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Role of divalent metals Cu2+ and Zn2+ in Microcystis aeruginosa proliferation and production of toxic microcystins
Toxicon 170 (2019) 51–59
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Role of divalent metals Cu2+ and Zn2+ in Microcystis aeruginosa proliferation and production of toxic microcystins
T
Dong Aoa,b,c,∗, Zhen Leib,c, Mawuli Dzakpasub,c, Rong Chenb,c a
School of Environmental and Chemical Engineering, Xi' an Polytechnic University, Xi'an, 710048, PR China International S&T Cooperation Centre for Urban Alternative Water Resources Development, No.13 Yanta Road, Xi'an, 710055, PR China c Key Lab of Environmental Engineering, Xi'an University of Architecture and Technology, No.13 Yanta Road, Xi'an, 710055, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cell proliferation Divalent metal M. aeruginosa Microcystins
Cu2+ and Zn2+, two ubiquitous metals in water environments, can widely trigger algae blooms at favourable environmental conditions. This paper elucidates the roles of Cu2+ and Zn2+ in the proliferation of Microcystis aeruginosa (M. aeruginosa) and synthesis of Microcystins (MCs). Findings indicate significant influences of increasing Cu2+ and Zn2+ concentrations on cell proliferation at limited available phosphorus concentrations of less than 0.1 mg/L. By contrast, Cu2+ and Zn2+ notably affected MCs production at all the inoculated phosphorus concentrations. The critical concentrations of 1 μg/L and 5 μg/L for Cu2+ and Zn2+, respectively, are determined to trigger rapid cell proliferation and MCs production. Furthermore, the impacts of Cu2+ and Zn2+ on nitrogen absorption and, subsequently, on amino acids (AAs) formation in cells, is likely key in MCs synthesis. The two AAs Alanine (Ala) and glutamic acid (Glu) demonstrate the most notable variations with the concentrations of Cu2+ & Zn2+.
1. Introduction Water eutrophication and algae blooms have become increasingly common, which cause the loss of environmental functions of scenic water. Microcystis is known as one of the major genera involved in cyanobacterial blooms around the world (Wu et al., 2007). Microcystis spp. are particularly problematic because they have the potential to produce Microcystins (MCs) that reduce water quality and adversely affect aquatic ecosystems, livestock, water supplies, and recreational activities (Nakamura et al., 2003; Svircev et al., 2017; Xu et al., 2012). MCs are a group of cyclic heptapeptide compounds having a variety of forms with distinct toxicity levels, which cause reproductive, developmental, and immune toxicities, especially to the liver (Chen et al., 2016; Lone et al., 2016; McLellan and Manderville, 2017; Welker and Von, 2006; Singh et al., 2012). However, the physiological and ecological roles of MCs, as well as whether they are a major or secondary biochemical product of the cells, is still unclear (Lyck, 2004). Both nitrogen (N) and phosphorus (P) are essential nutrients for algae, and most studies have shown that N and P, especially nitratenitrogen (NO3–N) and orthophosphate-phosphorus (PO4–P) are the critical nutrients causing algal blooms (Baek et al., 2015; Xu et al., 2010; Moisander et al., 2009). The effects of PO4–P and NO3–N on the growth of M. aeruginosa also have attracted extensive attention,
∗
especially PO4–P, which is often considered the limiting nutrient for algae proliferation (Horst et al., 2014; Lurling and Faassen, 2012). Microcystins production in batch and continuous cultures has been widely studied as a function of physicochemical changes in nutrients (e.g., phosphorus, nitrogen) (Baek et al., 2015; Xu et al., 2010; Moisander et al., 2009). On the other hand, research results suggest that functional nutrients are also important limiting factors for phytoplankton under certain conditions (Brand, 1983). Both Cu and Zn are essential nutrients for phytoplankton growth and generally exist as divalent ions in aquatic environments. Cu plays a crucial role in the activity of dinitrogenase and nitrate reductase. Appropriate concentrations of Cu can facilitate the absorption of nitrate by cyanobacteria. Conversely, excess Cu inhibits the synthesis of carbohydrates in algal cells, as well as the synthesis of some amino acids and, thus, restrict algae growth (Kashyap and Gupta, 1982; Kováčik et al., 2010). In addition, as a cofactor of various enzymes in algae respiration and photosynthesis, Cu can significantly enhance the expression of enzymes and reproduction of algae (Brand, 1983; Lukač and Aegerter, 1993). Furthermore, Zn is a cofactor of many enzymes such as carbonic anhydrase, superoxide dismutase, and RNA polymerase. Suitable concentrations of Zn in the surroundings promote several biochemical processes in algal cells (Bertrand and Poirier, 2005). However, when present in excessive concentrations, Zn
Corresponding author. School of Environmental and Chemical Engineering, Xi' an Polytechnic University, Xi'an, 710048, PR China. E-mail address: [email protected] (D. Ao).
https://doi.org/10.1016/j.toxicon.2019.09.012 Received 6 May 2019; Received in revised form 9 September 2019; Accepted 11 September 2019 Available online 14 September 2019 0041-0101/ © 2019 Elsevier Ltd. All rights reserved.
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intracellular nutrients. Subsequently, the centrifugation process was repeated to remove the nutrients released from the cells. Finally, the isolated M. aeruginosa was added to a series of solutions in 1000 mL flatbottomed bottles containing 500 mL of the medium with different concentrations of divalent metals and phosphate. CZn in the Cu experiments was similar to the concentration of Zn in the BG-11 medium, which is 50.1 μg/L. Similarly, CCu in the Zn experiments was 20.2 μg/L pH was adjusted to 7.1 with 1 mol/L hydrochloric acid, whereas the initial cell density was set at 2 × 105 cells/mL by adding the prepared M. aeruginosa. All bottles were placed in an illuminated incubator (Memmert I, Germany). The temperature was maintained at 25 ± 0.5 °C, and the luminance was set at 2000 lux with a light/dark cycle of 12 h each. Samples were set in triplicates, and each bottle was shaken three times, and its position changed every day.
becomes toxic to phytoplankton and decreases its growth rate, oxygen production, photosynthetic electron transport, and chlorophyll a content (Chaloub et al., 2005; Sayyed, 2010). Therefore, considering the critical roles of Cu and Zn in algae proliferation, it is necessary that attention is paid to functional nutrients in the research about algae blooms. Nonetheless, the effects of functional nutrients on cell growth is not always subsistent but rely on specific nutrients conditions. Burgeoning research notes the occurrence of functional nutrients (especially iron and Zn) in oceanic algal blooms and demonstrates its prevalence under conditions of high nitrate and low chlorophyll (HNLC). The prevalence of the HNLC phenomena in the equatorial Pacific, Southern Ocean, and Subarctic Pacific have puzzled oceanographers for decades and has finally been attributed to the extremely low levels of functional nutrients (Ellwood, 2004; Boyd et al., 2004). The research, as mentioned above, considerably propelled marine science research in the last decades, but the implications of this conclusion for urban water systems have not been confirmed. Furthermore, there is no consensus on whether the content and production of MCs are altered directly by environmental factors, or whether there is an indirect regulation of the toxin by factors that affect growth (Wiedner et al., 2003). In urban water systems, especially where reclaimed water from urban wastewater treatment plants is used as the main replenishing water resource, it is common that TN concentrations remain high because of the low denitrification capacity of conventional aerobic biological processes. Thus, the HNLC theory may also be applied to interpret the algae blooms induced by Cu and Zn in urban water systems. This study evaluates the effects of Cu and Zn on cell growth and MCs synthesis under constant nitrate but varying phosphate concentrations. The aims are to explore their effects on cell proliferation and MCs synthesis, determine the mechanism by which Cu and Zn influences the biochemical processes involving M. aeruginosa, ascertain the conditions of functional nutrients to be emphasized in urban water, and finally provide a theoretical basis for preventing and controlling algal blooms as well as the accompanying toxic blooms.
2.2. Cell density and chlorophyll a measurement 1 mL samples of the solutions were taken every 2 days to measure cell density using a cell counter (Cellometer Auto T4, Dakewe, China). The duration of this experiment was determined according to the method proposed by Wang et al.(2014). Samples of 15 mL solutions were taken every 2 days and centrifuged (6000 rad/min, 10 min). The residual M. aeruginosa cells were collected and used to extract chlorophyll a (abbreviated as Chl a) with 90% alcohol. OD was then measured using a spectrophotometer. The concentration of chlorophyll a was calculated using Eq. (1),
C = 11.64OD663 − 2.16OD645 + 0.1(OD630 − OD750)
(1)
Cell Chl a means the Chl a content per algae cell, which can be calculated by the Chl a divided by cell density. 2.3. Microcystins detection For MCs analysis, 20 mL of the algae suspension in stationary phase (which means the cell density reached the maximum values and the variation of specific growth rate was less than 5%) was centrifugated to extract MCs by ultrasonic crushing, which was then filtered with 0.2 μm glass fibre filter (Whatman GF/C) to purify it. The sample was then passed through an SPE (Agilent SampliQC18; 3 mL, 500 mg) at a flow rate of 5 mL/min to extract MCs. It was purified with 10 mL 5% methanol solution and then eluted with 4 mL methanol. A nitrogen evaporator (Sample Concentrator MD 200) was used to evaporate the eluent. The extracted MCs were finally dissolved in 5% methanol solution. Details of the sample preparation and analysis methods are presented in Long (2010). Standard MCs were obtained from Sigma (95% purity, ENZO, US) and samples of MCs were analyzed with an HPLC (LC-2010HT, Japan) equipped with a reverse ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm Agilent Co., USA) and a diode array detector at 238 nm. The mobile phase was a mixture of PBS (0.05 mol/L) and methanol solution containing 0.1% (v/v) of trifluoroacetic acid in a volume ratio of 43:57 (Fisher, MS, US). The flow rate and injection volume were set at 1.0 mL/min and 20 μL, respectively. The run time in the microcystins detection was 20 min and, the peak of MC-RR and MC-LR was flowing out at 8.15min and 10.20 min, respectively.
