth parameters of M. aeruginosa were analyzed and the changes in Total P (TP) in culture solutions were monitored under different P sources. Relationships between different DOP compounds and alkaline phosphatase activity (APA) were investigated during M. aeruginosa growth. Changes in APA at different orthophosphate (PO3 4 –P) concentrations in the culture media were also studied. 2. Materials and methods 2.1. Preparation of M. aeruginosa culture M. aeruginosa (FACHB-905) was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. The culture was centrifuged at 4000 rpm for 8 min at the exponential growth stage; the pelleted cells were washed three times with sterile P-free BG11 medium (Rippka et al., 1979). Subsequently, the cells were incubated in P-free medium from 5 d to 7 d to deplete the intracellular P stores. 2.2. Experimental set up After P starvation, the cells were re-inoculated into a 500 mL glass flasks filled with 250 mL BG11 medium, which contained three different phosphorus compounds: b-glycero-phosphate (bGP), adenosine triphosphate (ATP–P), and orthophosphate (Table 1). Among the three P compounds, b-GP and ATP–P are two different DOP sources while orthophosphate represents DIP. Every P compound was added at different five concentrations with a gradient of 0.1, 0.5, 1.0, 2.0, and 4.0 mg L1. Each experiment was conducted in triplicate. Hence, a total of 45 flasks were used in this study. To provide the maximal possible sterility and avoid any contamination of samples, the procedures were conducted under laminar air-flow chambers. All materials were sterilised by autoclaving before assembly and operation. To investigate the potential ecological significance of DIP and DOP for algal blooms, the growth of M. aeruginosa at the same initial algae concentration (3.0 106
cells mL1) was studied. The cultures were incubated in a light–dark cycle of 12 h:12 h with an intensity of 1600 lx provided by cool white fluorescent tubes at 25 ± 1 °C. All compounds were obtained from the Sigma Chemical Company (St. Louis, MO, USA). The culture flasks were shaken twice every day and their position was changed randomly.
2.3. Cell counts, APA, TP, and PO3 4 concentrations analyses All the experiments were conducted in triplicates. During the incubation period, 30 mL samples were collected every 2 d and used for test indicators analyses. Cell counts were performed on a compound microscope equipped with a hemocytometer after the samples were preserved with acid Lugol’s solution (Ma et al., 2009). Each sample was measured at least three times, with a maximum deviation of approximately 20%. TP and PO3 4 concentrations in the culture medium were measured using molybdenum–antimony spectrophotometric method (Li et al., 2015). APA was assayed according to the method described by Kruskopf and Du Plessis (2004). Specifically, a sample was harvested by centrifugation (8000 rpm) and homogenized at 4 °C in sodium phosphate buffer (0.2 mol/L, pH 7.4). The mixture was incubated for 5 min at 4 °C and centrifuged at 10,000 rpm for 5 min at 4 °C. Subsequently, the APA in the supernatant was measured using the colorimetric method with p-nitrophenyl phosphate (pNPP, Sigma) as the substrate.
2.4. Growth rates, P utilization efficiency and data analysis Specific growth rate (l) and mean doubling time (G) were calculated according to the equation, l ¼ ðInN2 InN1 Þðt2 t1 Þ1 G ¼ In2l1 , where N1 and N2 are the cell density at times t2 and t1, respectively. The maximum cell number (Nmax) is the cell density at t1 when the growth rate was lower than 5%. P utilization efficiency (g) was analyzed according to the equation, g = (C0Ct)/C0 100%, where C0 and Ct are the P concentrations in the media at initial time and at the end time, respectively. The differences in the growth responses of M. aeruginosa to different P factors were analyzed by ANOVA. Minitab software was used to conduct statistical analyses. The level of significance used was p < 0.01.