2. Materials and methods 2.1. Preparation of nutrients solutions and M. aeruginosa inoculation A stock solution of 15 mg/L nitrate-nitrogen was prepared using NaNO3 (Aladdin, AR, China). Three concentrations of phosphorus (CP) of 0.04, 0.1, and 0.5 mg/L were prepared using K2HPO4 (Aladdin, AR, China). The concentrations of Cu2+ (CCu) were varied at 0.01, 0.1, 1, 10 and 100 μg/L by adding CuSO4 (Aladdin, AR, China), and that for Zn2+ (CZn) was varied at 0.05, 0.5, 5, 50 and 200 μg/L using ZnSO4 (Aladdin, AR, China). Nutrients concentrations in the culture solution are shown in Table 1. M. aeruginosa (FACHB-912) was purchased from the Institute of Hydrobiology, Chinese Academy of Science and the inoculation was carried out according to the following steps. First, M. aeruginosa was concentrated by centrifugation at 4500 rad/min for 10 min. The isolated M. aeruginosa was washed three times with ultrapure water to remove extracellular nutrients. Second, the M. aeruginosa was inoculated in a BG-11 medium without nitrogen, phosphorus, and corresponding divalent metal (Cu or Zn) sources for 7 days to exhaust the Table 1 Compositions of culture solution.
2.4. Analysis for elements composition
Components
Dose (mg/L)
Components
Dose (mg/L)
Components
Dose (mg/L)
C6H8FeNO7 Citric acid MnCl2·4H2O
6.0 6.0 1.86
CaCl2·2H2O EDTANa2 H3BO3
36 1.0 2.86
Na2CO3 Na2MoO4·2H2O Co(NO3)2·6 H2O
2.0 0.390 H2O
Cell suspension of 200 mL in the stationary phase was centrifugated (6000 rad/min, 10 min). The collected algae cells were washed with distilled water three times, and finally vacuum freeze-dried (Biocool, FD-2, China). Dry algae cells of 15 mg were sampled for elemental analysis by using an organic element analyzer (Thermo Fisher, FLASH, 2000; USA) according to JY/T017-1996. 52
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140.3 × 105 cells/mL, and the optimal CCu for cell growth is determined to be approximately 1 μg/L. Similar trends are also shown in Fig 1(d)–(e), with an optimal CZn for cell growth of approximately 5 μg/ L.
2.5. Amino acids detection Dry algae cells of 5 mg were dissolved in 10 mL of 0.1 M hydrochloric acid solution and then ultrasonically extracted for 60 min. The suspension liquid was filtered with a 0.45 μm filter. Treated sample solutions of 300 μL were added in either 300 μL of OPA derivative or 400 μL of buffer solution, well mixed and re-filtered with a 0.45 μm filter. After 15 min derivatization, an HPLC (LC-2010HT, Shimadzu, Japan) equipped with a fluorescence detector (RF-20A, Shimadzu, Japan) was used to analyse and determine the amino acid content. Chromatographic analysis conditions were set as follows: ZORBAX SBC18 column (250 mm × 4.6 mm, 5 μm Agilent Co., USA), mobile phase A: 25 mmol/L boric acid solution (adjust pH to 10.4 with potassium hydroxide) to B: tetrahydrofuran = 95: 5 (V: V). The flow rate was 1.0 mL/min, Em = 340 nm, Ex = 450 nm, and the injection volume was 1 μL. The ratio of mobile phase A to mobile phase B was varied at 90%, 72%, 70%, 65%, 63%, 53%, 45%, 35%, 90% and 90% at the gradient elution program of 0, 12, 20, 22, 31, 35, 40, 50, 52 and 60 min, respectively.
3.1.2. Chlorophyll a concentration Besides cell density, the concentration of Chl a is also a standard index used to estimate the algal biomass. When the specific cell growth rate in two days is less than 5%, algae growth is considered to have reached a stationary phase (Li et al., 2015). For example, in Fig. 1(a), the average growth rate of cell density from the 10th to 12th day was 3.1% at Ccu of 1.0 μg/L. Thus, it reached a stationary phase on the 10th day, albeit the cell density continued increasing. The time of the stationary phase for the various groups is highlighted with green circles in Fig. 1. Chl a in the stationary phase show the same trend as that of the cell density in the CP range of 0.04 and 0.1 mg/L (Fig. 1(a)), whereby increases in CCu lead to a vaulted trend of Chl a at CP of less than 0.1 mg/L. Notably, Chl a in the stationary phase is also significantly affected by CCu (P < 0.05) when CP equals to 0.5 mg/L, similar results of which is also obtained under Zn stress (Fig. 2). At the various CZn of 0.05, 0.5, 5, 50 and 200 μg/L, corresponding Chl a of 3.49, 3.99, 4.22, 2.78 and 1.91 mg/L, respectively (CP = 0.5 mg/L) were recorded. Cell Chl a of 2.85 × 10−7, 3.21 × 10−7, 3.12 × 10−7, 2.89 × 10−7 and 2.54 × 10−7 mg/cell, respectively, were recorded, corresponding to cell densities of 122.6 × 105, 124.3 × 105, 135.3 × 105, 117.0 × 105 and 75.0 × 105 cells/mL as CZn increases from 0.05 to 200 μg/L under adequate CP (CP = 0.5 mg/L) (Fig. 2(b)). This finding indicates that changes in Chl a should attribute to the effect of Zn on cell Chl a instead of cell density. Unlike the concentration of Chl-a, the specific Chl-a content per cell (Chla/cell) shows a weak variation pattern (supported in Fig. S1 of supplementary materials), which might be due to the differences in cell sizes under the stress of different trace element concentrations. Further in-depth research is required to confirm.
2.6. Other analytic methods Nitrate-nitrogen (NO3–N), nitrite-nitrogen (NO2–N), and ammonianitrogen (NH4–N) were measured by using ultraviolet spectrophotometry, N-(1-naphthyl)-ethylenediamine spectrophotometry and Nessler's reagent spectrophotometry, respectively. Dissolved total nitrogen (DTN) was measured by using persulfate digestion spectrophotometry. The concentration of dissolved organic nitrogen (DON) was calculated as the difference between DTN, NO3–N, NO2–N, and NH4–N. Deionized water of 2 mL was added to a centrifuge tube with 2 mg dry algae and ultrasonicated with an ultrasonic cell disruptor for 10min. The suspension liquid was then filtered with a 0.45 μm filter, which was used to determine soluble protein content by Coomassie spectrophotometry.
3.2. Nitrogen absorption and elements composition 3.2.1. Nitrogen absorption DTN occurring in the solution comprised four components, namely, NO3–N, NO2–N, NH4–N, and DON. NO2–N and DON are derived from intermediate products and decomposition of dead algae cells. Both NO3–N and DON are the main components of DTN in the culture when phosphate is inadequate (CP ≤ 0.1 mg/L). Conversely, DON predominates the DTN when phosphate is adequate (CP = 0.5 mg/L) (Fig. 3). This result demonstrates that variations in CCu (or CZn) are key for cell growth when phosphate is insufficient due to the mass of residual NO3–N in the stable phase of the culture. However, NO3–N is exhausted when phosphate is adequate (CP = 0.5 mg/L). So, NO3–N also affects cell growth, and it is likely the dominant factor on account of the significance of nitrogen in algal biochemical processes. Under adequate phosphate concentrations, the minimum residual NO3–N concentration is achieved at 10 μg Cu/L and 50 μg Zn/L, which is the requirement for Cu and Zn to promote the absorption nitrate by algae. NO3–N, under high phosphate concentrations, is exhausted; this is the reason why cell density is not affected by CCu (or CZn) at adequate phosphate concentrations.
2.7. Experimental replication setting and data analysis The cell density, chl a, MCs, AAs content and the different forms of nitrogen were all measured in triplicate, while elements composition was analyzed in a composite of the triplicate samples. Data were processed using Excel 2007 and are presented as mean values ± standard deviations. Statistical analysis was carried out using SPSS version 20.0 (IBM Corp., USA). A value of P < 0.05 indicates a significant difference. 3. Results 3.1. Algae proliferation 3.1.1. Cell density Cell density appears to be significantly influenced (P < 0.05) by CCu and CZn when TP concentrations fall within the range of 0.04 and 0.1 mg/L (Fig. 1(a),(b),(d) and (e)). Nonetheless, this influence appears to diminish at CP beyond this range (i.e., CP = 0.5 mg/L). Taking the influence of Cu2+ as an example, cell density shows a vaulted trend with the increase of CCu, whereby maximum cell density in cultivation under different CCu treated groups of 4.2 × 105, 4.7 × 105, 6.7 × 105, 5.6 × 105 and 2.5 × 105 cells/mL were recorded at low CP (CP = 0.04 mg/L, Fig. 1(a)). Additionally, cell density exhibited significant differences among the various groups from the 2nd day of cultivation. Similar results are also shown in Fig. 1(b), but the difference is weakened at the CCu of 1 and 10 μg/L. Furthermore, the effect of Cu is weakened with increasing CP, which eventually disappear when CP equals to 0.5 mg/L, except for the group with adequate Cu. Cell density mainly falls within the rage of 80.0 × 105 and
3.2.2. Elemental composition of algae The composition of elements, especially the ratios of carbon (C), nitrogen (N), and phosphorus (P), are useful for determining the physiological condition of algae. So, element analysis was carried out with dried algae to verify the conclusion, as mentioned above, that nitrate concentrations limit cell density in the culture with adequate phosphate concentrations. The proportion of carbon progressively increases with the increase of CCu (or CZn) (Fig. 4). By contrast, the proportion of nitrate and phosphate exhibit a downtrend with increasing CCu (or CZn) when the concentration of Cu (or Zn) is moderate (CCu ≤ 10 μg/L and 53
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Fig. 1. Effects of Cu (ãc) and Zn (d ~ f) on cell density at different concentrations of phosphorus (CP). Data are means ± SD (n = 9, error bars denote the standard deviation). The green circles indicate the time of stationary phase for various groups.
Fig. 2. Chl a in stationary phase under different Cu (a) and Zn (b) stress. Columns marked with the same letter do not differ statistically from each other at different concentration of phosphate (CP) at P < 0.05. Data are means ± SD (n = 9, error bars denote the standard deviation).