Table 1 Molecular formula, weight and structure of the inorganic and organic P compounds used in the experiment. Compound class
Compound (acronym) (chemical formula)
Molecular weight (g mol1)
Inorganic
Orthophosphate (K2HPO4–P) (K2HPO4)
174
Organic
b-Glycero-phosphate (b-GP) (C3H7Na2O6P)
216
Organic
Adenosine triphos-phate (ATP–P) (C10H14N5Na2O13P3)
551
Structure
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J. Li et al. / Marine Pollution Bulletin 92 (2015) 52–58
3. Results 3.1. Growth of M. aeruginosa The effects of DIP and DOP sources under different P concentrations on the availability to M. aeruginosa after culturing for 14 d are shown in Fig. 1. Algal cells in the medium treated with DIP increased rapidly during the inoculation period (Fig. 1a). However, the cells of P0.1 reached a peak of approximately 8.91 106 cells mL1 on Day 10–Day 12 of the test. The l values and Nmax increased as P concentration increased (Table 2). In the P4.0 treatment, the maximum l value and Nmax were 0.164 d1 and 2.42 107 cells mL1, respectively. M. aeruginosa with different P source treatments of 0.1 mg L1 exhibited similar variations (Table 2). M. aeruginosa cultured in b-GP and ATP–P grew well, which suggests that M. aeruginosa can utilize DOP compounds in different molecular forms (Fig. 1 b and c). The growth changes in cell numbers under b-GP and ATP–P treatments displayed a similar pattern: increased on the Day 7 of incubation, and then remained at a constant level. The maximum l and Nmax with b-GP were 0.068 d1 and 6.93 106 cells mL1 when the concentration of b-GP was 0.5 mg L1. However, the maximum l values and Nmax were 0.078 d1 and 6.32 106 cells mL1 when the concentration of ATP–P was 1.0 mg L1 (Table 2). Table 3 shows that the growth response of P forms was significant (p < 0.01). However, the growth response was insignificant under the interaction between P concentration and P forms.
3.2. Changes in P concentration in culture medium The total P (TP) concentrations in the culture medium were monitored to determine the bioavailability of DIP and DOP to M.
aeruginosa. Fig. 2 shows the variations in TP in the solutions with DIP, ATP–P, and b-GP treatments. Among the DIP groups, the P concentrations of P0.1, P1.0, P2.0, and P4.0 systems decreased sharply during the first 3 d of incubation and remained low; the TP removal efficiencies were 89.58%, 54.35%, and 52.19%, respectively. However, the TP concentration in the P0.5 group decreased significantly until Day 7 of incubation; the TP removal efficiency in the culture medium was 56.54%. Similarly, the P concentrations of all ATP–P groups decreased significantly during the first 3 d of incubation and fluctuated within a narrow range afterwards. The removal efficiencies of P0.1, P0.5, P1.0, P2.0, and P4.0 systems were 89.58%, 54.35%, 52.19%, 59.07%, 56.29%, respectively. In the b-GP systems, the P concentrations of P0.1, P1.0, P2.0, and P4.0 groups decreased during Day 5 except that of the P0.5 group.
3.3. Alkaline phosphatase activity Fig. 4 shows the APA of M. aeruginosa under DOP and DIP of incubation. Overall, the changes in APA treated with DOP and DIP were different, except that all the initial values were low at the beginning of culture because of stock culture conditions. In the DIP systems, the APA of P0.1 system increased faster than the four other systems in 3 d; a decline in APA was observed on Day 7, but the APA values maintained a relatively high level afterwards. Among the five DIP systems, the APA of P0.5 group did not exhibit the tendency to increase until Day 7, which corresponded to the change in P concentrations in the culture medium. However, the APA values in all b-GP treatments demonstrated an increasing trend within 5 d and then displayed a significant decreasing trend before fluctuating within a narrow range. In the ATP–P systems, the APA values increased steadily in 5 d, followed by a gradual decline.