CZn ≤ 50 μg/L). On the other hand, the proportions of C, N, and P show rapid increases when Cu (or Zn) is adequate (CCu = 100 μg/L and CZn = 200 μg/L). This trend is seemingly inconsistent with the results presented in Fig. 3, albeit the N/C and N/P rate may explain this contradiction. The N/C rate shows a steady decline as the CCu increases from 0.01 to 10 μg/L, which then suddenly increase when CCu equals to 100 μg/L, so does the P/C rate. The N/P rate first slightly drops when CCu plummets to 0.1 μg/L and then increase steadily within the CCu range of 0.1–100 μg/L. The N/C, P/C, and N/P rates vary in the ranges of 0.091–0.113, 0.0031–0.0047 and 23.5–29.6, respectively, which are all lower than the Redfield ratio (Redfield, 1958). This finding demonstrates that both N and P are limiting nutrients for algae, and the effect of P is likely more significant than N. The same conclusion can
also be drawn under Zn stress. 3.3. Microcystins synthesis 3.3.1. MC-LR and MC-RR yield Two kinds of MCs were detected in M. aeruginosa (the chromatography of these are shown in Fig. S2 of supplementary materials), and the effects of Cu and Zn on the intracellular MCs are shown in Fig. 5(a) and (b), respectively. The intracellular MCs are positively related to CP, and the MCs are almost sextuple when CP increases from 0.04 to 0.5 mg/L, so does the proportion of MC-LR, but the effect of Cu and Zn on MCs is consistent. As shown in Fig. 5(a), the Intracellular MCs are significantly influenced by CCu when CP ≤ 0.1 mg/L (P < 0.05). This 54
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Fig. 3. Residual nitrogen in culture at stable phase under different Cu (a) and Zn (b) stress. Under the same concentration of phosphate (CP), five columns from left to right, respectively, represent the five concentrations of Cu (CCu) and Zn (CZn). Data are means ± SD (n = 9, error bars denote the standard deviation).
finding corroborates the effect of Cu on cell density and ρ (chl a), but the effect is more significant (P < 0.01) under adequate CP. The intracellular MCs equals to 104.2, 114.3, 108.7, 104.3 and 58.8 μg/L, respectively, corresponding to CCu increases from 0.01 to 100 μg/L when CP equals to 0.5 mg/L, and the optimal CCu of MCs (0.1 μg/L) is lower than that of cell density (1 μg/L). The optimal CZn for MCs synthesis (0.5 μg/L) is also lower than that for cell density (5 μg/L); this also demonstrates the similarity between Cu and Zn.
3.3.2. Protein and amino acid content Fig. 6 presents the content of soluble proteins at difference stresses of Cu (a) and Zn (b). The content of soluble proteins is significantly promoted when CP increases from 0.04 to 0.1 mg/L (P < 0.05), and it is nearly tripled when CP increases from 0.1 to 0.5 mg/L. Under a certain CP, the effect of Cu (or Zn) on the content of soluble proteins is also remarkable. For example, the content of soluble proteins increases from 15.37 to 18.84 mg/g when CCu increases from 0.01 to 1 μg/L under low CP (CP = 0.04 mg/L), which then sharply falls to 5.14 when CCu soar to
Fig. 4. Elements composition of algae at stable phase under high concentration of phosphate (CP). Data are means ± SD (n = 3, error bars denote the standard deviation). 55
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Fig. 5. Intracellular MCs in stationary phase under different Cu (a) and Zn (b) stress. Under the same concentration of phosphate (CP), five columns from left to right, respectively, represent the five concentrations of Cu (CCu) and Zn (CZn). Data are means ± SD (n = 9, error bars denote the standard deviation).
Fig. 6. Intracellular soluble protein at stable phase under different Cu (a) and Zn (b) stress. Under the same concentration of phosphate (CP), five columns from left to right respectively represent the five concentrations of Cu (CCu) and Zn (CZn). Columns marked with the same letter do not differ statistically from each other at different concentrations of phosphate (CP) at P < 0.05. Data are means ± SD (n = 9, error bars denote the standard deviation).
and 0.5–5 μg Zn/L, respectively. This means that adequate Cu (or Zn) will inhibit the content of these AAs. Glu, Ser, and Leu show another trend whereby the AAs content rack up with the increase of CCu even though under adequate concentrations of Cu, so does the content of Ser and Leu under Zn stress.
100 μg/L. By contrast, the effect of Cu on the soluble proteins content is likely more significant under high CP (P < 0.01). The content of soluble proteins achieves its maximum value (80.38 mg/g) at 1 μg Cu/L. The maximum content of soluble proteins of 88.0%, 98.9%,72.9%, and 38.0% correspond to 0.01 μg Cu/L, 0.1 μg Cu/L, 10 μg Cu/L and 100 μg Cu/L, respectively. A similar conclusion is also drawn under Zn stress except at the critical concentration. Moreover, the inhibition of these trace metals on intracellular soluble protein is also notable when the concentrations of metals are at the optimum values. Leucine (Leu), alanine (Ala), arginine (Arg), serine (Ser), glutamic acid (Glu), and aspartic acid (Asp) are involved in the synthesis of MCs. The contents of these AAs in M. aeruginosa under different cultivation conditions are shown in Fig. 7. Under inadequate CP (CP ≤ 0.1 mg/L), the content of Ala and Glu are lower than the other four types of AAs under Cu stress, but the content of Ala reaches a peak (1.57–2.31 μmol/ g). The same results are also shown under Zn stress. The effects of Cu (or Zn) on the AAs content shows two different trends with the concentrations of CCu (or CZn). The content of Asp, Arg, and Ala show a parabolic trend when CCu increases from 0.01 to 100 μg/L, so does Asp, Glu, Arg, and Ala under Zn stress. The AA contents peak at 1 μg Cu/L
4. Discussion The conditions required for micronutrients to be a predominant factor for algae growth in the ocean have been determined in studies of iron stimulated algae blooms, similar findings of which were also obtained for Zn-induced water blooms (Boyd et al., 2000; Brand, 1983). Previous laboratory studies and investigations of water blooms induced by micronutrients report of the coincident conclusion that under the condition of high nitrate and low chl a (HNLC), micronutrients are likely to be a non-negligible factor for algae growth (Boyd et al., 2004). Coale (2001) put forward an HNLC theory, which noted that trace metals should be noticed when CN ≥ 0.028 mg/L and ρ (chl a) ≤ 0.5 mg/m3 (CN/ρ (chl a) > 56). Though the CP was not specifically mentioned, it is commonly in the range of 1.5 × 10−6 to 10−5 M 56
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Fig. 7. Contents of dissociative amino acids in algae cells under different Cu (a) and Zn (b) stress. Under the same concentration of phosphate (CP), five columns from left to right, respectively, represent the five concentrations of Cu (CCu) and Zn (CZn). Data are means ± SD (n = 9, error bars denote the standard deviation).
adequate. Thus, not only the nutrients condition but also the biomass (cell density) has effects on cell proliferation. Nonetheless, attention should also be paid to the differences in the assessment index by which these two conclusions were drawn. When Chl a is used as the assessment index, CP can be ignored in both conclusions. Cellular MCs (microcystins content per cell) productions was closely related to the nitrogen uptake rate and positively correlated with the cellular N/C and N/P ratio (Downing et al., 2005). The algal elements composition shows substantial changes under different Cu2+/Zn2+ concentrations, especially N/C and N/P ratios (Fig. 5). According to Tillett et al. (2000), biosynthesis of microcystins refers to two putative operons (mcy à C and mcy D ~ J), whereby mcy A controls the condensation of Ala during the synthesis of microcystins and mcy D relates to the condensation of Glu. Qian et al. (2010) demonstrated that CuSO4 stimulated the transcription of mcy A in the initial stages when M. aeruginosa is exposed to CuSO4 solution with 0.5 μM Cu/L (exposed for 48 h). Conversely, the transcription of mcy D decreased to 59.8% and 46.5% of the control, respectively, after exposure to 0.1 and 0.5 μM CuSO4 for 96 h (Qian et al., 2010). This finding means that Cu2+ notably influences the synthesis of microcystins at the gene level.
(Boyd et al., 2000; Brand, 1983; Coale, 2001), whereas the CP/ρ (chl a) ranges between 9.4 × 10−5–1.86 × 10−4. Results from the current study show that the HNLC theory is also applicable to urban water when a similar nutrients condition (CN/ρ (chl a) ≥ 150, CP/ρ (chl a) ≤ 7.6 × 10−4) was set. More specifically, cell density, Chl a, MCs concentration, soluble proteins, and even the AAs content are significantly influenced by Cu (or Zn) concentrations when CP ≤ 0.1 mg/L at 15 mg N/L. By contrast, the effect of Cu (or Zn) on cell density was negligible at adequate P concentrations, which may be attributed to the population effect (Waters and Bassler, 2005). A logistic equation (Eq. (3)) for cell proliferation is used to determine the reason why the effect is weakened at adequate phosphate concentrations.
N = K /(1 + e ˆb (Xc − X ))
(3)
where, N is the biomass (cell density, 105 cells/mL); K is the environmental capacity of biomass (105 cells/mL); b is potential proliferation of algae. The fitting results of b and K shown in Table 2 indicate that cell density almost reaches the environmental capacity when phosphate is Table 2 K and b values of Logistic equation under Cu/Zn stress. Cu concentration b K (105 cells/mL)
0.01 μg/L 0.354 ± 0.015 166.4 ± 6.4
0.1 μg/L 0.392 ± 0.021 158.7 ± 6.4
1 μg/L 0.433 ± 0.031 158.4 ± 6.3
10 μg/L 0.445 ± 0.045 146.0 ± 8.7
Zn concentration b K (105 cells/mL)
0.05 μg/L 0.434 ± 0.057 145.5 ± 12.4
0.5 μg/L 0.429 ± 0.054 144.5 ± 10.7
5 μg/L 0.447 ± 0.026 149.8 ± 4.6
50 μg/L 0.469 ± 0.034 137.9 ± 9.4
Data are means ± SD (n = 3, error bars denote the standard deviation). 57
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Table 3 Correlations between microcystins and amino acids.
Asp Glu Ser Arg Ala Leu MC-RR MC-LR T-MCs
Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed)
Asp
Glu
1
−0.374 0.169 1
−0.232 0.406 0.133 0.637 0.657a 0.008 0.693a 0.004 −0.759a 0.001 −0.247 0.375 0.812a 0.000 0.366 .180
0.885a 0.000 0.473 0.075 0.412 0.127 0.097 0.730 −0.604b 0.017 0.296 0.284 −0.540b .038
Ser 0.319 0.246 0.359 0.189 1 0.732a 0.002 0.759a 0.001 −0.353 0.197 −0.871a 0.000 0.627b 0.012 −0.585b .022
Arg
Ala a
0.719 0.003 0.124 0.661 0.623b 0.013 1 0.941a 0.000 −0.536b 0.039 −0.639b 0.010 0.914a 0.000 −0.059 .834
Leu a
0.813 0.000 0.013 0.964 0.748a 0.001 0.912a 0.000 1 −0.740a 0.002 −0.783a 0.001 0.947a 0.000 −0.190 .497
MC-RR
−0.784 0.001 0.342 0.212 −0.392 0.149 −0.657a 0.008 −0.802a 0.000 1 a
0.659a 0.008 −0.708a 0.003 0.221 .428
-.0294 0.287 −0.122 0.665 −0.867a 0.000 −0.561b 0.029 −0.719a 0.003 0.575b 0.025 1 −0.615b 0.015 0.730a .002
MC-LR a
0.961 0.000 0.879a 0.000 0.469 0.078 0.821a 0.000 0.908a 0.000 −0.778a 0.001 −0.434 0.106 1 0.057 .839
T-MCs 0.378 0.165 0.504 0.055 −0.592b 0.020 −0.010 0.972 −0.116 0.680 0.056 0.844 0.760a 0.001 0.255 0.358 1
a
Means correlation is significant at the 0.01 level (2-tailed). Means correlation is significant at the 0.05 level (2-tailed). The lower-left portion is correlations analysis results under Cu stress, the upper-right portion is correlations analysis results under Zn stress. b
Fig. 8. Possible mechanism for Cu/Zn impact on algae proliferation and MCs synthesis.
considered the characteristic AA under Cu (or Zn) stress and this attributes to its low content (Fig. 7(b and d)). Due to the close correlations between AAs and MCs, Cu (or Zn) induced AAs changes will inevitably affect the synthesis of MCs, especially Glu and Ala, which should be the limiting AAs in the MCs synthesis process. Based on the results and discussion presented above, the possible mechanism for Cu (or Zn) impacts on algal proliferation and MCs synthesis can be summarized as shown in Fig. 8. The proposed mechanism shows that cell proliferation is restricted by the absence of Cu (or Zn) and that Cu (or Zn) impacts on algal proliferation and MCs synthesis in two ways. On the one hand, both Cu and Zn are essential components of many enzymes. Thus, the lack of these trace metals will restrict the synthesis of essential enzymes, which further induces adverse effects on the associated biochemical processes required for cell proliferation and MCs synthesis. On the other hand, Cu (or Zn) play a
Furthermore, the contents of AAs, which constitute MCs affected by Cu and Zn, also has immediate effects on the synthesis of MCs, as demonstrated in this paper. Correlation analysis between MCs content and AAs content shown in Table 3 indicates that the content of Ser is closely related to the TMCs content under both Cu and Zn stress (the original data is shown in Tables S1 and S2 of supplementary materials). This finding indicates that Ser, a kind of AA, is the restrictive AA in MCs synthesis. Furthermore, under Cu stress, Ser, Arg, and Ala are negatively correlated with the MC-RR content, whereas the content of Leu positively correlates with the MC-RR content. The correlations between the AAs, as mentioned above, and the MC-LR was opposite to that with MC-RR. The same correlations are also shown under Zn stress. In particular, Glu shows strong correlations with MC-RR under Cu stress and also shows close correlations with MC-LR under Zn stress. Therefore, it is should be 58
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key role in the activity of some enzymes such as nitrogenase, and nitrate reductase (Kashyap and Gupta, 1982). Hence, the absence of Cu (or Zn) would reduce the capability of algae to absorb other nutrients. By comparing the two approaches to cell proliferation and MCs synthesis, the final algal biomass (cell density) is higher when there is the appropriate Cu (or Zn) concentration than that when Cu/Zn is adequate. Moreover, the protein, AAs and MCs content of dry algal cells and the cellular content are also restrained when Cu (or Zn) is deficient. These findings imply that the limitation of Cu (or Zn) on cell proliferation and MCs synthesis are not independent. Hence, MCs should be classed as a kind of cellular material in cell proliferation.
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Kováčik, J., Klejdus, B., Hedbavny, J., Bačkor, M., 2010. Effect of copper and salicylic acid on phenolic metabolites and free amino acids in Scenedesmus quadricauda (Chlorophyceae). Plant Sci. 178 (3), 307–311. Li, J.H., Wang, Z.W., Cao, X., Wang, Z.F., Zheng, Z., 2015. Effect of orthophosphate and bioavailability of dissolved organic phosphorous compounds to typically harmful cyanobacterium Microcystis aeruginosa. Mar. Pollut. Bull. 92 (1–2), 52–58. Lone, Y., Bhide, M., Koiri, R.K., 2016. Microcystin-LR induced immunotoxicity in mammals. J. Toxicol. 2, 1–5. Long, B.M., 2010. Evidence that sulfur metabolism plays a role in microcystin production by Microcystis aeruginosa. Harmful Algae 9 (1), 74–81. Lurling, M., Faassen, E.J., 2012. Controlling toxic cyanobacteria: effects of dredging and phosphorus-binding clay on cyanobacteria and microcystins. Water Res. 46 (5), 1447–1459. Lukač, M., Aegerter, R., 1993. Influence of trace metals on growth and toxin production of microcystis aeruginosa. Toxicon : Off. J. Int. Soci. Toxinol. 31 (3), 293–305. Lyck, S., 2004. Simultaneous changes in cell quotas of microcystin, chlorophyll a, protein and carbohydrate during different growth phases of a batch culture experiment with Microcystis aeruginosa. J. Plankton Res. 26 (7), 727–736. McLellan, N.L., Manderville, R.A., 2017. Toxic mechanisms of microcystins in mammals. Toxicol. Res 6 (4), 391–405. Moisander, P.H., Ochiai, M., Lincoff, A., 2009. Nutrient limitation of Microcystis aeruginosa in northern California Klamath River reservoirs. Harmful Algae 8 (6), 889–897. Nakamura, N., Nakano, K., Matsumura, M., Sugiura, N., 2003. A novel cyanobacteriolytic bacterium Bacillus Isolated from a Eutrophic Lake. J. Ferment. Technol. 95 (2), 179–184. Qian, H., Yu, S., Sun, Z., Xie, X., Liu, W., Fu, Z., 2010. Effects of copper sulfate, hydrogen peroxide and N-phenyl-2-naphthylamine on oxidative stress and the expression of genes involved photosynthesis and microcystin disposition in Microcystis aeruginosa. Aquat. Toxicol. 99 (3), 405–412. Redfield, A.C., 1958. The biological control of chemical factors in the environment. Am. Sci. 46, 205–221. Sayyed, J.A., 2010. The study of zinc metal concentration by spectrophotometric method from Godavari River at Nanded, Maharashatra. Chem. Sin. 1 (2), 104–109. Singh, S., Srivastava, A., Oh, H.M., Ahn, C.Y., Choi, G.G., Asthana, R.K., 2012. Recent trends in development of biosensors for detection of microcystin. Toxicon : Off. J. Int. Soci. Toxinol. 60 (5), 878–894. Svircev, Z., Drobac, D., Tokodi, N., Mijovic, B., Codd, G.A., Meriluoto, J., 2017. Toxicology of microcystins with reference to cases of human intoxications and epidemiological investigations of exposures to cyanobacteria and cyanotoxins. Arch. Toxicol. 91 (2), 621–650. Tillett, D., E.M., Von, D.H., Borner, T., Neilan, B.A., Dittmann, E., 2000. Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system. Chem. Biol. 7 (10), 753–764. Waters, C.M., Bassler, B.L., 2005. Quorum sensing cell-to-cell communication in bacteria. Annu. Rev. Cell Dev. Biol. 21, 319–346. Wang, Y., Wu, M., Yu, J., Zhang, J., Zhang, R., Zhang, L., Chen, G., 2014. Differences in growth, pigment composition and photosynthetic rates of two phenotypes Microcystis aeruginosa strains under high and low iron conditions. Biochem. Syst. Ecol. 55, 112–117. Welker, M., Von, D.H., 2006. Cyanobacterial peptides - nature's own combinatorial biosynthesis. FEMS Microbiol. Rev. 30 (4), 530–563. Wiedner, C., Visser, P.M., Fastner, J., Metcalf, J.S., Codd, G.A., Mur, L.R., 2003. Effects of light on the microcystin content of microcystis strain PCC 7806. Appl. Environ. Microbiol. 69 (3), 1475–1481. Wu, Z.X., Gan, N.Q., Song, L.R., 2007. Genetic diversity: geographical distribution and toxin profiles of microcystis strains (cyanobacteria) in China. J. Integr. Plant Biol. 49 (3), 262–269. Xu, H., Paerl, H.W., Qin, B.Q., Zhu, G.W., Gao, G.A., 2010. Nitrogen and phosphorus inputs control phytoplankton growth in eutrophic Lake Taihu,China. Limnol. Oceanogr. 55 (1), 420–432. Xu, L., Qin, W., Zhang, H., Wang, Y., Dou, H., Yu, D., Ding, Y., Yang, L., Wang, Y., 2012. Alterations in microRNA expression linked to microcystin-LR-induced tumorigenicity in human WRL-68 Cells. Mutat. Res. 743 (1–2), 75–82.