(a) K2HPO4-P
6
-1
Cell numbers (10 cells ml )
24 P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
20 16 12 8 4 0
2
4
6
8
10
12
14
16
Days (d)
(b) 7.5
(c) -GP
6.5 6.0
6.0
ATP-P
5.5
6
5.5 5.0 4.5 P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
4.0 3.5 3.0 2.5
6.5
-1
Cell numbers (10 cells ml )
Cell numbers (106 cells ml-1)
7.0
0
2
4
6
8
Days (d)
10
12
5.0 4.5 P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
4.0 3.5 3.0
14
16
0
2
4
6
8
10
12
14
16
Days (d)
Fig. 1. Growth curve of M. aeruginosa under different phosphorus sources: (a) K2HPO4, (b) b-GP and (c) ATP–P with the phosphorus concentrations (0.1, 0.5, 1.0, 2.0 and 4.0 mg/L).
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J. Li et al. / Marine Pollution Bulletin 92 (2015) 52–58 Table 2 Mean values of specific growth rate (l), mean doubling time (G) in days and maximum cell numbers (Nmax) of M. aeruginosa under different phosphorus sources. Parameters
l G Nmax
DIP
ATP–P
b-GP
P0.1
P0.5
P1.0
P2.0
P4.0
P0.1
P0.5
P1.0
P2.0
P4.0
P0.1
P0.5
P1.0
P2.0
P4.0
0.083 3.2 8.91
0.135 2.7 18.53
0.146 2.6 20.56
0.154 2.6 22.41
0.164 2.5 24.17
0.047 3.8 5.60
0.068 3.4 6.93
0.043 3.8 4.97
0.051 3.7 6.03
0.052 3.7 5.85
0.076 3.3 6.19
0.045 3.8 5.35
0.078 3.2 6.32
0.075 3.3 5.94
0.069 3.4 5.87
Table 3 Two-way ANOVA: cell number versus P form and P concentration. Source
df
SS
MS
F
P
P form P concentration Interaction Error Total
2 4 8 75 89
523.76 25.09 77.44 1059.56 1685.85
261.882 6.272 9.680 14.127 –
18.54 0.44 0.69 – –
0.000 0.776 0.703 – –
4. Discussion The roles of the bioavailability of various P compounds in phytoplankton have been studied in recent years (Herbes et al., 1974; Ammerman and Azam, 1991; Scanlan et al., 1997). Although DIP can be directly utilized by algal cells and is considered the most important P source for microalgae (Herbes et al., 1974), many authors have demonstrated that phytoplankton is also capable of utilizing DOP via enzymatic hydrolysis (Cembella et al., 1984; Huang and Hong, 1999). The present study has shown that the cyanobacteria, M. aeruginosa, can utilize DOP apart from DIP even
under low P conditions (Fig. 1). The capability of M. aeruginosa to use two different DOP compounds by inducing AP might be an important characteristic that allows the algae to sustain growth in DIP-deficient environments (Huang et al., 2005). The abilities that M. aeruginosa uses to utilize DOP under DIP-deficient conditions may be the principal features that allow the algae to form blooms in aquatic systems. In the present study, P forms may be the key factor in the growth of M. aeruginosa (Table 3). For example, M. aeruginosa in the b-GP and ATP–P treatments exhibited similar growth curves compared with that treated. However, the l and Nmax were higher in media treated with ATP–P than those treated with b-GP, suggesting that M. aeruginosa can easily utilize DOP with low molecular weight (Table 2). It has been reported that algae could accumulate and store large quantities of P in phosphate-rich environments (Watanabe et al., 1987). Huang et al. (2005) have shown that algae can accumulate excess phosphorus in the first 2 d to 3 d of incubation (Huang et al., 2005). Our results revealed that M. aeruginosa exhibited a rapid uptake when cultured in orthophosphate media, consistent with the report by Wu et al. (2009). Changes in TP concentration in the culture medium indicate the bioavailability of DIP and
(a) 4.5 -1
P concentration (mg L )
4.0
K2HPO4-P
P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0
2
4
6
8
10
Days (d)
(b) 4.5
(c) 4.5 -GP
4.0
P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
3.0 2.5
-1
3.5
P concentration (mg L )
-1
P concentration (mg L )
4.0
2.0 1.5 1.0 0.5
ATP-P
P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
0.0 0
2
4
6
Days (d)
8
10
0
2
4
6
8
10
Days (d)
Fig. 2. Variation of TP in culture media under different phosphorus sources: (a) K2HPO4, (b) b-GP and (c) ATP–P with the phosphorus concentrations (0.1, 0.5, 1.0, 2.0 and 4.0 mg/L).