5. Conclusion This paper elucidated the effects of Cu/Zn on algae cells proliferation and MCs biosynthesis in aquatic systems under high nitrate and variable CP concentrations. The results show that cell proliferation is significantly influenced by Cu (or Zn) concentration when CP is less than 0.1 mg/L, but no significant effect occurs when CP is adequate (CP = 0.5 mg/L). The critical concentrations of Cu and Zn for cell proliferation are 1 μg/L and 5 μg/L, respectively, and the biosynthesis of MCs occur at almost the same critical concentrations of Cu and Zn for cell proliferation. Nonetheless, MCs yield was still affected by functional nutrients concentrations under high CP (CP = 0.5 mg/L), which means that Cu/Zn is still efficacious under high CP (CP = 0.5 mg/L). Acknowledgement This work was supported by the National Key Research and Development Program of China (SQ2017YFGH001891), Shaanxi Program for Overseas Returnees (No. 2018012) and Xi'an Polytechnic University Ph.D. Research Startup Fund (No. BS201921). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.toxicon.2019.09.012. References Baek, S.H., Kim, D., Son, M., Yun, S.M., Kim, Y.O., 2015. Seasonal distribution of phytoplankton assemblages and nutrient-enriched bioassays as indicators of nutrient limitation of phytoplankton growth in Gwangyang Bay, Korea. Estuar. Coast Shelf Sci. 163, 265–278. Bertrand, M., Poirier, I., 2005. Photosynthetic organisms and adequate of metals. Photosynthetica 43 (3), 345–353. Boyd, P.W., Watson, A.J., Law, C.S., Abraham, E.R., Trull, T., Murdoch, R., Bakker, D.C.E., Bowie, A.R., Buesseler, K.O., Chang, H., Charette, M., Croot, P., Downing, K., Frew, R., Gall, M., Hadfield, M., Hall, J., Harvey, M., Jameson, G., LaRoche, J., Liddicoat, M., Ling, R., Maldonado, M.T., McKay, R.M., Nodder, S., Pickmere, S., Pridmore, R., Rintoul, S., Safi, K., Sutton, P., Strzepek, R., Tanneberger, K., Turner, S., Waite, A., Zeldis, J., 2000. A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization. Nature 407 (6805), 695–702. Boyd, P.W., Law, C.S., Wong, C.S., Nojiri, Y., Tsuda, A., Levasseur, M., Takeda, S., Rivkin, R., Harrison, P.J., Strzepek, R., Gower, J., McKay, R.M., Abraham, E., Arychuk, M., Clarke, J.B., Crawford, W., Crawford, D., Hale, M., Harada, K., Johnson, K., Kiyosawa, H., Kudo, I., Marchetti, A., Miller, W., Needoba, J., Nishioka, J., Ogawa, H., Page, J., Robert, M., Saito, H., Sastri, A., Sherry, N., Soutar, T., Sutherland, N., Taira, Y., Whitney, F., Wong, S.K.E., Yoshimura, T., 2004. The decline and fate of an iron-induced subarctic phytoplankton bloom. Nature 428 (6982), 549–553. Brand, L.E., 1983. Limitation of marine phytoplankton reproductive rates by zinc, manganese, and iron. Limnol. Oceanogr. 28 (6), 1182–1198. Chaloub, R.M., Magalhães, C.C.P., Dos Santos, C.P., 2005. Early toxic effects of zinc on psii of Synechocystis aquatilis F. Aquatilis (Cyanophyceae)1. J. Phycol. 41 (6), 1162–1168. Chen, L., Chen, J., Zhang, X., Xie, P., 2016. A review of reproductive toxicity of
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Role of divalent metals Cu2+ and Zn2+ in Microcystis aeruginosa proliferation and production of toxic microcystins
T
Dong Aoa,b,c,∗, Zhen Leib,c, Mawuli Dzakpasub,c, Rong Chenb,c a
School of Environmental and Chemical Engineering, Xi' an Polytechnic University, Xi'an, 710048, PR China International S&T Cooperation Centre for Urban Alternative Water Resources Development, No.13 Yanta Road, Xi'an, 710055, PR China c Key Lab of Environmental Engineering, Xi'an University of Architecture and Technology, No.13 Yanta Road, Xi'an, 710055, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cell proliferation Divalent metal M. aeruginosa Microcystins
Cu2+ and Zn2+, two ubiquitous metals in water environments, can widely trigger algae blooms at favourable environmental conditions. This paper elucidates the roles of Cu2+ and Zn2+ in the proliferation of Microcystis aeruginosa (M. aeruginosa) and synthesis of Microcystins (MCs). Findings indicate significant influences of increasing Cu2+ and Zn2+ concentrations on cell proliferation at limited available phosphorus concentrations of less than 0.1 mg/L. By contrast, Cu2+ and Zn2+ notably affected MCs production at all the inoculated phosphorus concentrations. The critical concentrations of 1 μg/L and 5 μg/L for Cu2+ and Zn2+, respectively, are determined to trigger rapid cell proliferation and MCs production. Furthermore, the impacts of Cu2+ and Zn2+ on nitrogen absorption and, subsequently, on amino acids (AAs) formation in cells, is likely key in MCs synthesis. The two AAs Alanine (Ala) and glutamic acid (Glu) demonstrate the most notable variations with the concentrations of Cu2+ & Zn2+.
1. Introduction Water eutrophication and algae blooms have become increasingly common, which cause the loss of environmental functions of scenic water. Microcystis is known as one of the major genera involved in cyanobacterial blooms around the world (Wu et al., 2007). Microcystis spp. are particularly problematic because they have the potential to produce Microcystins (MCs) that reduce water quality and adversely affect aquatic ecosystems, livestock, water supplies, and recreational activities (Nakamura et al., 2003; Svircev et al., 2017; Xu et al., 2012). MCs are a group of cyclic heptapeptide compounds having a variety of forms with distinct toxicity levels, which cause reproductive, developmental, and immune toxicities, especially to the liver (Chen et al., 2016; Lone et al., 2016; McLellan and Manderville, 2017; Welker and Von, 2006; Singh et al., 2012). However, the physiological and ecological roles of MCs, as well as whether they are a major or secondary biochemical product of the cells, is still unclear (Lyck, 2004). Both nitrogen (N) and phosphorus (P) are essential nutrients for algae, and most studies have shown that N and P, especially nitratenitrogen (NO3–N) and orthophosphate-phosphorus (PO4–P) are the critical nutrients causing algal blooms (Baek et al., 2015; Xu et al., 2010; Moisander et al., 2009). The effects of PO4–P and NO3–N on the growth of M. aeruginosa also have attracted extensive attention,
∗
especially PO4–P, which is often considered the limiting nutrient for algae proliferation (Horst et al., 2014; Lurling and Faassen, 2012). Microcystins production in batch and continuous cultures has been widely studied as a function of physicochemical changes in nutrients (e.g., phosphorus, nitrogen) (Baek et al., 2015; Xu et al., 2010; Moisander et al., 2009). On the other hand, research results suggest that functional nutrients are also important limiting factors for phytoplankton under certain conditions (Brand, 1983). Both Cu and Zn are essential nutrients for phytoplankton growth and generally exist as divalent ions in aquatic environments. Cu plays a crucial role in the activity of dinitrogenase and nitrate reductase. Appropriate concentrations of Cu can facilitate the absorption of nitrate by cyanobacteria. Conversely, excess Cu inhibits the synthesis of carbohydrates in algal cells, as well as the synthesis of some amino acids and, thus, restrict algae growth (Kashyap and Gupta, 1982; Kováčik et al., 2010). In addition, as a cofactor of various enzymes in algae respiration and photosynthesis, Cu can significantly enhance the expression of enzymes and reproduction of algae (Brand, 1983; Lukač and Aegerter, 1993). Furthermore, Zn is a cofactor of many enzymes such as carbonic anhydrase, superoxide dismutase, and RNA polymerase. Suitable concentrations of Zn in the surroundings promote several biochemical processes in algal cells (Bertrand and Poirier, 2005). However, when present in excessive concentrations, Zn
Corresponding author. School of Environmental and Chemical Engineering, Xi' an Polytechnic University, Xi'an, 710048, PR China. E-mail address: [email protected] (D. Ao).
https://doi.org/10.1016/j.toxicon.2019.09.012 Received 6 May 2019; Received in revised form 9 September 2019; Accepted 11 September 2019 Available online 14 September 2019 0041-0101/ © 2019 Elsevier Ltd. All rights reserved.
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intracellular nutrients. Subsequently, the centrifugation process was repeated to remove the nutrients released from the cells. Finally, the isolated M. aeruginosa was added to a series of solutions in 1000 mL flatbottomed bottles containing 500 mL of the medium with different concentrations of divalent metals and phosphate. CZn in the Cu experiments was similar to the concentration of Zn in the BG-11 medium, which is 50.1 μg/L. Similarly, CCu in the Zn experiments was 20.2 μg/L pH was adjusted to 7.1 with 1 mol/L hydrochloric acid, whereas the initial cell density was set at 2 × 105 cells/mL by adding the prepared M. aeruginosa. All bottles were placed in an illuminated incubator (Memmert I, Germany). The temperature was maintained at 25 ± 0.5 °C, and the luminance was set at 2000 lux with a light/dark cycle of 12 h each. Samples were set in triplicates, and each bottle was shaken three times, and its position changed every day.
becomes toxic to phytoplankton and decreases its growth rate, oxygen production, photosynthetic electron transport, and chlorophyll a content (Chaloub et al., 2005; Sayyed, 2010). Therefore, considering the critical roles of Cu and Zn in algae proliferation, it is necessary that attention is paid to functional nutrients in the research about algae blooms. Nonetheless, the effects of functional nutrients on cell growth is not always subsistent but rely on specific nutrients conditions. Burgeoning research notes the occurrence of functional nutrients (especially iron and Zn) in oceanic algal blooms and demonstrates its prevalence under conditions of high nitrate and low chlorophyll (HNLC). The prevalence of the HNLC phenomena in the equatorial Pacific, Southern Ocean, and Subarctic Pacific have puzzled oceanographers for decades and has finally been attributed to the extremely low levels of functional nutrients (Ellwood, 2004; Boyd et al., 2004). The research, as mentioned above, considerably propelled marine science research in the last decades, but the implications of this conclusion for urban water systems have not been confirmed. Furthermore, there is no consensus on whether the content and production of MCs are altered directly by environmental factors, or whether there is an indirect regulation of the toxin by factors that affect growth (Wiedner et al., 2003). In urban water systems, especially where reclaimed water from urban wastewater treatment plants is used as the main replenishing water resource, it is common that TN concentrations remain high because of the low denitrification capacity of conventional aerobic biological processes. Thus, the HNLC theory may also be applied to interpret the algae blooms induced by Cu and Zn in urban water systems. This study evaluates the effects of Cu and Zn on cell growth and MCs synthesis under constant nitrate but varying phosphate concentrations. The aims are to explore their effects on cell proliferation and MCs synthesis, determine the mechanism by which Cu and Zn influences the biochemical processes involving M. aeruginosa, ascertain the conditions of functional nutrients to be emphasized in urban water, and finally provide a theoretical basis for preventing and controlling algal blooms as well as the accompanying toxic blooms.