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J. Li et al. / Marine Pollution Bulletin 92 (2015) 52–58
-1
-1
4-Nitrophenol (ug mg Pro h )
(a) 0.35 P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
0.30 0.25
K2HPO4-P
0.20 0.15 0.10 0.05 0.00
0
1
2
3
4
5
6
7
8
9
10 11
Days (d)
(c) 0.40 -1
4-Nitrophenol (ug mg Pro h )
-1
P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
-GP
0.24 0.20 0.16 0.12 0.08 0.04 0.00
0.30 0.25 0.20 0.15 0.10 0.05 0.00
0
1
2
3
4
5
6
7
8
9
ATP-P
P 0.1 P 0.5 P 1.0 P 2.0 P 4.0
0.35
-1
-1
4-Nitrophenol (ug mg Pro h )
(b) 0.28
10 11
0
1
2
3
4
Days (d)
5
6
7
8
9
10 11
Days (d)
Fig. 3. Alkaline phosphatase activity of M. aeruginosa under different phosphorus sources.
(a) 100 P Utilization Efficiency (%)
P4.0 P2.0
90
P1.0 P0.5
80
R2=0.9884
70
60
P0.1
K2HPO4-P
50 8
10
12
14
16
18
20
22 6
24
26
-1
Maximum cell numbers X m (10 cells ml )
(c) 100
90 P4.0
80
70
P0.1
R2=0.8273
P Utilization Efficiency (%)
P Utilization Efficiency (%)
(b) 100
P0.5
P2.0
P1.0 P0.1
60
50
-GP
5.0
5.5
6.0
6.5
P4.0
-1
Maximum cell numbers X m (10 cells ml )
P2.0
P1.0
80 70 60 R2=0.7750 50 P0.5 40 30 5.2
7.0 6
90
ATP-P 5.4
5.6
5.8
6.0
6.2 6
6.4 -1
Maximum cell numbers X m (10 cells ml )
Fig. 4. Relationship between the P utilization efficiency and the maximum cell numbers of M. aeruginosa.
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J. Li et al. / Marine Pollution Bulletin 92 (2015) 52–58
(b)
-1
0.25 0.20 0.15 0.10 0.05 0.00 0-0.05(4) 0.05-0.1(4) 0.10-0.5(9) 0.5-1.0(4)
0.30
-GP
-1
0.30
4-Nitrophenol (ug mg Pro h )
K2HPO4-P
-1
-1
4-Nitrophenol (ug mg Pro h )
(a) 0.35
0.25 0.20 0.15 0.10 0.05 0.00 0-0.01(5)
> 1.0(4)
0.01-0.05(6)
4-Nitrophenol (ug mg Pro h )
0.40
-1
-1
0.45
> 0.1(8)
-1
PO4 -P(mg L )
PO4 -P(mg L )
(c)
0.05-0.1(6)
3-
-1
3-
ATP-P
0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00
0-0.005(4) 0.005-0.01(4) 0.01-0.05(8) 0.05-0.1(5) 3-
> 0.1(4)
-1
PO4 -P(mg L ) Fig. 5. Changes of alkaline phosphatase activity at different PO3 4 –P concentrations.