2.2. Cell density and chlorophyll a measurement 1 mL samples of the solutions were taken every 2 days to measure cell density using a cell counter (Cellometer Auto T4, Dakewe, China). The duration of this experiment was determined according to the method proposed by Wang et al.(2014). Samples of 15 mL solutions were taken every 2 days and centrifuged (6000 rad/min, 10 min). The residual M. aeruginosa cells were collected and used to extract chlorophyll a (abbreviated as Chl a) with 90% alcohol. OD was then measured using a spectrophotometer. The concentration of chlorophyll a was calculated using Eq. (1),
C = 11.64OD663 − 2.16OD645 + 0.1(OD630 − OD750)
(1)
Cell Chl a means the Chl a content per algae cell, which can be calculated by the Chl a divided by cell density. 2.3. Microcystins detection For MCs analysis, 20 mL of the algae suspension in stationary phase (which means the cell density reached the maximum values and the variation of specific growth rate was less than 5%) was centrifugated to extract MCs by ultrasonic crushing, which was then filtered with 0.2 μm glass fibre filter (Whatman GF/C) to purify it. The sample was then passed through an SPE (Agilent SampliQC18; 3 mL, 500 mg) at a flow rate of 5 mL/min to extract MCs. It was purified with 10 mL 5% methanol solution and then eluted with 4 mL methanol. A nitrogen evaporator (Sample Concentrator MD 200) was used to evaporate the eluent. The extracted MCs were finally dissolved in 5% methanol solution. Details of the sample preparation and analysis methods are presented in Long (2010). Standard MCs were obtained from Sigma (95% purity, ENZO, US) and samples of MCs were analyzed with an HPLC (LC-2010HT, Japan) equipped with a reverse ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm Agilent Co., USA) and a diode array detector at 238 nm. The mobile phase was a mixture of PBS (0.05 mol/L) and methanol solution containing 0.1% (v/v) of trifluoroacetic acid in a volume ratio of 43:57 (Fisher, MS, US). The flow rate and injection volume were set at 1.0 mL/min and 20 μL, respectively. The run time in the microcystins detection was 20 min and, the peak of MC-RR and MC-LR was flowing out at 8.15min and 10.20 min, respectively.
2. Materials and methods 2.1. Preparation of nutrients solutions and M. aeruginosa inoculation A stock solution of 15 mg/L nitrate-nitrogen was prepared using NaNO3 (Aladdin, AR, China). Three concentrations of phosphorus (CP) of 0.04, 0.1, and 0.5 mg/L were prepared using K2HPO4 (Aladdin, AR, China). The concentrations of Cu2+ (CCu) were varied at 0.01, 0.1, 1, 10 and 100 μg/L by adding CuSO4 (Aladdin, AR, China), and that for Zn2+ (CZn) was varied at 0.05, 0.5, 5, 50 and 200 μg/L using ZnSO4 (Aladdin, AR, China). Nutrients concentrations in the culture solution are shown in Table 1. M. aeruginosa (FACHB-912) was purchased from the Institute of Hydrobiology, Chinese Academy of Science and the inoculation was carried out according to the following steps. First, M. aeruginosa was concentrated by centrifugation at 4500 rad/min for 10 min. The isolated M. aeruginosa was washed three times with ultrapure water to remove extracellular nutrients. Second, the M. aeruginosa was inoculated in a BG-11 medium without nitrogen, phosphorus, and corresponding divalent metal (Cu or Zn) sources for 7 days to exhaust the Table 1 Compositions of culture solution.
2.4. Analysis for elements composition
Components
Dose (mg/L)
Components
Dose (mg/L)
Components
Dose (mg/L)
C6H8FeNO7 Citric acid MnCl2·4H2O
6.0 6.0 1.86
CaCl2·2H2O EDTANa2 H3BO3
36 1.0 2.86
Na2CO3 Na2MoO4·2H2O Co(NO3)2·6 H2O
2.0 0.390 H2O
Cell suspension of 200 mL in the stationary phase was centrifugated (6000 rad/min, 10 min). The collected algae cells were washed with distilled water three times, and finally vacuum freeze-dried (Biocool, FD-2, China). Dry algae cells of 15 mg were sampled for elemental analysis by using an organic element analyzer (Thermo Fisher, FLASH, 2000; USA) according to JY/T017-1996. 52
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140.3 × 105 cells/mL, and the optimal CCu for cell growth is determined to be approximately 1 μg/L. Similar trends are also shown in Fig 1(d)–(e), with an optimal CZn for cell growth of approximately 5 μg/ L.
2.5. Amino acids detection Dry algae cells of 5 mg were dissolved in 10 mL of 0.1 M hydrochloric acid solution and then ultrasonically extracted for 60 min. The suspension liquid was filtered with a 0.45 μm filter. Treated sample solutions of 300 μL were added in either 300 μL of OPA derivative or 400 μL of buffer solution, well mixed and re-filtered with a 0.45 μm filter. After 15 min derivatization, an HPLC (LC-2010HT, Shimadzu, Japan) equipped with a fluorescence detector (RF-20A, Shimadzu, Japan) was used to analyse and determine the amino acid content. Chromatographic analysis conditions were set as follows: ZORBAX SBC18 column (250 mm × 4.6 mm, 5 μm Agilent Co., USA), mobile phase A: 25 mmol/L boric acid solution (adjust pH to 10.4 with potassium hydroxide) to B: tetrahydrofuran = 95: 5 (V: V). The flow rate was 1.0 mL/min, Em = 340 nm, Ex = 450 nm, and the injection volume was 1 μL. The ratio of mobile phase A to mobile phase B was varied at 90%, 72%, 70%, 65%, 63%, 53%, 45%, 35%, 90% and 90% at the gradient elution program of 0, 12, 20, 22, 31, 35, 40, 50, 52 and 60 min, respectively.
3.1.2. Chlorophyll a concentration Besides cell density, the concentration of Chl a is also a standard index used to estimate the algal biomass. When the specific cell growth rate in two days is less than 5%, algae growth is considered to have reached a stationary phase (Li et al., 2015). For example, in Fig. 1(a), the average growth rate of cell density from the 10th to 12th day was 3.1% at Ccu of 1.0 μg/L. Thus, it reached a stationary phase on the 10th day, albeit the cell density continued increasing. The time of the stationary phase for the various groups is highlighted with green circles in Fig. 1. Chl a in the stationary phase show the same trend as that of the cell density in the CP range of 0.04 and 0.1 mg/L (Fig. 1(a)), whereby increases in CCu lead to a vaulted trend of Chl a at CP of less than 0.1 mg/L. Notably, Chl a in the stationary phase is also significantly affected by CCu (P < 0.05) when CP equals to 0.5 mg/L, similar results of which is also obtained under Zn stress (Fig. 2). At the various CZn of 0.05, 0.5, 5, 50 and 200 μg/L, corresponding Chl a of 3.49, 3.99, 4.22, 2.78 and 1.91 mg/L, respectively (CP = 0.5 mg/L) were recorded. Cell Chl a of 2.85 × 10−7, 3.21 × 10−7, 3.12 × 10−7, 2.89 × 10−7 and 2.54 × 10−7 mg/cell, respectively, were recorded, corresponding to cell densities of 122.6 × 105, 124.3 × 105, 135.3 × 105, 117.0 × 105 and 75.0 × 105 cells/mL as CZn increases from 0.05 to 200 μg/L under adequate CP (CP = 0.5 mg/L) (Fig. 2(b)). This finding indicates that changes in Chl a should attribute to the effect of Zn on cell Chl a instead of cell density. Unlike the concentration of Chl-a, the specific Chl-a content per cell (Chla/cell) shows a weak variation pattern (supported in Fig. S1 of supplementary materials), which might be due to the differences in cell sizes under the stress of different trace element concentrations. Further in-depth research is required to confirm.
2.6. Other analytic methods Nitrate-nitrogen (NO3–N), nitrite-nitrogen (NO2–N), and ammonianitrogen (NH4–N) were measured by using ultraviolet spectrophotometry, N-(1-naphthyl)-ethylenediamine spectrophotometry and Nessler's reagent spectrophotometry, respectively. Dissolved total nitrogen (DTN) was measured by using persulfate digestion spectrophotometry. The concentration of dissolved organic nitrogen (DON) was calculated as the difference between DTN, NO3–N, NO2–N, and NH4–N. Deionized water of 2 mL was added to a centrifuge tube with 2 mg dry algae and ultrasonicated with an ultrasonic cell disruptor for 10min. The suspension liquid was then filtered with a 0.45 μm filter, which was used to determine soluble protein content by Coomassie spectrophotometry.
3.2. Nitrogen absorption and elements composition 3.2.1. Nitrogen absorption DTN occurring in the solution comprised four components, namely, NO3–N, NO2–N, NH4–N, and DON. NO2–N and DON are derived from intermediate products and decomposition of dead algae cells. Both NO3–N and DON are the main components of DTN in the culture when phosphate is inadequate (CP ≤ 0.1 mg/L). Conversely, DON predominates the DTN when phosphate is adequate (CP = 0.5 mg/L) (Fig. 3). This result demonstrates that variations in CCu (or CZn) are key for cell growth when phosphate is insufficient due to the mass of residual NO3–N in the stable phase of the culture. However, NO3–N is exhausted when phosphate is adequate (CP = 0.5 mg/L). So, NO3–N also affects cell growth, and it is likely the dominant factor on account of the significance of nitrogen in algal biochemical processes. Under adequate phosphate concentrations, the minimum residual NO3–N concentration is achieved at 10 μg Cu/L and 50 μg Zn/L, which is the requirement for Cu and Zn to promote the absorption nitrate by algae. NO3–N, under high phosphate concentrations, is exhausted; this is the reason why cell density is not affected by CCu (or CZn) at adequate phosphate concentrations.