DOP compounds to M. aeruginosa (Fig. 2). In the current study, the TP concentrations in DOP source treatments decreased significantly in the initial three days, but TP concentrations in the media treated with DIP decreased faster, which indicates a higher uptake and storage capacity compared with those in the DOP treatments. When P was exhausted in the culture media, M. aeruginosa utilized the internal P storage to maintain its growth. The constantly low TP concentrations in the solutions indicate that the cell numbers will reach the highest level in a few days. Hence, the changes in TP in the culture media showed the existence of a relationship with the Nmax of M. aeruginosa during incubation (Zhao et al., 2012). We further tested the relationship between the g and the Nmax of M. aeruginosa through nonlinear fit (Fig. 4). Nmax of M. aeruginosa exhibited a good linear response to DIP (p < 0.01, R2 = 0.9884), indicating that P dominated the primary productivity in the system (Liang et al., 2014). Moreover, Nmax and b-GP as well as ATP–P, obtained a significantly positive fit (p < 0.01, R2 = 0.8273; and p < 0.01, R2 = 0.7750, respectively). These results are supported by those of previous studies, particularly those conducted on P-limited rivers (Heiskary and Markus, 2001; Rier and Stevenson, 2006; Royer et al., 2008). The production of AP has been proposed as a mechanism to allow cyanobacteria to overcome serious phosphorus depletion (Scanlan and Wilson, 1999; Hoppe, 2003). When P content in cells decreases, phytoplankton can produce AP in the substrate quickly to hydrolyze DOP. The APA is generally recommended as an indicator of P status to compensate for the ambient P deficiency (Jamet et al., 2001). In the present study, intracellular APA was low under all of the three P source treatments during the adaptation period. Subsequently, the activities of AP increased significantly in 3 d,
which promoted DOP decomposition by M. aeruginosa during the incubation period. These data indicated that M. aeruginosa could utilize DOP because DOP was hydrolyzed to DIP gradually via AP. The APA also remained low before P was depleted in the DOP treatments, a result that is consistent with that of Huang and Hong (1999). The values of APA in the DIP treatment remained relatively high when the P concentration was very low (Fig. 3). Some authors have proven that DIP negatively correlates with algal APA (Huang et al., 2005). However, studies on the relationship between DOP and APA during the growth of M. aeruginosa are few, particularly those on the relationship between extracellular DOP and intracellular APA. Fig. 5 shows the changes in APA at different PO3 4 –P concentrations in the culture media. Our results revealed negative correlations between the APA of M. aeruginosa and DIP. Conversely, the values of APA decreased as the DIP concentrations increased in the media. Nausch (1998) pointed out that P in cells might inhibit the induction of APA when its concentration reaches a certain level, but P-deficiency may cause M. aeruginosa to induce APA. The chemical composition of P is also crucial for phosphatase generation (Hoppe, 2003). In the current study, the APA in the b-GP treatment increased within the PO3 4 –P concentration range of 0 mg L1–0.1 mg L1 and decreased afterwards. However, other variation patterns of APA were observed in the ATP–P system. The APA increased when the PO3 4 –P concentration was in the range of 0 mg L1–0.05 mg L1 and > 0.1 mg L1, but decreased in 0.05 mg L1–0.1 mg L1. In general, a negative correlation between APA and DOP under low P concentrations was observed. Although there is no conclusive evidence that smaller molecular DOP is transported directly into algal cells, a report has demonstrated that the transportation and metabolism of DOP by phytoplankton was
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apparently not mediated by AP; other results also have revealed the complex correlations between APA and DOP (Hernandez et al., 2002; Huang et al., 2005). In summary, the present study has suggested the following: (1) M. aeruginosa is capable of utilizing both DIP and DOP even under low P conditions to sustain its growth; (2) M. aeruginosa can accumulate and store cellular P to ensure that its growth rates do not immediately decline; (3) The l values and Nmax in the ATP–P treatments can be higher than those in b-GP systems possibly because of the low molecular weight of ATP–P; (4) The changes in APA at different PO3 4 –P concentrations in the culture medium can reveal negative correlations between APA and DIP, although a positive relationship can be observed between APA and DOP under low P concentrations. However, the relationship between APA and different DOP compounds needs to be further studied. The findings from future studies, together with those from the present study, may be important to the full understanding of the HABs mechanism, particularly in DIP-deficient lakes.
Acknowledgements The authors thank the students from Fudan University for helping with lab experiments. This study is financially supported by the Major Science and Technology Program for Water Pollution Control and Treatment (No. 2012ZX07102-004).
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