2.7. Experimental replication setting and data analysis The cell density, chl a, MCs, AAs content and the different forms of nitrogen were all measured in triplicate, while elements composition was analyzed in a composite of the triplicate samples. Data were processed using Excel 2007 and are presented as mean values ± standard deviations. Statistical analysis was carried out using SPSS version 20.0 (IBM Corp., USA). A value of P < 0.05 indicates a significant difference. 3. Results 3.1. Algae proliferation 3.1.1. Cell density Cell density appears to be significantly influenced (P < 0.05) by CCu and CZn when TP concentrations fall within the range of 0.04 and 0.1 mg/L (Fig. 1(a),(b),(d) and (e)). Nonetheless, this influence appears to diminish at CP beyond this range (i.e., CP = 0.5 mg/L). Taking the influence of Cu2+ as an example, cell density shows a vaulted trend with the increase of CCu, whereby maximum cell density in cultivation under different CCu treated groups of 4.2 × 105, 4.7 × 105, 6.7 × 105, 5.6 × 105 and 2.5 × 105 cells/mL were recorded at low CP (CP = 0.04 mg/L, Fig. 1(a)). Additionally, cell density exhibited significant differences among the various groups from the 2nd day of cultivation. Similar results are also shown in Fig. 1(b), but the difference is weakened at the CCu of 1 and 10 μg/L. Furthermore, the effect of Cu is weakened with increasing CP, which eventually disappear when CP equals to 0.5 mg/L, except for the group with adequate Cu. Cell density mainly falls within the rage of 80.0 × 105 and
3.2.2. Elemental composition of algae The composition of elements, especially the ratios of carbon (C), nitrogen (N), and phosphorus (P), are useful for determining the physiological condition of algae. So, element analysis was carried out with dried algae to verify the conclusion, as mentioned above, that nitrate concentrations limit cell density in the culture with adequate phosphate concentrations. The proportion of carbon progressively increases with the increase of CCu (or CZn) (Fig. 4). By contrast, the proportion of nitrate and phosphate exhibit a downtrend with increasing CCu (or CZn) when the concentration of Cu (or Zn) is moderate (CCu ≤ 10 μg/L and 53
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Fig. 1. Effects of Cu (ãc) and Zn (d ~ f) on cell density at different concentrations of phosphorus (CP). Data are means ± SD (n = 9, error bars denote the standard deviation). The green circles indicate the time of stationary phase for various groups.
Fig. 2. Chl a in stationary phase under different Cu (a) and Zn (b) stress. Columns marked with the same letter do not differ statistically from each other at different concentration of phosphate (CP) at P < 0.05. Data are means ± SD (n = 9, error bars denote the standard deviation).
CZn ≤ 50 μg/L). On the other hand, the proportions of C, N, and P show rapid increases when Cu (or Zn) is adequate (CCu = 100 μg/L and CZn = 200 μg/L). This trend is seemingly inconsistent with the results presented in Fig. 3, albeit the N/C and N/P rate may explain this contradiction. The N/C rate shows a steady decline as the CCu increases from 0.01 to 10 μg/L, which then suddenly increase when CCu equals to 100 μg/L, so does the P/C rate. The N/P rate first slightly drops when CCu plummets to 0.1 μg/L and then increase steadily within the CCu range of 0.1–100 μg/L. The N/C, P/C, and N/P rates vary in the ranges of 0.091–0.113, 0.0031–0.0047 and 23.5–29.6, respectively, which are all lower than the Redfield ratio (Redfield, 1958). This finding demonstrates that both N and P are limiting nutrients for algae, and the effect of P is likely more significant than N. The same conclusion can
also be drawn under Zn stress. 3.3. Microcystins synthesis 3.3.1. MC-LR and MC-RR yield Two kinds of MCs were detected in M. aeruginosa (the chromatography of these are shown in Fig. S2 of supplementary materials), and the effects of Cu and Zn on the intracellular MCs are shown in Fig. 5(a) and (b), respectively. The intracellular MCs are positively related to CP, and the MCs are almost sextuple when CP increases from 0.04 to 0.5 mg/L, so does the proportion of MC-LR, but the effect of Cu and Zn on MCs is consistent. As shown in Fig. 5(a), the Intracellular MCs are significantly influenced by CCu when CP ≤ 0.1 mg/L (P < 0.05). This 54
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Fig. 3. Residual nitrogen in culture at stable phase under different Cu (a) and Zn (b) stress. Under the same concentration of phosphate (CP), five columns from left to right, respectively, represent the five concentrations of Cu (CCu) and Zn (CZn). Data are means ± SD (n = 9, error bars denote the standard deviation).
finding corroborates the effect of Cu on cell density and ρ (chl a), but the effect is more significant (P < 0.01) under adequate CP. The intracellular MCs equals to 104.2, 114.3, 108.7, 104.3 and 58.8 μg/L, respectively, corresponding to CCu increases from 0.01 to 100 μg/L when CP equals to 0.5 mg/L, and the optimal CCu of MCs (0.1 μg/L) is lower than that of cell density (1 μg/L). The optimal CZn for MCs synthesis (0.5 μg/L) is also lower than that for cell density (5 μg/L); this also demonstrates the similarity between Cu and Zn.
3.3.2. Protein and amino acid content Fig. 6 presents the content of soluble proteins at difference stresses of Cu (a) and Zn (b). The content of soluble proteins is significantly promoted when CP increases from 0.04 to 0.1 mg/L (P < 0.05), and it is nearly tripled when CP increases from 0.1 to 0.5 mg/L. Under a certain CP, the effect of Cu (or Zn) on the content of soluble proteins is also remarkable. For example, the content of soluble proteins increases from 15.37 to 18.84 mg/g when CCu increases from 0.01 to 1 μg/L under low CP (CP = 0.04 mg/L), which then sharply falls to 5.14 when CCu soar to
Fig. 4. Elements composition of algae at stable phase under high concentration of phosphate (CP). Data are means ± SD (n = 3, error bars denote the standard deviation). 55
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Fig. 5. Intracellular MCs in stationary phase under different Cu (a) and Zn (b) stress. Under the same concentration of phosphate (CP), five columns from left to right, respectively, represent the five concentrations of Cu (CCu) and Zn (CZn). Data are means ± SD (n = 9, error bars denote the standard deviation).
Fig. 6. Intracellular soluble protein at stable phase under different Cu (a) and Zn (b) stress. Under the same concentration of phosphate (CP), five columns from left to right respectively represent the five concentrations of Cu (CCu) and Zn (CZn). Columns marked with the same letter do not differ statistically from each other at different concentrations of phosphate (CP) at P < 0.05. Data are means ± SD (n = 9, error bars denote the standard deviation).
and 0.5–5 μg Zn/L, respectively. This means that adequate Cu (or Zn) will inhibit the content of these AAs. Glu, Ser, and Leu show another trend whereby the AAs content rack up with the increase of CCu even though under adequate concentrations of Cu, so does the content of Ser and Leu under Zn stress.
100 μg/L. By contrast, the effect of Cu on the soluble proteins content is likely more significant under high CP (P < 0.01). The content of soluble proteins achieves its maximum value (80.38 mg/g) at 1 μg Cu/L. The maximum content of soluble proteins of 88.0%, 98.9%,72.9%, and 38.0% correspond to 0.01 μg Cu/L, 0.1 μg Cu/L, 10 μg Cu/L and 100 μg Cu/L, respectively. A similar conclusion is also drawn under Zn stress except at the critical concentration. Moreover, the inhibition of these trace metals on intracellular soluble protein is also notable when the concentrations of metals are at the optimum values. Leucine (Leu), alanine (Ala), arginine (Arg), serine (Ser), glutamic acid (Glu), and aspartic acid (Asp) are involved in the synthesis of MCs. The contents of these AAs in M. aeruginosa under different cultivation conditions are shown in Fig. 7. Under inadequate CP (CP ≤ 0.1 mg/L), the content of Ala and Glu are lower than the other four types of AAs under Cu stress, but the content of Ala reaches a peak (1.57–2.31 μmol/ g). The same results are also shown under Zn stress. The effects of Cu (or Zn) on the AAs content shows two different trends with the concentrations of CCu (or CZn). The content of Asp, Arg, and Ala show a parabolic trend when CCu increases from 0.01 to 100 μg/L, so does Asp, Glu, Arg, and Ala under Zn stress. The AA contents peak at 1 μg Cu/L
4. Discussion The conditions required for micronutrients to be a predominant factor for algae growth in the ocean have been determined in studies of iron stimulated algae blooms, similar findings of which were also obtained for Zn-induced water blooms (Boyd et al., 2000; Brand, 1983). Previous laboratory studies and investigations of water blooms induced by micronutrients report of the coincident conclusion that under the condition of high nitrate and low chl a (HNLC), micronutrients are likely to be a non-negligible factor for algae growth (Boyd et al., 2004). Coale (2001) put forward an HNLC theory, which noted that trace metals should be noticed when CN ≥ 0.028 mg/L and ρ (chl a) ≤ 0.5 mg/m3 (CN/ρ (chl a) > 56). Though the CP was not specifically mentioned, it is commonly in the range of 1.5 × 10−6 to 10−5 M 56
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Fig. 7. Contents of dissociative amino acids in algae cells under different Cu (a) and Zn (b) stress. Under the same concentration of phosphate (CP), five columns from left to right, respectively, represent the five concentrations of Cu (CCu) and Zn (CZn). Data are means ± SD (n = 9, error bars denote the standard deviation).
adequate. Thus, not only the nutrients condition but also the biomass (cell density) has effects on cell proliferation. Nonetheless, attention should also be paid to the differences in the assessment index by which these two conclusions were drawn. When Chl a is used as the assessment index, CP can be ignored in both conclusions. Cellular MCs (microcystins content per cell) productions was closely related to the nitrogen uptake rate and positively correlated with the cellular N/C and N/P ratio (Downing et al., 2005). The algal elements composition shows substantial changes under different Cu2+/Zn2+ concentrations, especially N/C and N/P ratios (Fig. 5). According to Tillett et al. (2000), biosynthesis of microcystins refers to two putative operons (mcy à C and mcy D ~ J), whereby mcy A controls the condensation of Ala during the synthesis of microcystins and mcy D relates to the condensation of Glu. Qian et al. (2010) demonstrated that CuSO4 stimulated the transcription of mcy A in the initial stages when M. aeruginosa is exposed to CuSO4 solution with 0.5 μM Cu/L (exposed for 48 h). Conversely, the transcription of mcy D decreased to 59.8% and 46.5% of the control, respectively, after exposure to 0.1 and 0.5 μM CuSO4 for 96 h (Qian et al., 2010). This finding means that Cu2+ notably influences the synthesis of microcystins at the gene level.
(Boyd et al., 2000; Brand, 1983; Coale, 2001), whereas the CP/ρ (chl a) ranges between 9.4 × 10−5–1.86 × 10−4. Results from the current study show that the HNLC theory is also applicable to urban water when a similar nutrients condition (CN/ρ (chl a) ≥ 150, CP/ρ (chl a) ≤ 7.6 × 10−4) was set. More specifically, cell density, Chl a, MCs concentration, soluble proteins, and even the AAs content are significantly influenced by Cu (or Zn) concentrations when CP ≤ 0.1 mg/L at 15 mg N/L. By contrast, the effect of Cu (or Zn) on cell density was negligible at adequate P concentrations, which may be attributed to the population effect (Waters and Bassler, 2005). A logistic equation (Eq. (3)) for cell proliferation is used to determine the reason why the effect is weakened at adequate phosphate concentrations.
N = K /(1 + e ˆb (Xc − X ))
(3)
where, N is the biomass (cell density, 105 cells/mL); K is the environmental capacity of biomass (105 cells/mL); b is potential proliferation of algae. The fitting results of b and K shown in Table 2 indicate that cell density almost reaches the environmental capacity when phosphate is Table 2 K and b values of Logistic equation under Cu/Zn stress. Cu concentration b K (105 cells/mL)
0.01 μg/L 0.354 ± 0.015 166.4 ± 6.4
0.1 μg/L 0.392 ± 0.021 158.7 ± 6.4
1 μg/L 0.433 ± 0.031 158.4 ± 6.3
10 μg/L 0.445 ± 0.045 146.0 ± 8.7
Zn concentration b K (105 cells/mL)
0.05 μg/L 0.434 ± 0.057 145.5 ± 12.4
0.5 μg/L 0.429 ± 0.054 144.5 ± 10.7
5 μg/L 0.447 ± 0.026 149.8 ± 4.6
50 μg/L 0.469 ± 0.034 137.9 ± 9.4
Data are means ± SD (n = 3, error bars denote the standard deviation). 57
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Table 3 Correlations between microcystins and amino acids.
Asp Glu Ser Arg Ala Leu MC-RR MC-LR T-MCs
Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed) Pearson Correlation Sig. (2-tailed)
Asp
Glu
1
−0.374 0.169 1
−0.232 0.406 0.133 0.637 0.657a 0.008 0.693a 0.004 −0.759a 0.001 −0.247 0.375 0.812a 0.000 0.366 .180
0.885a 0.000 0.473 0.075 0.412 0.127 0.097 0.730 −0.604b 0.017 0.296 0.284 −0.540b .038
Ser 0.319 0.246 0.359 0.189 1 0.732a 0.002 0.759a 0.001 −0.353 0.197 −0.871a 0.000 0.627b 0.012 −0.585b .022
Arg
Ala a
0.719 0.003 0.124 0.661 0.623b 0.013 1 0.941a 0.000 −0.536b 0.039 −0.639b 0.010 0.914a 0.000 −0.059 .834
Leu a
0.813 0.000 0.013 0.964 0.748a 0.001 0.912a 0.000 1 −0.740a 0.002 −0.783a 0.001 0.947a 0.000 −0.190 .497
MC-RR
−0.784 0.001 0.342 0.212 −0.392 0.149 −0.657a 0.008 −0.802a 0.000 1 a
0.659a 0.008 −0.708a 0.003 0.221 .428
-.0294 0.287 −0.122 0.665 −0.867a 0.000 −0.561b 0.029 −0.719a 0.003 0.575b 0.025 1 −0.615b 0.015 0.730a .002
MC-LR a
0.961 0.000 0.879a 0.000 0.469 0.078 0.821a 0.000 0.908a 0.000 −0.778a 0.001 −0.434 0.106 1 0.057 .839
T-MCs 0.378 0.165 0.504 0.055 −0.592b 0.020 −0.010 0.972 −0.116 0.680 0.056 0.844 0.760a 0.001 0.255 0.358 1
a
Means correlation is significant at the 0.01 level (2-tailed). Means correlation is significant at the 0.05 level (2-tailed). The lower-left portion is correlations analysis results under Cu stress, the upper-right portion is correlations analysis results under Zn stress. b
Fig. 8. Possible mechanism for Cu/Zn impact on algae proliferation and MCs synthesis.
considered the characteristic AA under Cu (or Zn) stress and this attributes to its low content (Fig. 7(b and d)). Due to the close correlations between AAs and MCs, Cu (or Zn) induced AAs changes will inevitably affect the synthesis of MCs, especially Glu and Ala, which should be the limiting AAs in the MCs synthesis process. Based on the results and discussion presented above, the possible mechanism for Cu (or Zn) impacts on algal proliferation and MCs synthesis can be summarized as shown in Fig. 8. The proposed mechanism shows that cell proliferation is restricted by the absence of Cu (or Zn) and that Cu (or Zn) impacts on algal proliferation and MCs synthesis in two ways. On the one hand, both Cu and Zn are essential components of many enzymes. Thus, the lack of these trace metals will restrict the synthesis of essential enzymes, which further induces adverse effects on the associated biochemical processes required for cell proliferation and MCs synthesis. On the other hand, Cu (or Zn) play a
Furthermore, the contents of AAs, which constitute MCs affected by Cu and Zn, also has immediate effects on the synthesis of MCs, as demonstrated in this paper. Correlation analysis between MCs content and AAs content shown in Table 3 indicates that the content of Ser is closely related to the TMCs content under both Cu and Zn stress (the original data is shown in Tables S1 and S2 of supplementary materials). This finding indicates that Ser, a kind of AA, is the restrictive AA in MCs synthesis. Furthermore, under Cu stress, Ser, Arg, and Ala are negatively correlated with the MC-RR content, whereas the content of Leu positively correlates with the MC-RR content. The correlations between the AAs, as mentioned above, and the MC-LR was opposite to that with MC-RR. The same correlations are also shown under Zn stress. In particular, Glu shows strong correlations with MC-RR under Cu stress and also shows close correlations with MC-LR under Zn stress. Therefore, it is should be 58
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key role in the activity of some enzymes such as nitrogenase, and nitrate reductase (Kashyap and Gupta, 1982). Hence, the absence of Cu (or Zn) would reduce the capability of algae to absorb other nutrients. By comparing the two approaches to cell proliferation and MCs synthesis, the final algal biomass (cell density) is higher when there is the appropriate Cu (or Zn) concentration than that when Cu/Zn is adequate. Moreover, the protein, AAs and MCs content of dry algal cells and the cellular content are also restrained when Cu (or Zn) is deficient. These findings imply that the limitation of Cu (or Zn) on cell proliferation and MCs synthesis are not independent. Hence, MCs should be classed as a kind of cellular material in cell proliferation.
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5. Conclusion This paper elucidated the effects of Cu/Zn on algae cells proliferation and MCs biosynthesis in aquatic systems under high nitrate and variable CP concentrations. The results show that cell proliferation is significantly influenced by Cu (or Zn) concentration when CP is less than 0.1 mg/L, but no significant effect occurs when CP is adequate (CP = 0.5 mg/L). The critical concentrations of Cu and Zn for cell proliferation are 1 μg/L and 5 μg/L, respectively, and the biosynthesis of MCs occur at almost the same critical concentrations of Cu and Zn for cell proliferation. Nonetheless, MCs yield was still affected by functional nutrients concentrations under high CP (CP = 0.5 mg/L), which means that Cu/Zn is still efficacious under high CP (CP = 0.5 mg/L). Acknowledgement This work was supported by the National Key Research and Development Program of China (SQ2017YFGH001891), Shaanxi Program for Overseas Returnees (No. 2018012) and Xi'an Polytechnic University Ph.D. Research Startup Fund (No. BS201921). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.toxicon.2019.09.012. References Baek, S.H., Kim, D., Son, M., Yun, S.M., Kim, Y.O., 2015. Seasonal distribution of phytoplankton assemblages and nutrient-enriched bioassays as indicators of nutrient limitation of phytoplankton growth in Gwangyang Bay, Korea. Estuar. Coast Shelf Sci. 163, 265–278. Bertrand, M., Poirier, I., 2005. Photosynthetic organisms and adequate of metals. Photosynthetica 43 (3), 345–353. Boyd, P.W., Watson, A.J., Law, C.S., Abraham, E.R., Trull, T., Murdoch, R., Bakker, D.C.E., Bowie, A.R., Buesseler, K.O., Chang, H., Charette, M., Croot, P., Downing, K., Frew, R., Gall, M., Hadfield, M., Hall, J., Harvey, M., Jameson, G., LaRoche, J., Liddicoat, M., Ling, R., Maldonado, M.T., McKay, R.M., Nodder, S., Pickmere, S., Pridmore, R., Rintoul, S., Safi, K., Sutton, P., Strzepek, R., Tanneberger, K., Turner, S., Waite, A., Zeldis, J., 2000. A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization. Nature 407 (6805), 695–702. Boyd, P.W., Law, C.S., Wong, C.S., Nojiri, Y., Tsuda, A., Levasseur, M., Takeda, S., Rivkin, R., Harrison, P.J., Strzepek, R., Gower, J., McKay, R.M., Abraham, E., Arychuk, M., Clarke, J.B., Crawford, W., Crawford, D., Hale, M., Harada, K., Johnson, K., Kiyosawa, H., Kudo, I., Marchetti, A., Miller, W., Needoba, J., Nishioka, J., Ogawa, H., Page, J., Robert, M., Saito, H., Sastri, A., Sherry, N., Soutar, T., Sutherland, N., Taira, Y., Whitney, F., Wong, S.K.E., Yoshimura, T., 2004. The decline and fate of an iron-induced subarctic phytoplankton bloom. Nature 428 (6982), 549–553. Brand, L.E., 1983. Limitation of marine phytoplankton reproductive rates by zinc, manganese, and iron. Limnol. Oceanogr. 28 (6), 1182–1198. Chaloub, R.M., Magalhães, C.C.P., Dos Santos, C.P., 2005. Early toxic effects of zinc on psii of Synechocystis aquatilis F. Aquatilis (Cyanophyceae)1. J. Phycol. 41 (6), 1162–1168. Chen, L., Chen, J., Zhang, X., Xie, P., 2016. A review of reproductive toxicity of
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